Antimicrobial resistance in Gram-positive bacteria from Timorese River Buffalo (Bubalus bubalis) skin microbiota.

PubMed

Oliveira, Manuela; Monteiro, José L; Rana, Sílvia; Vilela, Cristina L

2010-06-01

The Timorese River Buffalo (Bubalus bubalis) plays a major role in the East Timor economy, as it is an important source of animal protein in human nutrition. They are widely spread throughout the country and are in direct contact with the populations. In spite of this proximity, information on their microbiota is scarce. This work aimed at characterizing the skin microbiota of the East Timorese River Buffalo and its antimicrobial resistance profile. Skin swab samples were taken from 46 animals in surveys conducted in three farms located in "Suco de Nairete", Lospalos district, during July and August 2006. Bacteria were isolated and identified according to conventional microbiological procedures. A total of 456 isolates were obtained, including Gram-positive (n = 243) and Gram-negative (n = 213) bacteria. Due to their importance as potential pathogens and as vehicles for antimicrobial resistance transmission, Gram-positive cocci (n = 27) and bacilli (n = 77) isolates were further characterized, and their antimicrobial resistance profile determined by the disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines. This study shows the high bacterial diversity of B. bubalis skin microbiota, representing an important first step towards understanding its importance and epidemiologic role in animal health. It also points out the potential role of these animals as vectors of antimicrobial resistant bacteria dissemination and the importance of antimicrobial resistance monitoring in developing countries.

Expression and characterization of constitutive heat shock protein 70.1 (HSPA-1A) gene in in vitro produced and in vivo-derived buffalo (Bubalus bubalis) embryos.

PubMed

Sharma, G T; Nath, A; Prasad, S; Singhal, S; Singh, N; Gade, N E; Dubey, P K; Saikumar, G

2012-12-01

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA-1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA-1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo-derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA-1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA-1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8-16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA-1A revealed that it shares 91-98% identity with other mammalian sequences. It can be concluded that higher level of HSPA-1A mRNA in IVP embryos in comparison with in vivo-derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA-1A gene could be used as a stress biomarker during pre-implantation development. © 2012 Blackwell Verlag GmbH.

Genomic structural differences between cattle and river buffalo identified through a combination and genomic and transcriptomic analysis

USDA-ARS?s Scientific Manuscript database

Water buffalo (Bubalus bubalis L.) is an important livestock species worldwide. Like many other livestock species, water buffalo lacks high quality and continuous reference genome assembly required for fine-scale comparative genomics studies. In this work, we present a dataset, which characterizes g...

Design and validation of a 90K SNP genotyping assay for the Water Buffalo (Bubalus bubalis)

USDA-ARS?s Scientific Manuscript database

The completion of the human genome sequence in 2001 was a major step forward in knowledge necessary to understand the variations between individuals. For farmed species, genomic information will facilitate the selection of animals optimised to live, and be productive in particular environments. The ...

Re-evaluation of endogenous development of Eimeria bareillyi Gill, Chhabra and Lall, 1963 in water buffalo (Bubalus bubalis).

PubMed

Dubey, J P

2018-04-25

Water buffalo (Bubalus bubalis) is important for the economy of Asia, South America and parts of Europe. Coccidiosis is an important cause of neonatal mortality in livestock, including buffalo. Of more than 12 species of Eimeria in buffalo, Eimeria bareillyi is the most pathogenic. There are uncertainties concerning its asexual and sexual development. During a previously reported outbreak of fatal enteritis associated with E. bareillyi in buffaloes in the Netherlands, sections of small intestine were re-evaluated histologically and by transmission electron microscopy (TEM) to seek details of endogenous development. Profuse asexual multiplication occurred in the jejunum and ileum. Light microscopic examination revealed that parasites divided in two (probably endodyogeny) or more organisms. There were two or more generations of morphologically different merozoites; some of these observations were confirmed by TEM. Details of gametogonic development, including oocyst wall formation are provided. Schizogonic and gametogonic development described in the present study can serve as a guide for differential diagnosis of Eimeria species in histological sections of intestines of buffaloes.

Production of nuclear transfer embryos by using somatic cells isolated from milk in buffalo (Bubalus bubalis).

PubMed

Golla, K; Selokar, N L; Saini, M; Chauhan, M S; Manik, R S; Palta, P; Singla, S K

2012-10-01

Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p < 0.01), than for MDF-NT embryos (16.44 ± 0.75%, 170.57 ± 4.50 respectively). We conclude that somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos. © 2012 Blackwell Verlag GmbH.

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up.

PubMed

Liang, X W; Lu, Y Q; Chen, M T; Zhang, X F; Lu, S S; Zhang, M; Pang, C Y; Huang, F X; Lu, K H

2008-04-15

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.

Evaluation of indirect TaSP enzyme-linked immunosorbent assay for diagnosis of tropical theileriosis in cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in Egypt.

PubMed

Mohamed, Amr M; Abdel-Rady, Ahmed; Ahmed, Laila S; El-Hosary, Amira

2012-05-25

The aim of the present study was to evaluate the validity of Theileria annulata surface protein (TaSP)-ELISA, in comparison with traditional microscopic test, for the diagnosis of T. annulata infection among Egyptian baladi cattle (Bos taurus) and water buffaloes (Bubalus bubalis). Molecular confirmation of infection using T. annulata merozoite surface (Tams-1) target amplification by PCR was used as a gold standard. A total of 76 clinically suspected animals including 64 baladi cattle and 12 water buffaloes were investigated in the current study by the three methods. Based on the PCR-confirmed results, the evaluation study revealed higher sensitivity of TaSP-ELISA (72.9% and 75%) as compared to microscopic examination (58.3% and 50%) among cattle and buffaloes, respectively. On the other hand, the specificity of TaSP-ELISA in diagnosis of T. annulata infection was higher (87.5%) in baladi cattle as compared to water buffaloes (37.5%). In conclusion, TaSP-ELISA was shown to be suitable for the diagnosis of T. annulata infection in cattle under field conditions. Copyright © 2011 Elsevier B.V. All rights reserved.

Baccharis megapotamica var. weirii poisoning in water buffalo (Bubalus bubalis).

PubMed

Oliveira-Filho, José C; Carmo, Priscila M S; Lucena, Ricardo B; Pierezan, Felipe; Barros, Claudio S L

2011-05-01

An outbreak of an acute disease in buffalo (Bubalus bubalis) caused by the ingestion of Baccharis megapotamica var. weirii occurred in the southern region of Brazil. Ten out of 50 buffalo died 24-48 hr after being introduced into a pasture containing abundant amounts of the plant. Factors influencing the ingestion of the plant and consequent toxicosis included hunger, stress caused by shipment, and unfamiliarity with the plant. Clinical signs included serous ocular discharge, incoordination, mild bloat, and muscle trembling. One buffalo was necropsied. Gross findings included dehydration, abundant liquid in the rumen, reddening of the mucosa of forestomachs, abomasum, and intestine, and edema of the wall of the rumen. The main histologic lesions were superficial to full thickness degeneration and necrosis of the stratified epithelium lining the forestomachs, necrosis of the intestinal mucosa, and widespread lymphoid necrosis. A calf (Bos taurus) was fed a single dose of 5 g/kg/body weight of B. megapotamica var. weirii harvested from the same site where the buffalo died. Twenty hours after the administration of the plant this calf died with clinical signs and lesions similar to those observed in the naturally poisoned buffalo. © 2011 The Author(s)

Expression, Localization of SUMO-1, and Analyses of Potential SUMOylated Proteins in Bubalus bubalis Spermatozoa

PubMed Central

Brohi, Rahim Dad; Wang, Li; Hassine, Najla Ben; Cao, Jing; Talpur, Hira Sajjad; Wu, Di; Huang, Chun-Jie; Rehman, Zia-Ur; Bhattarai, Dinesh; Huo, Li-Jun

2017-01-01

Mature spermatozoa have highly condensed DNA that is essentially silent both transcriptionally and translationally. Therefore, post translational modifications are very important for regulating sperm motility, morphology, and for male fertility in general. Protein sumoylation was recently demonstrated in human and rodent spermatozoa, with potential consequences for sperm motility and DNA integrity. We examined the expression and localization of small ubiquitin-related modifier-1 (SUMO-1) in the sperm of water buffalo (Bubalus bubalis) using immunofluorescence analysis. We confirmed the expression of SUMO-1 in the acrosome. We further found that SUMO-1 was lost if the acrosome reaction was induced by calcium ionophore A23187. Proteins modified or conjugated by SUMO-1 in water buffalo sperm were pulled down and analyzed by mass spectrometry. Sixty proteins were identified, including proteins important for sperm morphology and motility, such as relaxin receptors and cytoskeletal proteins, including tubulin chains, actins, and dyneins. Forty-six proteins were predicted as potential sumoylation targets. The expression of SUMO-1 in the acrosome region of water buffalo sperm and the identification of potentially SUMOylated proteins important for sperm function implicates sumoylation as a crucial PTM related to sperm function. PMID:28659810

Birth of cloned calves from vitrified-warmed zona-free buffalo (Bubalus bubalis) embryos produced by hand-made cloning.

PubMed

Saha, Ambikaprasanna; Panda, Sudeepta K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K

2013-01-01

The availability of techniques for the vitrification of cloned blastocysts can improve their effective use. The present study compared the developmental competence of buffalo cloned embryos derived from adult (BAF), newborn (BNF) and fetal fibroblast (BFF) before and after vitrification. Despite similar cleavage rates among the three groups, the blastocyst rate was lower for BAF- than BNF- and BFF-derived embryos (30.2±2.2% vs 41.7±1.7% and 39.1±2.1%, respectively; P<0.01). The total cell number of BNF-derived blastocysts was significantly higher (P<0.01) than that of BFF-derived blastocysts, which, in turn, was higher (P<0.01) than that of BAF-derived blastocysts. Following transfer of vitrified-warmed blastocysts to recipients, no pregnancy was obtained with fresh (n=8) or vitrified-warmed (n=18) BAF-derived blastocysts, whereas transfer of fresh BNF- (n=53) and BFF-derived (n=32) blastocysts resulted in four and three pregnancies, respectively, which aborted within 90 days of gestation. The transfer of vitrified-warmed BNF-derived blastocysts (n=39) resulted in the live birth of a calf weighing 41kg, which is now 23 months old and has no apparent abnormality, whereas the transfer of vitrified-warmed BFF-derived blastocysts (n=18) resulted in one live birth of a calf that died within 6h. These results demonstrate that cloned buffalo embryos cryopreserved by vitrification can be used to obtain live offspring.

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

PubMed

Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

2012-12-01

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures.

PubMed

Kitiyanant, Y; Saikhun, J; Chaisalee, B; White, K L; Pavasuthipaisit, K

2001-01-01

Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p < 0.05) percentage of blastocyst development was observed in the NT embryos activated by calcium ionophore and 6-DMAP when compared with 6-DMAP alone (33% versus 17%). The results indicate that the somatic nuclei from buffalo can be reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.

Identification and genetic characterization of rabies virus from Egyptian water buffaloes (Bubalus bubalis) bitten by a fox.

PubMed

El-Tholoth, Mohamed; El-Beskawy, Mohamed; Hamed, Mohamed F

2015-09-01

Rabies is caused by negative strand RNA-virus classified in the genus Lyssavirus, family Rhabdoviridae of the order Mononegavirales. The aim of the present study was to identify and analyze nucleotides sequence of nucleoprotein (N) gene of rabies virus (RABV) from two cases of water buffaloes (Bubalus bubalis) bitten by a fox in Egypt, 2013. The diseased buffaloes showed nervous manifestations with fever. Specimens from brains of the buffaloes with suspected rabies were collected. RABV in collected samples was identified using direct fluorescent antibody (dFA) technique, histopathological examination and reverse transcription-polymerase chain reaction (RT-PCR). Also, nucleotides sequence of partially amplified nucleoprotein (N) gene was compared with the other street strains of RABV available on GenBank. The results revealed that RABV antigen was identified in the brains of diseased buffaloes by dFA technique and the characteristic intracytoplasmic inclusions (Negri bodies) and RABV nucleic acid were detected by histopathology and RT-PCR, respectively. The identified virus showed close genetic relationship with street strains identified previously from dogs in different Governorates in Egypt and with strains identified in Israel and Jordan indicating transmission of the virus between Egyptian Governorates with a potential transmission from and/or to our neighboring countries.

Molecular Cloning, Identification, and Expression Patterns of Myostatin Gene in Water Buffalo (Bubalus Bubalis).

PubMed

Zhu, Peng; Li, Haiyang; Huang, Guiting; Cui, Jiayu; Zhang, Ruimen; Cui, Kuiqing; Yang, Sufang; Shi, Deshun

2018-01-02

Myostatin (MSTN), also named growth differentiation factor 8 (GDF8), is a transforming growth factor-β (TGF-β) family member with a key role in the negative regulation of skeletal muscle growth. However, its role in ovarian folliculogenesis remains unclear. To provide us with a basis for understanding this role, we cloned MSTN and examined its expression patterns in water buffalo (Bubalus bubalis). The complete ORF of the water buffalo MSTN gene is 1,128 nucleotides, which encode a 375 amino acid protein and sharing 99% identity at the deducted amino acid level with that of Bos taurus. Protein sequence analysis showed that MSTN is a weakly acerbic extracellular protein, consisting of signal peptides at 18-19 sites, a TGF-β propeptide, and a TGF-β domain. RT-PCR analyses demonstrated that water buffalo MSTN was expressed in multiple tissues but not limited to muscle. Immunohistochemistry staining confirmed the presence of MSTN in oocytes and granulosal cells. To our knowledge, this is the first study to confirm the expression of MSTN in the water buffalo ovary, suggesting an additional role of MSTN in water buffalo folliculogenesis, along with its role in skeletal muscle growth regulation. Further study of the regulatory mechanism of MSTN in water buffalo reproduction is warranted. MSTN, myostatin; ORF, open reading frame.

Production of cloned and transgenic embryos using buffalo (Bubalus bubalis) embryonic stem cell-like cells isolated from in vitro fertilized and cloned blastocysts.

PubMed

George, Aman; Sharma, Ruchi; Singh, Karn P; Panda, Sudeepta K; Singla, Suresh K; Palta, Prabhat; Manik, Radhaysham; Chauhan, Manmohan S

2011-06-01

Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned blastocysts, generated using fibroblasts derived from an adult buffalo (BAF). These nuclear transfer embryonic stem cell-like cells (NT-ES) grew in well-defined and dome-shaped colonies. The expression pattern of pluripotency marker genes was similar in both NT-ES and in vitro fertilization (IVF) embryo-derived embryonic stem cell-like cells (F-ES). Upon spontaneous differentiation via embryoid body formation, cells of different morphology were observed, among which predominant were endodermal-like and epithelial-like cell types. The ES cell-like cells could be passaged only mechanically and did not form colonies when plated as single cell suspension at different concentrations. When F-ES cell-like, NT-ES cell-like, and BAF cells of same genotype were used for hand-made cloning (HMC), no significant difference (p > 0.05) was observed in cleavage and blastocyst rate. Following transfer of HMC embryos to synchronized recipients, pregnancies were established only with F-ES cell-like and BAF cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES cell-like cells and BAF were used for HMC, no significant difference (p > 0.05) was observed between cleavage and blastocyst rate. In conclusion, here we report for the first time the derivation of ES cell-like cells from an adult buffalo, and its genetic modification. We also report the birth of a live cloned calf from buffalo ES cell-like cells.

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

PubMed Central

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF. PMID:26053554

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

PubMed

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

Equivalency of Buffalo (Bubalus Bubalis) Embryonic Stem Cells Derived From Fertilized, Parthenogenetic, and Hand-Made Cloned Embryos

PubMed Central

Muzaffar, Musharifa; Selokar, Naresh L.; Singh, Karn P.; Zandi, Mohammad; Singh, Manoj K.; Shah, Riaz A.; Chauhan, Manmohan S.; Singla, Suresh K.; Palta, Prabhat

2012-01-01

Abstract This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production. PMID:22582863

Equivalency of buffalo (Bubalus bubalis) embryonic stem cells derived from fertilized, parthenogenetic, and hand-made cloned embryos.

PubMed

Muzaffar, Musharifa; Selokar, Naresh L; Singh, Karn P; Zandi, Mohammad; Singh, Manoj K; Shah, Riaz A; Chauhan, Manmohan S; Singla, Suresh K; Palta, Prabhat; Manik, Radheysham

2012-06-01

This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production.

Effect of timing of development on total cell number and expression profile of HSP-70.1 and GLUT-1 in buffalo (Bubalus bubalis) oocytes and preimplantation embryos produced in vitro.

PubMed

Rajhans, Rajib; Kumar, G Sai; Dubey, Pawan K; Sharma, G Taru

2010-03-29

The present study was designed to compare the expression profile of two developmentally important genes (HSP-70.1 and GLUT-1) and TCN (total cell number) count in fast (group A) and slow (group B) cleaved buffalo embryos to access their in vitro developmental competence. Buffalo COCs (cumulus oocyte complexes) were collected from local abattoir ovaries and subjected to in vitro maturation in: TCM-199 supplemented with 10% FBS (fetal bovine serum), BSA (3 mg/ml), sodium pyruvate (0.25 mM) and 20 ng/ml EGF (epidermal growth factor) at 38.5 degrees C under 5% CO2. In vitro derived embryos were collected at 4-8, 8-16 cell, morula and blastocyst stages at specific time points for gene expression analysis and total cell count. A semiquantitative RT-PCR (reverse transcriptase-PCR) assay was used to determine the HSP-70.1 and GLUT-1 transcripts. Results showed that developmental competence and TCN count in fast (group A)-cleaving embryos was significantly (P<0.05) higher than in the slow group (group B). The gene transcript of HSP-70.1 and GLUT-1 was expressed in oocytes (immature and mature) and throughout the embryonic developmental stages in the fast group (group A), while in the slow (group B) cleaving embryos, the expression of HSP-70.1 was absent in all the embryonic developmental stages, and expression of GLUT-1 was absent after 8-16 cell stage. In conclusion, TCN count and expression profile of HSP-70.1 and GLUT-1 genes in buffalo embryos are different taking into account the cleavage rate. Quality of such embryos for research purposes, TCN and expression profiling of developmentally important genes should be employed to optimize the in vitro culture system to produce superior quality of embryos.

Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a retrospective study.

PubMed

Misra, A K; Rao, M M; Kasiraj, R; Reddy, N S; Pant, H C

1999-07-01

The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n = 52) or induced (n = 39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at -12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P < 0.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality.

The complete coding region sequence of river buffalo (Bubalus bubalis) SRY gene.

PubMed

Parma, Pietro; Feligini, Maria; Greppi, Gianfranco; Enne, Giuseppe

2004-02-01

The Y-linked SRY gene is responsible for testis determination in mammals. Mutations in this gene can lead to XY Gonadal Dysgenesis, an abnormal sexual phenotype described in humans, cattle, horses and river buffalo. We report here the complete river buffalo SRY sequence in order to enable the genetic diagnosis of this disease. The SRY sequence was also used to confirm the evolutionary divergence time between cattle and river buffalo 10 million years ago.

Ultrastructure changes in buffalo (Bubalus bubalis) oocytes before and after maturation in vitro with sericin.

PubMed

Gustina, Sri; Hasbi, Hasbi; Karja, Ni Wayan Kurniani; Setiadi, Mohamad Agus; Supriatna, Iman

2017-12-01

The aim of this research was to identify the changes in the cytoplasmic ultrastructure of immature and matured oocytes in buffalo (Bubalus bubalis). Oocytes were matured in vitro in tissue culture medium-199 with and without sericin, and then analyzed by light and transmission electron microscopy. The experiment result showed that the nuclear maturation rate of buffalo oocytes was significantly higher in the presence of sericin (80.6%) than without sericin (68.1%) (P < 0.05). The immature oocytes were characterized by cortical granule clusters in the ooplasm and the absence of perivitelline space (PVS). In contrast, the oocytes matured either with or without sericin showed the formation of PVS, erected microvilli, the migration of cortical granules to the cytoplasmic periphery, and the clear appearance of the mitochondria and vesicle in the oolemma. Interestingly, matured oocytes with sericin have smaller cortical granules than do immature oocytes (P < 0.05). In conclusion, supplementation of 0.05% sericin in the maturation medium can enhance the maturation rate of buffalo oocytes. Several cytoplasmic ultrastructures were relocated and modulated during the in vitro maturation process of buffalo oocytes: PVS development, cortical granules migration to periphery, and mitochondria and vesicles in the cortical region. The ultrastructure was similar between the groups with and without sericin. © 2017 Japanese Society of Animal Science.

Genetic characteristic of swamp buffalo (Bubalus bubalis) from Pampangan, South Sumatra based on blood protein profile

NASA Astrophysics Data System (ADS)

Windusari, Yuanita; Hanum, Laila; Wahyudi, Rizki

2017-11-01

Swamp buffalo (Bubalus bubalis) is an endemic species and one of the genetic wealth of South Sumatra with a distribution area in the district of Pampangan (OganIlir and OganOganIlir). Suspected inbreeding causes decreased phenotypic properties. Inbreeding among various swamp buffalo is certainly not only lower the qualities but also genotypes and phenotypes. It is of interest to determine kinship variants swamp buffaloes from Pampangan through the analysis of a blood protein profile. Blood protein profile of four variants swamps buffalo was studied by using five electrophoresis system i.e. pre-albumin (Palb), albumin (Alb), ceruloplasmin (Cp), transferrin (Tf) and transferrin post (Ptf). In this paper, it is obtained that there was no significant differences among the four variants of the buffaloes were used as a sample. Prealbumin has two alleles (Palb1 and Palb2), albumin has three alleles (Alba, AlbB, AlbC), ceruloplasmin has one allele (BPA), post-transferrin has one allele (PTFA) with an allele frequency 1.0000 at any time transferrin has two alleles (TFA and TFB) with the allele frequency of 0.7500 and 1.0000. Characteristics prealbumin (Palb), albumin (Alb), ceruloplasmin (Cp), and post-transferrin (P-tf) is monomorphic, while transferrin is polymorphic average heterozygosity values all loci (H) 0.1286. Based on average heterozygosity, the swamp buffalo (Bubalusbubalis) from Pampangan has low genetic variation and closest genetic relationship.

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 7 and comparative mapping to the cattle and human genomes

PubMed Central

Goldammer, T.; Weikard, R.; Miziara, M.N.; Brunner, R.M.; Agarwala, R.; Schäffer, A.A.; Womack, J.E.; Amaral, M.E.J.

2013-01-01

A preliminary radiation hybrid (RH) map containing 50 loci on chromosome 7 of the domestic river buffalo Bubalus bubalis (BBU; 2n = 50) was constructed based on a comparative mapping approach. The RH map of BBU7 includes thirty-seven gene markers and thirteen microsatellites. All loci have been previously assigned to Bos taurus (BTA) chromosome BTA6, which is known for its association with several economically important milk production traits in cattle. The map consists of two linkage groups spanning a total length of 627.9 cR5,000. Comparative analysis of the BBU7 RH5,000 map with BTA6 in cattle gave new evidence for strong similarity between the two chromosomes over their entire length and exposed minor differences in locus order. Comparison of the BBU7 RH5,000 map with the Homo sapiens (HSA) genome revealed similarity with a large chromosome segment of HSA4. Comparative analysis of loci in both species revealed more variability than previously known in gene order and several chromosome rearrangements including centromere relocation. The data obtained in our study define the evolutionary conserved segment on BBU7 and HSA4 to be between 3.5 megabases (Mb) and 115.8 Mb in the HSA4 (genome build 36) DNA sequence. PMID:18253035

Functional gene profiling through metaRNAseq approach reveals diet-dependent variation in rumen microbiota of buffalo (Bubalus bubalis).

PubMed

Hinsu, Ankit T; Parmar, Nidhi R; Nathani, Neelam M; Pandit, Ramesh J; Patel, Anand B; Patel, Amrutlal K; Joshi, Chaitanya G

2017-04-01

Recent advances in next generation sequencing technology have enabled analysis of complex microbial community from genome to transcriptome level. In the present study, metatranscriptomic approach was applied to elucidate functionally active bacteria and their biological processes in rumen of buffalo (Bubalus bubalis) adapted to different dietary treatments. Buffaloes were adapted to a diet containing 50:50, 75:25 and 100:0 forage to concentrate ratio, each for 6 weeks, before ruminal content sample collection. Metatranscriptomes from rumen fiber adherent and fiber-free active bacteria were sequenced using Ion Torrent PGM platform followed by annotation using MG-RAST server and CAZYmes (Carbohydrate active enzymes) analysis toolkit. In all the samples Bacteroidetes was the most abundant phylum followed by Firmicutes. Functional analysis using KEGG Orthology database revealed Metabolism as the most abundant category at level 1 within which Carbohydrate metabolism was dominating. Diet treatments also exerted significant differences in proportion of enzymes involved in metabolic pathways for VFA production. Carbohydrate Active Enzyme(CAZy) analysis revealed the abundance of genes encoding glycoside hydrolases with the highest representation of GH13 CAZy family in all the samples. The findings provide an overview of the activities occurring in the rumen as well as active bacterial population and the changes occurring through different dietary treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pheno- and genotyping of Brucella abortus biovar 5 isolated from a water buffalo (Bubalus bubalis) fetus: First case reported in the Americas.

PubMed

Martínez, Diana; Thompson, Carolina; Draghi, Graciela; Canavesio, Vilma; Jacobo, Roberto; Zimmer, Patricia; Elena, Sebastián; Nicola, Ana M; de Echaide, Susana Torioni

2014-09-17

An isolate of Brucella spp. from an aborted water buffalo (Bubalus bubalis) fetus was characterized based on its pheno- and genotype. The phenotype was defined by carbon dioxide requirement, hydrogen sulfide production, sensitivity to thionin and basic fuchsin and agglutination with Brucella A and M monospecific antisera. The genotype was based on the amplification of the following genes: bcsp31, omp2ab, and eri and the species-specific localization of the insertion sequence IS711 in the Brucella chromosome via B. abortus-B. melitensis-B. ovis-B. suis (AMOS)-PCR. Unexpectedly, the isolate showed a phenotype different from B. abortus bv 1, the most prevalent strain in cattle in Argentina, and from vaccine strain 19, currently used in bovines and water buffaloes. Genotyping supported the phenotypic results, as the analysis of the omp2ab gene sequence showed an identical pattern to either B. abortus bv 5 or B. melitensis. Finally, the AMOS PCR generated a 1700-bp fragment from the isolate, different than those amplified from B. abortus bv 1 (498bp) and B. melitensis (731bp), confirming the presence of B. abortus bv 5. The OIE/FAO Reference Laboratory for Brucellosis confirmed this typing. This is the first report of B. abortus bv 5 from a water buffalo in the Americas. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of peripheral blood for production of buffalo (Bubalus bubalis) embryos by handmade cloning.

PubMed

Jyotsana, Basanti; Sahare, Amol A; Raja, Anuj K; Singh, Karn P; Nala, Narendra; Singla, S K; Chauhan, M S; Manik, R S; Palta, P

2016-09-15

Buffalo embryos were produced by handmade cloning using peripheral blood-derived lymphocytes as donor cells. Although the blastocyst rate was lower (P < 0.01) for lymphocyte- than control skin fibroblast-derived embryos (6.6 ± 0.84% vs. 31.15 ± 2.97%), the total cell number (152.6 ± 23.06 vs. 160.1 ± 13.25) and apoptotic index (6.54 ± 0.95 vs. 8.45 ± 1.32) were similar. The global level of H3K9ac was higher (P < 0.05) in lymphocyte- than that in skin-derived blastocysts; whereas in IVF blastocysts, the level was not significantly different from the two cloned groups. The level of H3K27me3 was similar among the three groups. The expression level of DNMT1, DNMT3a, HDAC1, and IGF-1R was higher (P < 0.01) in lymphocytes than that in skin fibroblasts. The expression level of CDX2 was higher (P < 0.05) than that of DNMT3a, IGF-1R, OCT4, and NANOG was lower (P < 0.05) in lymphocyte-derived than in IVF blastocysts; that of DNMT1 and HDAC1 was similar in the two groups. The expression level of all these genes, except that of NANOG, was lower (P < 0.05) in lymphocyte- than in skin fibroblast-derived blastocysts. It is concluded that, peripheral blood-derived lymphocytes can be used for producing handmade cloning embryos in bubaline buffaloes. Copyright © 2016 Elsevier Inc. All rights reserved.

Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

PubMed

Sood, Tanushri Jerath; Lagah, Swati Viviyan; Sharma, Ankita; Singla, Suresh Kumar; Mukesh, Manishi; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2017-10-01

We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.

Verocytotoxin-producing Escherichia coli O26 in raw water buffalo (Bubalus bubalis) milk products in Italy.

PubMed

Lorusso, Vanessa; Dambrosio, Angela; Quaglia, Nicoletta Cristiana; Parisi, Antonio; La Salandra, Giovanna; Lucifora, Giuseppe; Mula, Giuseppina; Virgilio, Sebastiano; Carosielli, Leonardo; Rella, Addolorata; Dario, Marco; Normanno, Giovanni

2009-08-01

Escherichia coli 026 is known as a verocytotoxin-producing E. coli (VTEC) organism that causes severe foodborne diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Although cattle are the most important reservoir of VTEC, only a few reports on the role of water buffalo (Bubalus bubalis) as a reservoir of VTEC and on the presence of these organisms in their milk are available. However, in Southern Italy, where water buffalo are intensively reared, an outbreak of hemolytic uremic syndrome due to E. coli 026 has recently been reported, in which the consumption of typical dairy products was considered to be a common risk factor. The aims of this work were to assess the prevalence of E. coli O26 in raw water buffalo milk, to characterize the virulence gene profiles of the isolates, and to evaluate their phenotypic antimicrobial resistance pattern. Of 160 analyzed samples, 1 (0.6%) tested positive for E. coli O26, and the isolate showed the stx1+/stx2+/eae-/hlyA+ genotypic profile. The strain showed resistance against glycopeptides, macrolides, and penicillins. The presence of VTEC organisms in raw water buffalo milk could be considered to be a potential threat to consumers; however, the strict adherence to the processes used in the preparation of the most common buffalo dairy products could strongly mitigate the foodborne risk. To our knowledge, this article reports the first isolation and characterization of E. coli O26 VTEC in raw water buffalo milk.

Disposition of a long-acting oxytetracycline formulation in Thai swamp buffaloes (Bubalus bubalis).

PubMed

Poapolathep, S; Wongpanit, K; Imsilp, K; Tanhan, P; Klangkaew, N; Giorgi, M; Poapolathep, A

2017-04-01

The present study aimed to characterize the pharmacokinetic profile of oxytetracycline long-acting formulation (OTC-LA) in Thai swamp buffaloes, Bubalus bubalis, following single intramuscular administration at two dosages of 20 and 30 mg/kg body weight (b.w.). Blood samples were collected at assigned times up to 504 h. The plasma concentrations of OTC were measured by high-performance liquid chromatography (HPLC). The concentrations of OTC in the plasma were determined up to 264 h and 432 h after i.m. administration at doses of 20 and 30 mg/kg b.w., respectively. The C max values of OTC were 12.11 ± 1.87 μg/mL and 12.27 ± 1.92 μg/mL at doses of 20 and 30 mg/kg, respectively. The AUC last values increased in a dose-dependent fashion. The half-life values were 52.00 ± 14.26 h and 66.80 ± 10.91 h at doses of 20 and 30 mg/kg b.w, respectively. Based on the pharmacokinetic data and PK-PD index (T > MIC), i.m. administration of OTC at a dose of 30 mg/kg b.w once per week might be appropriate for the treatment of susceptible bacterial infection in Thai swamp buffaloes. © 2016 John Wiley & Sons Ltd.

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

PubMed

Lu, F; Jiang, J; Li, N; Zhang, S; Sun, H; Luo, C; Wei, Y; Shi, D

2011-09-15

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation. Copyright © 2011 Elsevier Inc. All rights reserved.

Handmade Cloned Buffalo (Bubalus bubalis) Embryos Produced from Somatic Cells Isolated from Milk and Ear Skin Differ in Their Developmental Competence, Epigenetic Status, and Gene Expression.

PubMed

Jyotsana, Basanti; Sahare, Amol A; Raja, Anuj K; Singh, Karn P; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat

2015-10-01

We compared the cloning efficiency of buffalo embryos produced by handmade cloning (HMC) using ear skin- and milk-derived donor cells. The blastocyst rate was lower (p < 0.05) for milk-derived than that for skin-derived embryos, whereas the total cell number and apoptotic index were similar. The global level of H3K9ac was higher (p < 0.05) in skin- than in milk-derived cells, whereas the level of H3K27me3 was similar in the two groups. The global level of H3K9ac was similar between milk-derived and in vitro-fertilized (IVF) blastocysts, which was higher (p < 0.05) than that in skin-derived blastocysts. The level of H3K27me3 was similar among the three groups. The expression level of IGF-1R and G6PD was higher (p < 0.05) in skin- than in milk-derived cells, whereas DNMT1, DNMT3a, and HDAC1 expression level was similar. In the blastocysts, the expression level of DNMT1, HDAC1, OCT4, and CDX2 was higher (p < 0.05) in skin-derived than that in IVF blastocysts. The expression level of DNMT3a and IGF-1R, was in the order (p < 0.05) skin-derived and IVF > milk-derived blastocysts and that of NANOG was (p < 0.05) IVF-> milk-derived > skin-derived blastocysts. The expression level of all these genes, except NANOG, was lower (p < 0.05) in milk- than in skin-derived or IVF blastocysts. In conclusion, milk-derived cells can be used for producing HMC embryos of quality similar to that of skin-derived embryos, although with a lower blastocyst rate.

First molecular characterisation of Cryptosporidium and Giardia from Bubalus bubalis (water buffalo) in Victoria, Australia.

PubMed

Abeywardena, Harshanie; Jex, Aaron R; von Samson-Himmelstjerna, Georg; Haydon, Shane R; Stevens, Melita A; Gasser, Robin B

2013-12-01

We conducted a molecular epidemiological survey of Cryptosporidium and Giardia from Bubalus bubalis (water buffalo) on two extensive farms (450 km apart) in Victoria, Australia. Faecal samples (n=476) were collected from different age groups of water buffalo at two time points (six months apart) and tested using a PCR-based mutation scanning-targeted sequencing-phylogenetic approach, employing markers within the small subunit of ribosomal RNA (designated pSSU) and triose phosphate isomerase (ptpi) genes. Based on pSSU data, Cryptosporidium parvum, Cryptosporidium bovis and Cryptosporidium genotypes 1, 2 (each 99% similar genetically to Cryptosporidium ryanae) and 3 (99% similar to Cryptosporidium suis) were detected in two (0.4%), one (0.2%), 38 (8.0%), 16 (3.4%) and one (0.2%) of the 476 samples tested, respectively. Using ptpi, Giardia duodenalis assemblages A and E were detected in totals of 56 (11.8%) and six (1.3%) of these samples, respectively. Cryptosporidium was detected on both farms, whereas Giardia was detected only on farm B, and both genera were detected in 1.5% of all samples tested. The study showed that water buffaloes on these farms excreted C. parvum and/or G. duodenalis assemblage A, which are consistent with those found in humans, inferring that these particular pathogens are of zoonotic significance. Future work should focus on investigating, in a temporal and spatial manner, the prevalence and intensity of such infections in water buffaloes in various geographical regions in Australia and in other countries. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of Sex of Embryo on Developmental Competence, Epigenetic Status, and Gene Expression in Buffalo (Bubalus bubalis) Embryos Produced by Hand-Made Cloning.

PubMed

Sandhu, Anjit; Mohapatra, Sushil K; Agrawal, Himanshu; Singh, Manoj K; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S

2016-10-01

Buffalo embryos were produced by hand-made cloning using skin fibroblasts from male and female buffaloes (n = 4 each) as donor cells for examining the effect of sex. Although the rate of blastocyst formation (43.8% ± 1.31% vs. 42.2% ± 1.22%) was similar, the total cell number (333 ± 10.4 vs. 270 ± 10.9) was higher (p < 0.05) whereas the apoptotic index (6.39 ± 0.25 vs. 8.52 ± 0.38) was lower (p < 0.05) for male than for female blastocysts. In the blastocysts, the global level of H3K18ac was found to be in the following order: male>female>IVF (in vitro fertilization) blastocysts (p < 0.05). The global level of H3K9me2 was not significantly different between male and female blastocysts and was higher (p < 0.05) compared with that in their IVF counterparts. The relative mRNA abundance of X-chromosome-linked (XIST, HPRT, PGK, and G6PD), apoptosis- (CASPASE3) and pregnancy-related genes (IFN-τ) was significantly higher (p < 0.05) whereas that of DNMT1 was significantly lower (p < 0.05) in female than in male blastocysts; however, in the case of apoptosis- (BCL-XL) and developmental competence-related genes (IGF1R and OCT4), the expression level was similar between the two groups. The gene expression level of OCT4 and IFN-τ but not of IGF1R was significantly lower (p < 0.05) in cloned than in IVF blastocysts. This study demonstrates that the epigenetic status, quality, and expression level of several genes but not the developmental competence are affected by the sex of cloned embryos.

Epigenetic Alteration of Donor Cells with Histone Deacetylase Inhibitor m-Carboxycinnamic Acid Bishydroxymide Improves the In Vitro Developmental Competence of Buffalo (Bubalus bubalis) Cloned Embryos.

PubMed

Agrawal, Himanshu; Selokar, Naresh Lalaji; Saini, Monika; Singh, Manoj Kumar; Chauhan, Manmohan Singh; Palta, Prabhat; Singla, Suresh Kumar; Manik, Radhey Sham

2018-02-01

Epigenetic reprogramming is an indispensable process during the course of mammalian development, but aberrant in cloned embryos. The aim of this study was to examine the effect of donor cell treatment with histone deacetylase (HDAC) inhibitor m-carboxycinnamic acid bishydroxymide (CBHA) on cloned embryo development and establish its optimal concentration. Different concentrations of CBHA (2.5, 5.0, 10.0, and 20.0 μM) were used to treat buffalo adult fibroblast cells for 24 hours and effect on cell proliferation, gene expression, and histone modifications was analyzed. Based on these experiments, the best concentration was chosen to determine the effect of enhanced gene activation mark on developmental rates. Among the different concentrations, CBHA at higher concentration (20 μM) shows the sign of apoptosis and stress as indicated by proliferation rate and gene expression data. CBHA treatment significantly decreased the activity of HDACs and increased the level of gene activation mark H3K9ac and H3K4me3, but could not alter the level of H3K27ac. Based on these experiments, 5 μM CBHA was chosen for treatment of donor cells used for the production of cloned embryos. There was no significant difference in cleavage rate between the control and CBHA treatment group (98.5% ± 1.5% vs. 99.0% ± 1.0%), whereas, blastocyst rate markedly improved (46.65% ± 1.94% vs. 57.18% ± 2.68%). The level of H3K9ac and H3K27me3 did not differ significantly in cloned blastocyst produced from either control or CBHA-treated cells. Altogether, these results suggested that donor cell treatment with CBHA supports the reprogramming process and improves the cloned preimplantation development.

Sustained delivery of exogenous melatonin influences biomarkers of oxidative stress and total antioxidant capacity in summer-stressed anestrous water buffalo (Bubalus bubalis).

PubMed

Kumar, Ashok; Mehrotra, S; Singh, G; Narayanan, K; Das, G K; Soni, Y K; Singh, Mahak; Mahla, A S; Srivastava, N; Verma, M R

2015-06-01

High ambient temperature during summer in tropical and subtropical countries predisposes water buffaloes (Bubalus bubalis) to develop oxidative stress having antigonadotropic and antisteroidogenic actions. Melatonin is a regulator of seasonal reproduction in photoperiodic species and highly effective antioxidant and free radical scavenger. Therefore, a study was designed to evaluate the effect of sustained-release melatonin on biomarkers of oxidative stress i.e., the serum malondialdehyde (MDA) and nitric oxide (NO), and the total antioxidant capacity (TAC). For the study, postpartum buffaloes diagnosed as summer anestrus (absence of overt signs of estrus, concurrent rectal examination, and RIA for serum progesterone) were grouped as treated (single subcutaneous injection of melatonin at 18 mg/50 kg body weight dissolved in sterilized corn oil as vehicle, n = 20) and untreated (subcutaneous sterilized corn oil, n = 8). Blood sampling for estimation of serum TAC and MDA (mmol/L) and NO (μmol/L) was carried out at 4 days of interval from 8 days before treatment till 28 days after treatment or for the ensuing entire cycle length. Results showed serum TAC concentration was higher in the treatment group with a significant (P < 0.05) increasing trend, whereas MDA and NO revealed a significant (P < 0.05) decline. Serum MDA and NO were higher in control compared with those of treatment group. Moreover, buffaloes in the treatment group showed 90% estrus induction with 18.06 ± 1.57 days mean interval from treatment to the onset of estrus. These results report that melatonin has a protective effect by elevating antioxidant status and reducing oxidative stress resulting in the induction of cyclicity in summer-stressed anestrous buffaloes. Copyright © 2015 Elsevier Inc. All rights reserved.

Detection of pseudocowpox virus in water buffalo (Bubalus bubalis) with vesicular disease in the state of São Paulo, Brazil, in 2016.

PubMed

Laguardia-Nascimento, Mateus; de Oliveira, Ana Paula Ferreira; Fernandes, Fernanda Rodas Pires; Rivetti, Anselmo Vasconcelos; Camargos, Marcelo Fernandes; Fonseca Júnior, Antônio Augusto

2017-12-01

Parapoxviruses are zoonotic viruses that infect cattle, goats and sheep; there have also been reports of infections in camels, domestic cats and seals. The objective of this report was to describe a case of vesicular disease caused by pseudocowpox virus (PCPV) in water buffalo (Bubalus bubalis) in Brazil. Sixty buffalo less than 6 months old exhibited ulcers and widespread peeling of the tongue epithelium. There were no cases of vesicular disease in pigs or horses on the same property. Samples were analysed by PCR and sequencing. Phylogenetic analysis in MEGA 7.01 was reconstructed using major envelope protein (B2L) by the Tamura three-parameter nucleotide substitution model and the maximum likelihood and neighbor joining models, both with 1000 bootstrap replicates. The genetic distance between the groups was analysed in MEGA using the maximum composite likelihood model. The rate variation among sites was modeled using gamma distribution. The presence of PCPV in the buffalo herd could be demonstrated in epithelium and serum. The minimum genetic distance between the isolated PCPV strain (262-2016) and orf virus and bovine papular stomatitis virus was 6.7% and 18.4%, respectively. The maximum genetic distance calculated was 4.6% when compared with a PCPV detected in a camel. Conclusions/Clinical Importance: The peculiar position of the isolated strain in the phylogenetic trees does not necessarily indicate a different kind of PCPV that infects buffalo. More samples from cattle and buffalo in Brazil must be sequenced and compared to verify if PCPV from buffalo are genetically different from samples derived from cattle.

m-carboxycinnamic acid bishydroxamide improves developmental competence, reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos.

PubMed

Agrawal, H; Selokar, N L; Saini, M; Singh, M K; Chauhan, M S; Palta, P; Singla, S K; Manik, R S

2018-05-07

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m-carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand-made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency-related genes OCT-4 and NANOG, and anti-apoptotic gene BCL-XL, and decreased (p < .05) that of pro-apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis-related genes p53 and CASPASE3 and epigenetics-related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern. © 2018 Blackwell Verlag GmbH.

Expression stability of two housekeeping genes (18S rRNA and G3PDH) during in vitro maturation of follicular oocytes in buffalo (Bubalus bubalis).

PubMed

Aswal, Ajay Pal Singh; Raghav, Sarvesh; De, Sachinandan; Thakur, Manish; Goswami, Surender Lal; Datta, Tirtha Kumar

2008-01-15

The present study was undertaken to evaluate the expression stability of two housekeeping genes (HKGs), 18S rRNA and G3PDH during in vitro maturation (IVM) of oocytes in buffalo, which qualifies their use as internal controls for valid qRT-PCR estimation of other oocyte transcripts. A semi quantitative RT-PCR system was used with optimised qRT-PCR parameters at exponential PCR cycle for evaluation of temporal expression pattern of these genes over 24 h of IVM. 18S rRNA was found more stable in its expression pattern than G3PDH.

Meat species identification and Halal authentication analysis using mitochondrial DNA.

PubMed

Murugaiah, Chandrika; Noor, Zainon Mohd; Mastakim, Maimunah; Bilung, Lesley Maurice; Selamat, Jinap; Radu, Son

2009-09-01

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.

'Soya milk Tris-based phytoextender reduces apoptosis in cryopreserved buffalo (Bubalus bubalis) spermatozoa'.

PubMed

Mohan, R; Atreja, S K

2014-10-01

The objective of this study was to investigate the effect of newly developed soya milk Tris (SMT)-based phytoextender as an alternative to egg yolk Tris (EYT) extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of apoptotic proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS-PAGE followed by immunoblotting against caspase-8, caspase-9, caspase-3, poly(ADP-ribose)polymerase (PARP), cytochrome c and apoptosis inducing factor (AIF). In addition, fluorescence microscopy was used for the detection of mitochondrial membrane potential (JC-1 assay) and apoptotic cells (annexin V-FITC/PI assay). The results obtained clearly indicate the significant (p < 0.05) reduction in the expression of caspase-3 (27 kDa), caspase-8 (53 kDa), caspase-9 (50 kDa) precursor and cytochrome c (17 kDa) in semen cryopreserved in SMT extender in comparison with EYT extender. A non-significant (p > 0.05) reduction in expression of PARP-DNA-binding subunit (24 kDa) was observed in SMT extender. No expression of AIF was found in cryopreserved semen samples. A significant (p < 0.05) increase in the mean percentage of cells having high mitochondrial membrane potential and a non-significant (p > 0.05) decrease in late apoptotic cells (AN+/PI+) was observed in SMT extender when compared to EYT extender. The results demonstrated that cryopreservation of buffalo semen in SMT-based phytoextender can replace the traditional egg yolk extenders as it reduces the expression of apoptotic proteins maintaining high mitochondrial membrane potential and gives better protection to sperms in terms of its non-animal origin. © 2014 Blackwell Verlag GmbH.

Effect of bypass fat supplementation on productive performance and blood biochemical profile in lactating Murrah (Bubalus bubalis) buffaloes.

PubMed

Ranjan, Amit; Sahoo, Biswanath; Singh, Vijay Kumar; Srivastava, Susant; Singh, Suresh Pratap; Pattanaik, Ashok Kumar

2012-10-01

The study investigated the effect of dietary supplementation of bypass fat on productive performance and blood biochemical profile of lactating Murrah buffaloes (Bubalus bubalis). Fifteen multiparous buffaloes (2-4 lactation) of early to mid lactation were divided in three homogenous groups T(1) (control), T(2), and T(3) of five each. The animals in T(1) were fed with a basal diet consisting of a concentrate mixture, green sorghum, and wheat straw as per requirements, while the animals in group T(2) and T(3) were fed with same ration supplemented with 0.7 % (100 g/day) and 1.4 % (200 g/day) bypass fat (on dry matter intake (DMI) basis), respectively. The feed intake, milk yield, and milk composition were not influenced by supplemental bypass fat. However, fat-corrected milk (6.5 %) yield was higher (P < 0.05) in T(3) (14.21) than that of T(1) (9.83) and similar with T(2) (11.05). Feed efficiency (milk yield/kg DMI) was higher (P < 0.05) in group T(3) (0.51) than that of T(1) (0.38) and T(2) (0.41) indicating that buffaloes fed with bypass fat which is 1.4 % (200 g/day) of the diet were economically more efficient. The serum cholesterol level was higher (P < 0.01) in bypass fat-supplemented group (T(2) and T(3)) of animals. Serum high density lipoprotein (HDL) cholesterol (good cholesterol) level was more (P < 0.05) than LDL cholesterol (bad cholesterol) level with higher dose of bypass fat in T(3) than T(2). It was concluded that bypass fat supplementation with 1.4 % of the diet (200 g/day) increased the fat-corrected milk production and feed efficiency along with serum HDL cholesterol level in lactating Murrah buffaloes.

The behaviour and welfare of buffaloes (Bubalus bubalis) in modern dairy enterprises.

PubMed

Napolitano, F; Pacelli, C; Grasso, F; Braghieri, A; De Rosa, G

2013-10-01

This review deals with the behaviour of river buffaloes (Bubalus bubalis), in confinement and in extensive conditions, also focusing on the effects of different housing and rearing conditions on their welfare. The behavioural repertoire expressed by buffaloes in extensive and intensive conditions is similar to those displayed by other domestic ruminants. However, through natural selection, buffaloes have also acquired several morphological, physiological and behavioural (i.e. wallowing) adaptations to hot climatic conditions. Buffaloes kept in intensive conditions and having no access to pasture and water for wallowing extend their periods of idling and are less often involved in investigative activities. Confinement is also associated with a reduction of space; however, no specific studies have been carried out to determine the specific requirements of this species. Space restriction can adversely affect various aspects of buffalo welfare, such as health (increased levels of lesions and injuries), social behaviour (increased number of agonistic interactions) and heat dissipation. The buffaloes, originating from tropical areas, are well adapted to large variations in food availability and quality, and to dietetic unbalances. As to human animal relationship, it has been observed that the incidence of stepping and kicking behaviour of buffaloes in the milking parlour is positively correlated with the frequency of oxytocin injections, whereas the frequency of positive stockperson interactions with the animals such as talking quietly, petting and gentle touching are negatively correlated with the number of kicks during milking. Data from farms where both dairy cattle and buffaloes are present show that avoidance distance measured in the pen is lower in buffaloes than in cattle. This may be attributed to the fact that buffaloes are generally recognised to be curious animals. Finally, the effects of different farming practices on animal-related indicators are described. However, these measures should be integrated into a monitoring protocol, such as the Welfare Quality® scheme, to reliably assess buffalo welfare in the current intensive farming conditions.

Prevalence of antibodies against Bubaline herpesvirus (BuHV-1) among Mediterranean water buffalo (Bubalus bubalis) with implications in buffalo trade.

PubMed

Caruso, C; Prato, R; Ingravalle, F; Vecchio, D; Sciarra, A; Ternavasio, M; Ceccarelli, L; Martucciello, A; Galiero, G; De Carlo, E; Masoero, L

2016-12-01

Both Bovine herpesvirus (BoHV-1) and Bubaline herpesvirus (BuHV-1) have been reported to cross the species barrier. Antibody seroconversion in glycoprotein E (gE) blocking ELISA during BuHV-1 infection has been documented. Recent diagnostic efforts have focused on the development and application of discriminatory tests to distinguish between infections with BoHV-1 and BuHV-1. To evaluate the impact and distribution of these two infections in water buffalo farms in two regions (Piedmont (n = 3) and Campania (n = 10), Italy) where infectious bovine rhinotracheitis control programs have been implemented. Sampling was carried out on 13 buffalo farms comprising 1089 animals using specific gE-indirect ELISA's test able to discriminate among BoHV-1 and BuHV-1 infections. 59.0% of animals reacted positive to ELISA (irrespective of whether BoHV-1 or BuHV-1 antigen was used) and 86.4% of these were reactive to BuHV-1 only, whereas 11.8% showed absorbance values for both antigens and were classified as inconclusive. There was a statistically significant age-related difference in BuHV-1 infection rates but not in overall individual (47% vs. 58%) or herd prevalence (100% vs. 90%) of infection between the two regions. The low percentage of sera reactive to BoHV-1 (1.8%, 12/643) indicates that BuHV-1 may be the main circulating alphaherpesvirus infection in Mediterranean water buffalo in the two study areas. Since Bubalus bubalis is included in Directive 64/432/EEC on animal health problems affecting intra-community trade in bovine animals, diagnostic testing with nonspecific ELISA for BoHV-1 infection in buffalo may yield false-positive reactions. This scenario could lead to economic losses and hamper buffalo trade and movement, particularly for reproduction purposes.

Effect of different seasons on concentration of plasma luteinizing hormone and seminal quality vis-à-vis freezability of buffalo bulls ( Bubalus bubalis)

NASA Astrophysics Data System (ADS)

Bahga, C. S.; Khokar, B. S.

1991-12-01

Seasonal variations in semen quality, freezability and plasma luteinizing hormone (LH) levels were studied between summer and spring. Semen volume, density and initial sperm motility did not differ significantly between different seasons. Plasma LH decreased between summer and spring but the differences were, however, not significant. Pre-freezing motility did not differ significantly but post-freezing motility varied significantly ( P<0.01) between seasons. Post-freezing motility was lowest during summer and highest during winter. It can be concluded that summer spermatozoa may be fragile and cannot withstand freezing stress. To increase reproductive efficiency in buffalo during summer, semen should be frozen during winter and spring and used during hot weather conditions. Seasonal variations in plasma LH levels were insignificant.

Strategies to overcome seasonal anestrus in water buffalo.

PubMed

de Carvalho, Nelcio Antonio Tonizza; Soares, Julia Gleyci; Baruselli, Pietro Sampaio

2016-07-01

Reproductive seasonality in buffalo (Bubalus bubalis) is characterized by behavioral, endocrine, and reproductive changes that occur over distinct periods of the year. During the nonbreeding season (spring and summer), the greater light-dark ratio (long days) suppresses estrus behavior and the occurrence of ovulation. Anestrous buffaloes have insufficient pulsatile of LH to support the final stages of follicular development, and subsequently, estrus behavior and ovulation do not occur, limiting reproductive efficiency, especially in artificial insemination (AI) programs. A number of therapeutic strategies designed to synchronize follicular wave emergence and ovulation have allowed for the use of AI throughout the year, overcoming seasonal anestrus in buffalo. These therapies also improve reproductive performance by increasing the service rate and pregnancy per AI in buffalo herds, regardless of reproductive seasonality. Copyright © 2016 Elsevier Inc. All rights reserved.

Water Buffalo (Bubalus bubalis) as a spontaneous animal model of Vitiligo.

PubMed

Singh, Vijay Pal; Motiani, Rajender K; Singh, Archana; Malik, Garima; Aggarwal, Rangoli; Pratap, Kunal; Wani, Mohan R; Gokhale, Suresh B; Natarajan, Vivek T; Gokhale, Rajesh S

2016-07-01

Vitiligo is a multifactorial acquired depigmenting disorder. Recent insights into the molecular mechanisms driving the gradual destruction of melanocytes in vitiligo will likely lead to the discovery of novel therapies, which need to be evaluated in animal models that closely recapitulate the pathogenesis of human vitiligo. In humans, vitiligo is characterized by a spontaneous loss of functional melanocytes from the epidermis, but most animal models of vitiligo are either inducible or genetically programmed. Here, we report that acquired depigmentation in water buffalo recapitulates molecular, histological, immunohistochemical, and ultrastructural changes observed in human vitiligo and hence could be used as a model to study vitiligo pathogenesis and facilitate the discovery and evaluation of therapeutic interventions for vitiligo. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Carcass Characteristics and Meat Quality of Swamp Buffaloes (Bubalus bubalis) Fattened at Different Feeding Intensities.

PubMed

Lambertz, C; Panprasert, P; Holtz, W; Moors, E; Jaturasitha, S; Wicke, M; Gauly, M

2014-04-01

Twenty-four male 1-year old swamp buffaloes (Bubalus bubalis) were randomly allocated to 4 groups. One group grazed on guinea grass (GG) and another on guinea grass and the legume Stylosanthes guianensis (GL). The other two groups were kept in pens and fed freshly cut guinea grass and concentrate at an amount of 1.5% (GC1.5) and 2.0% (GC2.0) of body weight, respectively. The effect of the different feeding intensities on carcass characteristics and meat quality were assessed. The mean body weight at slaughter was 398 (±16) kg. Average daily gain was higher in concentrate-supplemented groups (570 and 540 g/d in GC1.5 and GC2.0, respectively) when compared to GG (316 g/d) and GL (354 g/d) (p<0.01). Likewise, the warm carcass weight was higher in GC1.5 and GC2.0 compared to GG and GL. Dressing percentage was 48.1% and 49.5% in GC1.5 and GC2.0 in comparison to 42.9% and 44.8% observed in GG and GL, respectively. Meat of Longissimus throracis from GC1.5 and GC2.0 was redder in color (p<0.01), while water holding capacity (drip and thawing loss) was improved in pasture-fed groups (p<0.05). Protein and fat content of Longissimus thoracis was higher in animals supplemented with concentrate (p<0.01), as was cholesterol content (p<0.05), whereas PUFA:SFA ratio was higher and n-6/n-3 ratio lower (p<0.01) in pasture-fed buffaloes. Results of the present study showed that the supplementation of pasture with concentrate enhances the growth and carcass characteristics of swamp buffaloes expressed in superior dressing percentage, better muscling, and redder meat with a higher content of protein and fat, whereas animals grazing only on pasture had a more favorable fatty acid profile and water holding capacity. In conclusion, the supplementation of concentrate at a rate of about 1.5% of body weight is recommended to improve the performance and carcass quality of buffaloes.

Carcass Characteristics and Meat Quality of Swamp Buffaloes (Bubalus bubalis) Fattened at Different Feeding Intensities

PubMed Central

Lambertz, C.; Panprasert, P.; Holtz, W.; Moors, E.; Jaturasitha, S.; Wicke, M.; Gauly, M.

2014-01-01

Twenty-four male 1-year old swamp buffaloes (Bubalus bubalis) were randomly allocated to 4 groups. One group grazed on guinea grass (GG) and another on guinea grass and the legume Stylosanthes guianensis (GL). The other two groups were kept in pens and fed freshly cut guinea grass and concentrate at an amount of 1.5% (GC1.5) and 2.0% (GC2.0) of body weight, respectively. The effect of the different feeding intensities on carcass characteristics and meat quality were assessed. The mean body weight at slaughter was 398 (±16) kg. Average daily gain was higher in concentrate-supplemented groups (570 and 540 g/d in GC1.5 and GC2.0, respectively) when compared to GG (316 g/d) and GL (354 g/d) (p<0.01). Likewise, the warm carcass weight was higher in GC1.5 and GC2.0 compared to GG and GL. Dressing percentage was 48.1% and 49.5% in GC1.5 and GC2.0 in comparison to 42.9% and 44.8% observed in GG and GL, respectively. Meat of Longissimus throracis from GC1.5 and GC2.0 was redder in color (p<0.01), while water holding capacity (drip and thawing loss) was improved in pasture-fed groups (p<0.05). Protein and fat content of Longissimus thoracis was higher in animals supplemented with concentrate (p<0.01), as was cholesterol content (p<0.05), whereas PUFA:SFA ratio was higher and n-6/n-3 ratio lower (p<0.01) in pasture-fed buffaloes. Results of the present study showed that the supplementation of pasture with concentrate enhances the growth and carcass characteristics of swamp buffaloes expressed in superior dressing percentage, better muscling, and redder meat with a higher content of protein and fat, whereas animals grazing only on pasture had a more favorable fatty acid profile and water holding capacity. In conclusion, the supplementation of concentrate at a rate of about 1.5% of body weight is recommended to improve the performance and carcass quality of buffaloes. PMID:25049987

Protein Modeling and Molecular Dynamics Simulation of Cloned Regucalcin (RGN) Gene from Bubalus bubalis.

PubMed

Pillai, Harikrishna; Yadav, Brijesh Singh; Chaturvedi, Navaneet; Jan, Arif Tasleem; Gupta, Girish Kumar; Baig, Mohammad Hassan; Bhure, Sanjeev Kumar

2017-01-01

Regucalcin (RGN), a calcium regulating protein having anti-prolific, antiapoptotic functions, plays important part in the biosynthesis of ascorbic acid. It is a highly conserved protein that has been reported from many tissue types of various vertebrate species. Employing its effect of regulating enzyme activities through reaction with sulfhydryl group (-SH) and calcium, structural level study believed to offer a better understanding of binding properties and regulatory mechanisms of RGN, was performed. Using sample from testis of Bubalus bubalis, amplification of regucalcin (RGN) gene was subjected to characterization by performing digestion using different restriction endonucleases (RE). Alongside, cDNA was cloned into pPICZαC vector and transformed in DH5α host for custom sequencing. To get a better insight of its structural characteristics, three dimensional (3D) structure of protein sequence was generated using in silico molecular modelling approach. The full trajectory analysis of structure was achieved by the Molecular Dynamics (MD) that explains the stability, flexibility and robustness of protein during simulation in a time of 50ns. Molecular docking against 1,5-anhydrosorbitol was performed for functional characterization of RGN. Preliminary screening of amplified products on Agarose gel showed expected size of ~893 bp of PCR product corresponding to RGN. Following sequencing, BLASTp search of the target sequence revealed that it shares 91% similarity score with human senescence marker protein-30 (pdb id: 3G4E). Molecular docking of 1,5-anhydrosorbitol reveals information regarding important binding site residues of RGN. 1,5-anhydrosorbitol was found to interact with binding free energy of - 6.01 Kcal/mol. RMSD calculation of subunits A, B and D-F might be responsible for functional and conserved regions of modeled protein. Three dimensional structure of RGN was generated and its interactions with 1,5- anhydrosorbitol, demonstrates the role of key binding residues. Until now, no structural details were available for buffalo RGN proteins, hence this study will broaden the horizon towards understanding the structural and functional aspects of different proteins in cattle. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro–Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos

PubMed Central

Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Zandi, Mohammad; Manik, Radhey Sham; Singla, Suresh Kumar; Palta, Prabhat

2015-01-01

Abstract We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro–fertilized, somatic cell nuclear–transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro–produced blastocysts. Most of the ICMs (45–55%) resulted in formation of primary colonies that were subcultured after 8–10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture–derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture–derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium. PMID:26168169

Plasma hormonal and electrolyte alterations in cycling buffaloes ( Bubalus bubalis) during hot summer months

NASA Astrophysics Data System (ADS)

Singh, Narinder; Chaudhary, K. C.

1992-09-01

Plasma levels of progesterone, prolactin, luteinizing hormone, and electrolytes were monitored by radioimmunoassay in ten cycling buffaloes maintained at Punjab Agricultural University, Ludhiana during the hot summer months of June July. The plasma progesterone concentration ranged from 0.28±0.04 to 3.09±0.03 ng/ml at various stages of the oestrous cycle. Prolactin values ranged from 319±23 to 371±25 ng/ml and LH levels from 0.95±0.05 to 1.35±0.08 ng/ml. Concentrations differed significantly ( P⩽0.05) at various stages of the cycle. Levels of electrolytes, viz. Ca+ +, Na+ and K+, were well within the normal range. The high levels of prolactin, progesterone and LH during the hot summer were assessed in relation to poor reproductive efficiency in buffaloes.

Cloning of Buffalo, a Highly Valued Livestock Species of South and Southeast Asia: Any Achievements?

PubMed

Selokar, Naresh L; Saini, Monika; Palta, Prabhat; Chauhan, Manmohan S; Manik, Radhey S; Singla, Suresh K

2018-04-01

Buffalo (Bubalus bubalis) is a major source of milk, meat, and draught power in many developing countries in Asia. Animal cloning holds a lot of potential for fast multiplication of elite buffaloes and conservation of their valuable germplasm. Although the progress of buffalo cloning has been slow in comparison to cattle or pig, several breakthroughs were reported in buffalo cloning such as the production of cloned calves from somatic cells isolated from over one-decade old frozen-thawed semen or from urine-derived cells. Since the initiation of buffalo cloning, several approaches have been tried to refine nuclear transfer protocols. This has resulted in increasing the blastocyst production rate and improving their quality leading to an increase in live birth rate. In this review, we discuss current developments in buffalo cloning, its challenges, and the future roadmap.

Trace elements in raw milk of buffaloes (Bubalus bubalis) from Campania, Italy.

PubMed

Esposito, Mauro; Miedico, Oto; Cavallo, Stefania; Pellicanò, Roberta; Rosato, Guido; Baldi, Loredana; Chiaravalle, A Eugenio

2017-10-15

The profile of 18 trace elements was traced in 68 milk samples collected from buffalo farms in the territory known as the "Land of Fires" in the Campania region (Italy). This area has been polluted by the illegal dumping in fields of industrial or domestic waste, wich is sometimes then burned spreading toxic contaminants. Milk from buffaloes raised on rural farms might be a good indicator of environmental contamination risk in the human food chain. Trace element analysis in milk was performed using mass spectrometry. One milk sample was found to be non-compliant due to high Pb concentration. In the absence of threshold values for the elements, established through legislation, the results were compared with similar studies from other countries, and in most cases the content determined in this study was in agreement with values reported elsewhere and do not represent a risk to human health. Copyright © 2017 Elsevier Ltd. All rights reserved.

Authentication of meat from game and domestic species by SNaPshot minisequencing analysis.

PubMed

La Neve, Fabio; Civera, Tiziana; Mucci, Nadia; Bottero, Maria Teresa

2008-10-01

The aim of the present study is to develop an assay for the specific identification of meat from Capreolus capreolus, Cervus elaphus, Capra ibex, Rupicapra rupicapra, targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The assay is also intended to enable differentiation between meat from these wild species as well as Ovis aries, Capra hircus, Bubalus bubalis, Bos taurus and Sus scrofa domestic species. The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 232bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the multiplex PER (primer extension reaction) test were confirmed by fragment sequencing. The assay offers the possibility of discriminating nine species at the same time.

Long term effect of Ovum Pick-up in buffalo species.

PubMed

Neglia, Gianluca; Gasparrini, Bianca; Vecchio, Domenico; Boccia, Lucia; Varricchio, Ettore; Di Palo, Rossella; Zicarelli, Luigi; Campanile, Giuseppe

2011-02-01

The aim of this study was to evaluate the effect of an Ovum Pick-up (OPU) treatment carried out for 9 months in buffalo (Bubalus bubalis) species. Eight pluriparous non-lactating buffalo cows underwent OPU for 9 months. Recovered cumulus enclosed oocytes (COCs) were classified and COCs suitable for in vitro embryo production (IVEP) were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the blastocyst (Bl) stage. Animals were monitored for a total period of 270 days, but at the summer solstice, follicular turnover decreased and at the 68-day of the trial, we decided to increase the OPU sampling interval from 3-4 to 7 days. It was therefore possible to distinguish two phases: a first phase (18 sessions), during which OPU was carried out twice weekly and a second phase (16 sessions) during which OPU sessions were performed weekly. This reduction did not modify the percentage of good quality COCs, while the incidence of grade D COCs decreased (P<0.01). Furthermore, embryo production was higher in the second phase, either if embryos were calculated on the total recovered COCs (8.3% vs. 21.4%; P<0.01) and on grade A+B COCs (13.0% vs. 32.1%; P<0.01), that supposedly should have given similar blastocyst yield. During the total period of the trial it was possible to distinguish a first period of 6 months (34 sessions) characterized by blastocyst production (0.36 blastocyst/buffalo/session), followed by an unproductive period of 3 months (12 sessions), during which embryos were not produced. During the first 6 months a higher (P<0.01) number of follicles (5.06 vs. 3.71), small follicles (3.38 vs. 2.07), total COCs (2.58 vs. 1.56) and good quality (A+B) COCs (1.51 vs. 0.94) per subject/session were recorded compared to the last 3 months. No Blastocyst were produced during the second period, even if the percentage of grade A+B COCs was similar to that recorded during the first period. In conclusion, buffalo cows submitted to repeated OPU sampling for a 9-month period, showed a decline of follicle recruitment and oocyte collection after the first two months of samplings. After 6-month of samplings, in spite of the quality grade of the collected oocytes, we found a drop in their developmental competence. Copyright © 2011 Elsevier B.V. All rights reserved.

Congenital Malformations in River Buffalo (Bubalus bubalis)

PubMed Central

Albarella, Sara; Ciotola, Francesca; D’Anza, Emanuele; Coletta, Angelo; Zicarelli, Luigi; Peretti, Vincenzo

2017-01-01

Simple Summary Congenital malformations (due to genetic causes) represent a hidden danger for animal production, above all when genetic selection is undertaken for production improvements. These malformations are responsible for economic losses either because they reduce the productivity of the farm, or because their spread in the population would decrease the total productivity of that species/breed. River buffalo is a species of increasing interest all over the world for its production abilities, as proved by the buffalo genome project and the genetic selection plans that are currently performed in different countries. The aim of this review is to provide a general view of different models of congenital malformations in buffalo and their world distribution. This would be useful either for those who performed buffalo genetic selection or for researchers in genetic diseases, which would be an advantage to their studies with respect to the knowledge of gene mutations and interactions in this species. Abstract The world buffalo population is about 168 million, and it is still growing, in India, China, Brazil, and Italy. In these countries, buffalo genetic breeding programs have been performed for many decades. The occurrence of congenital malformations has caused a slowing of the genetic progress and economic loss for the breeders, due to the death of animals, or damage to their reproductive ability or failing of milk production. Moreover, they cause animal welfare reduction because they can imply foetal dystocia and because the affected animals have a reduced fitness with little chances of survival. This review depicts, in the river buffalo (Bubalus bubalis) world population, the present status of the congenital malformations, due to genetic causes, to identify their frequency and distribution in order to develop genetic breeding plans able to improve the productive and reproductive performance, and avoid the spreading of detrimental gene variants. Congenital malformations most frequently reported in literature or signaled by breeders to the Department of Veterinary Medicine and Animal Production of the University Federico II (Naples, Italy) in river buffalo are: musculoskeletal defects (transverse hemimelia, arthrogryposis, umbilical hernia) and disorders of sexual development. In conclusion this review put in evidence that river buffalo have a great variety of malformations due to genetic causes, and TH and omphalocele are the most frequent and that several cases are still not reported, leading to an underestimation of the real weight of genetic diseases in this species. PMID:28208595

Trehalose improves semen antioxidant enzymes activity, post-thaw quality, and fertility in Nili Ravi buffaloes (Bubalus bubalis).

PubMed

Iqbal, Sajid; Andrabi, Syed Murtaza Hassan; Riaz, Amjad; Durrani, Aneela Zameer; Ahmad, Nasim

2016-03-15

Our objectives were to study the effect of trehalose in extender on (1) antioxidant enzymes profile during cryopreservation (after dilution, before freezing, and after thawing), (2) in vitro quality (after thawing), and (3) in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 20) from four buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of trehalose (0.0, 15, 30, 45, and 60 mM) and frozen in French straws. At post dilution, profile of sperm catalase (U/mL) was higher (P < 0.05) in extenders containing 15, 30, and 45 mM of trehalose as compared to control. Although profiles of superoxide dismutase (U/mL) and total glutathione (μM) were higher (P < 0.05) in extenders containing 15 and 30 mM of trehalose as compared to control. At prefreezing, sperm catalase, superoxide dismutase, and total glutathione profiles were higher (P < 0.05) in all the treatment groups as compared to control. At post thawing, the profiles of catalase and total glutathione were higher (P < 0.05) in extender containing 30-mM trehalose as compared to other treatment groups and control. Whereas, profile of superoxide dismutase was higher (P < 0.05) in extenders containing 30, 45, and 60 mM of trehalose as compared to control and 15mM group. Post thaw total sperm motility (%) was higher (P < 0.05) in extender containing 30-mM trehalose as compared to control and 15 and 60-mM groups. Although sperm progressive motility (%), rapid velocity (%), average path velocity (μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), plasma membrane (structural and functional, %), acrosome (%), and DNA (%) integrity were higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment groups and control. The fertility rates (61% vs. 43%) were higher (P < 0.05) in buffaloes inseminated with semen doses cryopreserved in extender containing 30 mM of trehalose than the control. It is concluded that addition of 30-mM trehalose in extender improves the semen antioxidant enzymes activity, post thaw quality, and fertility in Nili Ravi buffaloes. Copyright © 2016 Elsevier Inc. All rights reserved.

First molecular characterization of Cryptosporidium and Giardia from bovines (Bos taurus and Bubalus bubalis) in Sri Lanka: unexpected absence of C. parvum from pre-weaned calves

PubMed Central

2014-01-01

Background The genetic characterization of Cryptosporidium and Giardia has important implications for investigating their epidemiology and underpins their control. We undertook the first molecular epidemiological survey of domestic bovids in selected regions of Sri Lanka to establish whether they excreted Cryptosporidium and/or Giardia with zoonotic potential. Methods Faecal samples were collected from dairy calves (n = 340; Bos taurus; < 3 months of age; weekly sampling for six weeks) and water buffaloes (n = 297; Bubalus bubalis; <6 months and ≥6 months of age; one sampling) from seven different farms in Sri Lanka. Genomic DNAs were extracted from individual faecal samples and then tested for the presence of parasite DNA using a PCR-based mutation scanning-targeted sequencing-phylogenetic approach, employing genetic markers within the small subunit of nuclear ribosomal RNA and 60 kDa glycoprotein genes (designated pSSU and pgp60, respectively) for Cryptosporidium, and within the triose phosphate isomerise (ptpi) gene for Giardia. Results Based on pSSU sequence data, C. bovis, C. ryanae and six new genotypes that were genetically similar but not identical to C. andersoni (n = 1), C. bovis (n = 1), C. ryanae (n = 3) and C. suis (n = 1) were recorded in cattle. For pSSU, two other, new genotypes were defined in water buffalo, which were genetically most similar to Cryptosporidium genotypes recorded previously in this host species in other countries including Australia. Consistent with the findings for pSSU, no species or genotypes of Cryptosporidium with zoonotic potential were detected using pgp60. Based on ptpi sequence data, G. duodenalis assemblages A and E were detected in four and 137 samples from cattle, respectively, and assemblage E in two samples from water buffaloes. Conclusions The present study showed that C. parvum, the most commonly reported zoonotic species of Cryptosporidium recognised in bovine calves globally, was not detected in any of the samples from pre-weaned calves tested in the present study. However, eight new genotypes were recorded. Future studies of different host species in various regions are required to investigate the molecular epidemiology of cryptosporidiosis and giardiasis in Sri Lanka and neighbouring countries in South Asia. PMID:24559043

Evaluation of lipid peroxidation and antioxidant status on fenvalerate, nitrate and their co-exposure in Bubalus bubalis.

PubMed

Gill, Kamalpreet Kaur; Sandhu, Harpal Singh; Kaur, Rajdeep

2015-09-01

The toxic effects of pesticides and minerals have been explored in different species, but still there is paucity of information regarding their combined toxicological effects. The present investigation reports oxidative stress induced by oral subacute exposure to fenvalerate (1 mg/kg) and sodium nitrate (20 mg/kg) alone, as well as in combination daily for 21 days in buffalo calves. Fenvalerate exposure produced significant elevation in lipid peroxidation (LPO), glutathione peroxidase (GPx), while it produced significant decline in blood glutathione (GSH) levels, superoxide dismutase (SOD) and catalase (CAT). No significant alteration was evidenced in nitric oxide (NOx) levels. Oral exposure to sodium nitrate produced significant inclination in LPO and NOx, while on the other hand significant depreciation in SOD and CAT with no significant change in GPx activity. Combined exposure to fenvalerate and sodium nitrate produced severe effects with an appreciably more prominent elevation in extent of LPO and decline in blood GSH levels. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular detection of Mycobacterium tuberculosis in cattle and buffaloes: a cause for public health concern.

PubMed

Abdel-Moein, Khaled A; Hamed, Osman; Fouad, Heba

2016-12-01

Tuberculosis is a re-emerging disease causing a growing public health burden. The current study was conducted to investigate the occurrence of Mycobacterium tuberculosis among cattle and buffaloes with tuberculous lesions. Typical tuberculous lesions were collected from 34 cattle and 34 buffaloes (Bubalus bubalis) through postmortem examination of slaughtered animals in abattoirs. DNAs were extracted from samples, and M. tuberculosis was identified by PCR. Positive samples were examined for resistance against rifampicin and isoniazid using GenoType MTBDRplus. Moreover, sera from 90 slaughterhouse workers, butchers, or meat inspectors were examined for the presence of M. tuberculosis antibodies using ELISA. Five cattle (14.7 %) and three buffaloes (8.8 %) tested positive. M. tuberculosis from one cattle was resistant to rifampicin and another was resistant to isoniazid. In addition, the seroprevalence of M. tuberculosis IgG among examined humans was 5.6 %. The occurrence of M. tuberculosis in cattle and buffaloes is a public health concern.

Bovine papillomavirus type 2 infects the urinary bladder of water buffalo (Bubalus bubalis) and plays a crucial role in bubaline urothelial carcinogenesis.

PubMed

Roperto, Sante; Russo, Valeria; Ozkul, Ayhan; Sepici-Dincel, Aylin; Maiolino, Paola; Borzacchiello, Giuseppe; Marcus, Ioan; Esposito, Iolanda; Riccardi, Marita Georgia; Roperto, Franco

2013-02-01

Bovine papillomavirus type 2 (BPV-2) has been shown to infect and play a role in urinary bladder carcinogenesis of buffaloes grazed on pastures with ferns from the Marmara and Black Sea Regions of Turkey. BPV-2 DNA has been found in both neoplastic and non-neoplastic lesions of the urinary bladder. Furthermore, this virus may be a normal inhabitant of the urinary bladder since BPV-2 DNA has also been detected in clinically normal buffaloes. The viral activation by fern immunosuppressant or carcinogen may trigger the urothelial cell transformation. The E5 oncoprotein was solely detected in urothelial tumours and appeared to be co-localized with the overexpressed and phosphorylated platelet derived growth factor (PDGF) β receptor in a double-colour immunofluorescence assay. Our results indicate that the E5-PDGF β receptor interaction also occurs in spontaneous tumours of the bubaline urinary bladder, revealing an additional role of BPV-2 in bladder carcinogenesis of buffaloes.

Utility of ultrasonography for diagnosis of superficial swellings in buffalo (Bubalus bubalis)

PubMed Central

ABOUELNASR, Khaled; EL-SHAFAEY, El-Sayed; MOSBAH, Esam; EL-KHODERY, Sabry

2016-01-01

We studied 72 buffalo with superficial swellings in the head (n=4), neck (n=5), chest wall (n=4), abdominal wall (n=28), limbs (n=16), gluteal region (n=8), perineal region (n=6) and udder (n=1). Ultrasonographically, the swellings varied according to type, duration, content and location. The clinical use of ultrasound to assess these superficial swellings allowed diagnosis of abscesses (n=21), hematomas (n=11), hernias (n=17), bursitis (n=13), urethral diverticula (n=6) and tumors (n=4). Ultrasonography could precisely discriminate each lesion type (sensitivity, 71–100%; specificity, 75–100%; odds ratio, 1.0–8.4; Confidence Interval, 74.2–20; and P value 0.001). The specificity for ultrasonographic evaluation of superficial swellings was 100% for hernias, urethral diverticula and tumors, whilst the lowest specificity was recorded for hematomas (75%) and abscesses (92%). In conclusion, ultrasonography provides a precise, non-invasive and fast technique for the evaluation, classification and subsequent treatment of a variety of superficial swellings in buffalo. PMID:27181085

Evaluation of fasting metabolism of growing water buffalo (Bubalus, Bubalis).

PubMed

Qin, Guangsheng; Zou, Caixia; Pang, Chunying; Yang, Bingzhuan; Liang, Xianwei; Liu, Jianxin; Xia, Zhongsheng; Wen, Qiuyan; Yan, Tianhai

2011-12-01

The objectives of the present study were to evaluate fasting metabolism (FM) of water buffalo (Bubalus, Bubalis) at three stages of growth (12, 18 and 24 months) in Guangxi, China. Five female water buffalo were used for each age group and their live weight was on average 254, 326 and 338 kg, respectively. All animals were of average body condition, healthy and de-wormed before start of the study. Prior to a 6-day fasting period, buffalo were offered a mixed diet of forage and concentrates (70% and 30%, dry matter basis) on a restricted nutritional level (419 kJ/kg(0.75) of metabolizable energy, ME) for 15 days. Gas exchanges for each animal were determined for 3 days from day 4 of starvation, using open-circuit respiratory head hoods. Fasting body weight was 0.918 of live weight (P < 0.001, r(2) = 0.99). Both fasting heat production (FHP) and FM (MJ/day) increased significantly with increased age of animals (P < 0.05). Linear regression analysis indicated a positive relationship between fasting body weight (kg(0.75)) and FHP (MJ/day, P < 0.01, r(2) = 0.49) or FM (MJ/day P < 0.01, r(2) = 0.52) when using individual animal data across three groups. However, when expressed as kJ/kg(0.75) of fasting body weight, the differences in FHP or FM between three groups of animals were not significant. The present average FHP and FM (322 and 347 kJ/kg(0.75) of fasting body weight) were compatible to those published in the literature for water buffalo, beef and dairy cattle. The present FM data were also used to estimate net energy (NE(m)) and ME (ME(m)) requirements for maintenance for water buffalo. The results for these two parameters were similar to those for FHP and FM. There was no significant difference between three groups of buffalo in NE(m) or ME(m) when expressed as kJ/kg(0.75) of live weight. The present average NE(m) and ME(m) values (347 and 506 kJ/kg(0.75) of live weight) are close to those proposed by the Agricultural and Food Research Council adopted in UK for beef and dairy cattle. The results indicate that the present FM data can be used as a basis for rationing water buffalo in China. © 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

Characterization of Smoc-1 uncovers two transcript variants showing differential tissue and age specific expression in Bubalus bubalis

PubMed Central

Srivastava, Jyoti; Premi, Sanjay; Kumar, Sudhir; Parwez, Iqbal; Ali, Sher

2007-01-01

Background Secreted modular calcium binding protein-1 (Smoc-1) belongs to the BM-40 family which has been implicated with tissue remodeling, angiogenesis and bone mineralization. Besides its anticipated role in embryogenesis, Smoc-1 has been characterized only in a few mammalian species. We made use of the consensus sequence (5' CACCTCTCCACCTGCC 3') of 33.15 repeat loci to explore the buffalo transcriptome and uncovered the Smoc-1 transcript tagged with this repeat. The main objective of this study was to gain an insight into its structural and functional organization, and expressional status of Smoc-1 in water buffalo, Bubalus bubalis. Results We cloned and characterized the buffalo Smoc-1, including its copy number status, in-vitro protein expression, tissue & age specific transcription/translation, chromosomal mapping and localization to the basement membrane zone. Buffalo Smoc-1 was found to encode a secreted matricellular glycoprotein containing two EF-hand calcium binding motifs homologous to that of BM-40/SPARC family. In buffalo, this single copy gene consisted of 12 exons and was mapped onto the acrocentric chromosome 11. Though this gene was found to be evolutionarily conserved, the buffalo Smoc-1 showed conspicuous nucleotide/amino acid changes altering its secondary structure compared to that in other mammals. In silico analysis of the Smoc-1 proposed its glycoprotein nature with a calcium dependent conformation. Further, we unveiled two transcript variants of this gene, varying in their 3'UTR lengths but both coding for identical protein(s). Smoc-1 evinced highest expression of both the variants in liver and modest to negligible in other tissues. The relative expression of variant-02 was markedly higher compared to that of variant-01 in all the tissues examined. Moreover, expression of Smoc-1, though modest during the early ages, was conspicuously enhanced after 1 year and remained consistently higher during the entire life span of buffalo with gradual increment in expression of variant-02. Immunohistochemically, Smoc-1 was localized in the basement membrane zones and extracellular matrices of various tissues. Conclusion These data added to our understandings about the tissue, age and species specific functions of the Smoc-1. It also enabled us to demonstrate varying expression of the two transcript variants of Smoc-1 amongst different somatic tissues/gonads and ages, in spite of their identical coding frames. Pursuance of these variants for their roles in various disease phenotypes such as hepatocellular carcinoma and angiogenesis is envisaged to establish broader biological significance of this gene. PMID:18042303

Preliminary Crystallographic Study of Hemoglobin from Buffalo (Bubalus bubalis): A Low Oxygen Affinity Species.

PubMed

Balasubramanian, Moovarkumudalvan; Moorthy, Ponnuraj Sathya; Neelagandan, Kamariah; Ponnuswamy, Mondikalipudur Nanjappa Gounder

2009-01-01

Hemoglobin is a tetrameric, iron-containing metalloprotein, which plays a vital role in the transportation of oxygen from lungs to tissues and carbon dioxide back to lungs. Though good amount of work has already been done on hemoglobins, the scarcity of data on three dimensional structures pertaining to low oxygen affinity hemoglobins from mammalian species, motivated our group to work on this problem specifically. Herein, we report the preliminary crystallographic analysis of buffalo hemoglobin, which belongs to low oxygen affinity species. The buffalo blood was collected, purified by anion exchange chromatography and crystallized with PEG 3350 using 50mM phosphate buffer at pH 6.7 as a precipitant by hanging drop vapor diffusion method. Data collection was carried out using mar345dtb image plate detector system. Buffalo hemoglobin crystallizes in orthorhombic space group P2(1)2(1)2(1) with one whole biological molecule (alpha2beta2) in the asymmetric unit with cell dimensions a=63.064A, b=74.677A, c=110.224A.

Empirical and bioinformatic characterization of buffalo (Bubalus bubalis) colostrum whey peptides & their angiotensin I-converting enzyme inhibition.

PubMed

Ashok, N R; Aparna, H S

2017-08-01

Whey based peptides are well known for their nutritional and multifunctional properties. In this context, whey proteins from buffalo colostrum & milk were digested by in vitro simulation digestion and analyzed by nano-LC-MS/MS. Functional protein association networks, gene annotations and localization of identified proteins were carried out. An ACE inhibitory peptide sorted from the library was custom synthesized and an in vitro ACE assay was performed. The study led to the identification of 74 small peptides which were clustered into 5 gene functional groups and majority of them were secretory proteins. Among the identified peptides, majority of them were found identical to angiotensin I-converting enzyme (ACE) inhibitors, antioxidant, antimicrobial, immunomodulatory and opioidal peptides. An octapeptide (m/z - 902.51, IQKVAGTW) synthesized was found to inhibit ACE with an IC 50 of 300±2µM. The present investigation thus establishes newer vista for food derived peptides having ACE inhibitory potential for nutraceutical or therapeutic applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic parameters for milk, fat and protein yields in Murrah buffaloes (Bubalus bubalis Artiodactyla, Bovidae)

PubMed Central

2010-01-01

The objective of the present study was to estimate genetic parameters for test-day milk, fat and protein yields and 305-day-yields in Murrah buffaloes. 4,757 complete lactations of Murrah buffaloes were analyzed. Co-variance components were estimated by the restricted maximum likelihood method. The models included additive direct genetic and permanent environmental effects as random effects, and the fixed effects of contemporary group, milking number and age of the cow at calving as linear and quadratic covariables. Contemporary groups were defined by herd-year-month of test for test-day yields and by herd-year-season of calving for 305-day yields. The heritability estimates obtained by two-trait analysis ranged from 0.15 to 0.24 for milk, 0.16 to 0.23 for protein and 0.13 to 0.22 for fat, yields. Genetic and phenotypic correlations were all positive. The observed population additive genetic variation indicated that selection might be an effective tool in changing population means in milk, fat and protein yields. PMID:21637608

Cholesterol and fatty acid composition of longissimus thoracis from water buffalo (Bubalus bubalis) and Brahman-influenced cattle raised under savannah conditions.

PubMed

Giuffrida-Mendoza, Maria; Arenas de Moreno, Lilia; Huerta-Leidenz, Nelson; Uzcátegui-Bracho, Sojan; Valero-Leal, Kutchynskaya; Romero, Sonia; Rodas-González, Argenis

2015-08-01

Male (n=66) water buffalo (Buffalo) and Brahman-influenced cattle (Brahman) were born, raised, weaned, fattened on grazing savannah and harvested at two different ages (19 and 24months) to compare lipid composition of the longissimus thoracis muscle. Half of the animals were castrated at seven months of age (MOA) to examine the castration effects. At 24 MOA Brahman steers showed the highest content of total lipids (P<0.05). No significant variation was detected in cholesterol content for either the main or interaction effects in the age groups. Some individual fatty acids varied with the species (P<0.05), however, interspecific similarities were found in fatty acid ratios. For health-related indices, only atherogenic index (AI) showed lower values in favor of Buffalo meat (P<0.05) at both harvesting ages. Although, meat derived from both bovid groups was leaner and showed lower cholesterol level, AI indicates that Buffalo meat might be beneficial from a human health standpoint. Copyright © 2015 Elsevier Ltd. All rights reserved.

Activation and Inhibition of The Wnt3A Signaling Pathway in Buffalo (Bubalus bubalis) Embryonic Stem Cells: Effects of WNT3A, Bio and Dkk1.

PubMed

Zandi, Mohammad; Shah, Syed Mohamad; Muzaffar, Musharifa; Kumar Singh, Manoj; Palta, Prabhat; Kumar Singla, Suresh; Sham Manik, Radhey; Chauhan, Manmohan Singh

2015-01-01

This research studies the effects of activation and inhibition of Wnt3A signaling pathway in buffalo (Bubalus bubalis) embryonic stem (ES) cell-like cells. To carry on this experimental study, the effects of activation and inhibition of Wnt3A signaling in buffalo ES cell-like cells were examined using Bio (0.5 mM) combined with WNT3A (200 ng/ml), as an activator, and Dickkopf-1 (Dkk1, 250 ng/ml), as an inhibitor, of the pathway. ES cells were cultured up to three weeks in ES cell medium without fibroblast growth factor-2 (FGF-2) and leukemia inhibitory factor (LIF), but in the presence of Bio, WNT3A, Bio+WNT3A and Dkk1. The effects of these supplements were measured on the mean area of ES cell colonies and on the expression levels of a number of important genes related to pluripotency (Oct4, Nanog, Sox2 and c-Myc) and the Wnt pathway (β-catenin). ES cell colonies cultured in ES cell medium that contained optimized quantities of LIF and FGF-2 were used as the control. Data were collected for week-1 and week-3 treated cultures. In addition, WNT3A-transfected ES cells were compared with the respective mock-transfected colonies, either alone or in combination with Dkk1 for expression of β-catenin and the pluripotency-related genes. Data were analyzed by ANOVA, and statistical significance was accepted at P<0.05. Among various examined concentrations of Bio (0.5-5 mM), the optimum effect was observed at the 0.5 mM dose as indicated by colony area and expressions of pluripotency-related genes at both weeks-1 and -3 culture periods. At this concentration,the expressions of Nanog, Oct3/4, Sox2, c-Myc and β-catenin genes were nonsignificantly higher compared to the controls. Expressions of these genes were highest in the Bio+WNT3A treated group, followed by the WNT3A and Bio-supplemented groups, and lowest in the Dkk1-treated group. The WNT-transfected colonies showed higher expressions compared to both mock and Dkk1-treated mock transfected colonies. WNT3A functions to maintain the pluripotency of ES cell-like cells both as an exogenous growth factor as well as an endogenously expressed gene. It complements the absence of FGF-2 and LIF, otherwise propounded essential for buffalo ES cell culture. WNT3A antagonizes the inhibitory effects of Dkk1 and acts in combination with its activator, Bio, to activate the Wnt signaling pathway.

Insight into Buffalo (Bubalus bubalis) RIG1 and MDA5 Receptors: A Comparative Study on dsRNA Recognition and In-Vitro Antiviral Response

PubMed Central

Singh, Manvender; Brahma, Biswajit; Maharana, Jitendra; Patra, Mahesh Chandra; Kumar, Sushil; Mishra, Purusottam; Saini, Megha; De, Bidhan Chandra; Mahanty, Sourav; Datta, Tirtha Kumar; De, Sachinandan

2014-01-01

RIG1 and MDA5 have emerged as important intracellular innate pattern recognition receptors that recognize viral RNA and mediate cellular signals controlling Type I interferon (IFN-I) response. Buffalo RIG1 and MDA5 genes were investigated to understand the mechanism of receptor induced antiviral response. Sequence analysis revealed that RIG1 and MDA5 maintain a domain arrangement that is common in mammals. Critical binding site residues of the receptors are evolutionary conserved among mammals. Molecular dynamics simulations suggested that RIG1 and MDA5 follow a similar, if not identical, dsRNA binding pattern that has been previously reported in human. Moreover, binding free energy calculation revealed that MDA5 had a greater affinity towards dsRNA compared to RIG1. Constitutive expressions of RLR genes were ubiquitous in different tissues without being specific to immune organs. Poly I:C stimulation induced elevated expressions of IFN-β and IFN-stimulated genes (ISGs) through interferon regulatory factors (IRFs) mediated pathway in buffalo foetal fibroblast cells. The present study provides crucial insights into the structure and function of RIG1 and MDA5 receptors in buffalo. PMID:24587036

Comparative antinociceptive and sedative effects of epidural romifidine and detomidine in buffalo (Bubalus bubalis).

PubMed

Marzok, M A; El-Khodery, S A

2017-07-01

In this study, comparative antinociceptive and sedative effects of epidural administration of romifidine and detomidine in buffalo were evaluated. Eighteen healthy adult buffalo, allocated randomly in three groups (two experimental and one control; n=6) received either 50 μg/kg of romifidine or detomidine diluted in sterile saline (0.9 per cent) to a final volume of 20 ml, or an equivalent volume of sterile saline epidurally. Antinociception, sedation and ataxia parameters were recorded immediately after drug administration. Epidural romifidine and detomidine produced mild to deep sedation and complete antinociception of the perineum, inguinal area and flank, and extended distally to the coronary band of the hindlimbs and cranially to the chest area. Times to onset of antinociception and sedation were significantly shorter with romifidine than with detomidine. The antinociceptive and sedative effects were significantly longer with romifidine than with detomidine. Romifidine or detomidine could be used to provide a reliable, long-lasting and cost-effective method for achieving epidural anaesthesia for standing surgical procedures in buffalo. Romifidine induces a longer antinociceptive effect and a more rapid onset than detomidine. Consequently, epidural romifidine may offer better therapeutic benefits in the management of acute postoperative pain. British Veterinary Association.

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50)

PubMed Central

Iamartino, Daniela; Pruitt, Kim D; Sonstegard, Tad; Smith, Timothy P L; Low, Wai Yee; Biagini, Tommaso; Bomba, Lorenzo; Capomaccio, Stefano; Castiglioni, Bianca; Coletta, Angelo; Corrado, Federica; Ferré, Fabrizio; Iannuzzi, Leopoldo; Lawley, Cynthia; Macciotta, Nicolò; McClure, Matthew; Mancini, Giordano; Matassino, Donato; Mazza, Raffaele; Milanesi, Marco; Moioli, Bianca; Morandi, Nicola; Ramunno, Luigi; Peretti, Vincenzo; Pilla, Fabio; Ramelli, Paola; Schroeder, Steven; Strozzi, Francesco; Thibaud-Nissen, Francoise; Zicarelli, Luigi; Ajmone-Marsan, Paolo; Valentini, Alessio; Chillemi, Giovanni; Zimin, Aleksey

2017-01-01

Abstract Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2n = 50) and the swamp (2n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1. PMID:29048578

Serological profile of buffalo (Bubalus bubalis) female calves vaccinated with standard Brucella abortus strain 19 vaccine using rose bengal, 2-mercaptoethanol and complement fixation tests.

PubMed

Nardi, G Júnior; Ribeiro, M G; Jorge, A M; Megid, J; Silva, L M P

2012-03-01

The serological profiles of 21 female buffaloes vaccinated between 3 and 8 months of age using Brucella abortus strain 19 (S19) were evaluated by rose bengal (RBT), 2-mercaptoethanol (2ME) and complement fixation (CFT) tests. The serum strains were collected in day zero, 15, 30, 45, 60th days and subsequently to each 30 months, until 720th day after vaccination. No animal showed reaction in day zero. In 15th day above 95% of animals revealed reaction in all tests. All the animals presented absence of reactions in CFT, RBT and 2ME tests at 270, 300 and 360 days after vaccination, respectively. Our finding highlighted early response in CFT compared than other conventional agglutination tests. None of animals presented oscillation of titers or reactions in any test after 360 day of study, which enables the use of these tests after this period without interference of antibodies from S19 vaccine origin between 3 and 8 months in buffalo heifers. Copyright © 2011 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Partial deoxygenation of extender improves sperm quality, reduces lipid peroxidation and reactive oxygen species during cryopreservation of buffalo (Bubalus bubalis) semen.

PubMed

Balamurugan, B; Ghosh, S K; Lone, S A; Prasad, J K; Das, G K; Katiyar, R; Mustapha, Abdul Rahman; Kumar, Ajay; Verma, M R

2018-02-01

The present study was designed to investigate the effect of partial deoxygenation of extender on sperm quality, lipid peroxidation (LPO) and reactive oxygen species (ROS) in buffalo (Bubalus bubalis) during cryopreservation of semen. Semen extender was prepared freshly and split into three sub-extenders [Extender I: control (non-deoxygenated), Extender II (partially deoxygenated by using LN 2 flushing) and Extender III (partially deoxygenated mechanically by vacuum pump)]. Amounts of dissolved oxygen (DO) were determined in all the three extenders and also in post-thaw semen. Ejaculates with mass motility of  ≥3+ and individual progressive motility of 70% or greater were collected from Murrah buffalo bulls and utilized in the study. Each semen sample was divided into Groups I (diluted with Extender I), II (diluted with Extender II) and III (diluted Extender III) with a maximum of 60 × 10 6 sperm/mL. French mini straws (0.25 mL) were filled with the extended semen samples, sealed with polyvinyl alcohol powder, kept for 3 h at 5 °C for equilibration and then stored in an automatic programmable freezer until the temperature of straws reached -145 °C followed by plunging the straws into liquid nitrogen (-196 °C). Semen samples were evaluated at pre-freeze and post-thaw stages for various variables [sperm motility, live sperm count, acrosomal integrity, hypo-osmotic swelling (HOS) response, LPO and ROS concentrations]. The mean DO was less (P < 0.05) in Extender II as compared to I and III. The DO was less (P < 0.05) in Group II (semen extended with Extender II) as compared with III (semen extended with Extender III) and I (semen extended with Extender I). The percentages for sperm motility, viability and intact acrosomes (PIA) were greater (P < 0.05) in Groups II and III as compared to the control group at the pre-freeze stage, while at the post-thaw stage, percentages of sperm motility, viability, PIA and HOS response were greater (P < 0.05) in Group II as compared with the control group and Group III. Pre-freeze HOS response (%) was greater (P < 0.05) in Group II as compared with the control and Group III. At the pre-freeze stage, sperm LPO and ROS were less (P < 0.05) in Groups II and III as compared with the control and at post-thaw stage, spermatic LPO and ROS concentrations were less (P < 0.05) in Group II than in the control group and Group III. In conclusion, partial deoxygenation of extender improves sperm quality, reduces sperm LPO and ROS concentrations in buffalo during cryopreservation. Partial deoxygenation of the extender with LN 2 flushing may be one of the ways for improving quality and fertility of frozen-thawed buffalo sperm. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescence in situ hybridization mapping of six loci containing genes involved in the dioxin metabolism of domestic bovids.

PubMed

Genualdo, Viviana; Spalenza, Veronica; Perucatti, Angela; Iannuzzi, Alessandra; Di Meo, Giulia Pia; Caputi-Jambrenghi, Annamaria; Vonghia, Gino; Rasero, Roberto; Nebbia, Carlo; Sacchi, Paola; Iannuzzi, Leopoldo

2011-05-01

Six loci containing genes involved in the dioxin metabolism (ARNT, AHR, CYP1A1, CYP1A2, CYP1B1 and AHRR) were assigned, for the first time, to cattle (Bos taurus, 2n = 60, BTA), river buffalo (Bubalus bubalis, 2n = 50, BBU), sheep (Ovis aries, 2n = 54, OAR) and goat (Capra hircus, 2n = 60, CHI) chromosomes by comparative FISH-mapping and R-banding using bovine BAC-clones. The following chromosome locations were found: ARNT to BTA3q21, BBU6q21, OAR1p21 and CHI3q21, AHR to BTA4q15, BBU8q15, OAR4q15 and CHI4q15; CYP1A1 and CYP1A2 to BTA21q17, BBU20q17, OAR18q17 and CHI21q17; CYP1B1 to BTA11q16, BBU12q22, OAR3p16 and CHI11q16, AHRR to BTA20q24, BBU19q24, OAR16q24 and CHI20q24. All loci were mapped at the same homoeologous chromosomes and chromosome bands of the four bovid species. Comparisons with corresponding human locations were also reported.

Association of a novel SNP in exon 10 of the IGF2 gene with growth traits in Egyptian water buffalo (Bubalus bubalis).

PubMed

Abo-Al-Ela, Haitham G; El-Magd, Mohammed Abu; El-Nahas, Abeer F; Mansour, Ali A

2014-08-01

Insulin-like growth factor 2 (IGF2) plays an important role in muscle growth and it might be used as a marker for the growth traits selection strategies in farm animals. The objectives of this study were to detect polymorphisms in exon 10 of IGF2 and to determine associations between these polymorphisms and growth traits in Egyptian water buffalo. PCR-single-strand conformation polymorphism (SSCP) and DNA sequencing methods were used to detect any prospective polymorphism. A novel single nucleotide polymorphism (SNP), C287A, was detected. It was a non-synonymous mutation and led to replacement of glutamine (Q) amino acid (aa) by histidine (H) aa. Three different SSCP patterns were observed: AA, AC, and CC, with frequencies of 0.540, 0.325, and 0.135, respectively. Association analyses revealed that the AA individuals had a higher average daily gain (ADG) than other individuals (CC and AC) from birth to 9 months of age. We conclude that the AA genotype in C287A SNP in the exon 10 of the IGF2 gene is associated with the ADG during the age from birth to 9 months and could be used as a potential genetic marker for selection of growth traits in Egyptian buffalo.

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50).

PubMed

Williams, John L; Iamartino, Daniela; Pruitt, Kim D; Sonstegard, Tad; Smith, Timothy P L; Low, Wai Yee; Biagini, Tommaso; Bomba, Lorenzo; Capomaccio, Stefano; Castiglioni, Bianca; Coletta, Angelo; Corrado, Federica; Ferré, Fabrizio; Iannuzzi, Leopoldo; Lawley, Cynthia; Macciotta, Nicolò; McClure, Matthew; Mancini, Giordano; Matassino, Donato; Mazza, Raffaele; Milanesi, Marco; Moioli, Bianca; Morandi, Nicola; Ramunno, Luigi; Peretti, Vincenzo; Pilla, Fabio; Ramelli, Paola; Schroeder, Steven; Strozzi, Francesco; Thibaud-Nissen, Francoise; Zicarelli, Luigi; Ajmone-Marsan, Paolo; Valentini, Alessio; Chillemi, Giovanni; Zimin, Aleksey

2017-10-01

Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2 n = 50) and the swamp (2 n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1. © The Author 2017. Published by Oxford University Press.

Molecular phylogenetic analysis of Fasciola flukes from eastern India.

PubMed

Hayashi, Kei; Ichikawa-Seki, Madoka; Mohanta, Uday Kumar; Singh, T Shantikumar; Shoriki, Takuya; Sugiyama, Hiromu; Itagaki, Tadashi

2015-10-01

Fasciola flukes from eastern India were characterized on the basis of spermatogenesis status and nuclear ITS1. Both Fasciola gigantica and aspermic Fasciola flukes were detected in Imphal, Kohima, and Gantoku districts. The sequences of mitochondrial nad1 were analyzed to infer their phylogenetical relationship with neighboring countries. The haplotypes of aspermic Fasciola flukes were identical or showed a single nucleotide substitution compared to those from populations in the neighboring countries, corroborating the previous reports that categorized them in the same lineage. However, the prevalence of aspermic Fasciola flukes in eastern India was lower than those in the neighboring countries, suggesting that they have not dispersed throughout eastern India. In contrast, F. gigantica was predominant and well diversified, and the species was thought to be distributed in the area for a longer time than the aspermic Fasciola flukes. Fasciola gigantica populations from eastern India were categorized into two distinct haplogroups A and B. The level of their genetic diversity suggests that populations belonging to haplogroup A have dispersed from the west side of the Indian subcontinent to eastern India with the artificial movement of domestic cattle, Bos indicus, whereas populations belonging to haplogroup B might have spread from Myanmar to eastern India with domestic buffaloes, Bubalus bubalis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Populations of domesticated cattle and buffalo in the Western Forest Complex of Thailand and their possible impacts on the wildlife community.

PubMed

Chaiyarat, Rattanawat; Srikosamatara, Sompod

2009-03-01

The Western Forest Complex (WEFCOM) of Thailand is comprised of many protected areas and has one of the highest wildlife populations in the country. Populations of wildlife in the WEFCOM have decreased dramatically over recent years. Rapid economic development has resulted in the conversion of forest into agricultural and pastoral land, which has directly and indirectly impacted the wildlife community. This research aimed to evaluate populations of domesticated cattle (Bos indicus) and buffalo (Bubalus bubalis) in the WEFCOM and their possible impacts on the wildlife community. Domesticated cattle and buffalo keepers from 1561 (or 3.3%) of houses in and near WEFCOM were interviewed. The average number of animals per household was 15.6 cattle and 8.5 buffalo. Most villagers released domesticated cattle and buffalo to forage in the protected areas. This tended to have a high impact on the wildlife community in Huai Kha Khaeng Wildlife Sanctuary and Tungyai Naresuan Wildlife Sanctuary. The least impacted areas were Luam Khlong Ngu National Park, Thong Pha Phum National Park and Chaleam Ratanakosin National Park. With a high risk to the wildlife community, law enforcement should be used in combination with a certain level of co-management with local communities.

Comparative study of the gut microbiome potentially related to milk protein in Murrah buffaloes (Bubalus bubalis) and Chinese Holstein cattle.

PubMed

Zhang, Jiachao; Xu, Chuanbiao; Huo, Dongxue; Hu, Qisong; Peng, Qiannan

2017-02-08

Previous studies suggested a close relationship between ruminant gut microbes and the mammary gland. In this study, shotgun metagenomic sequencing was used to reveal the differences in the intestinal microbiome potentially related to milk components in Murrah buffaloes and Chinese Holstein cattle. A PCoA based on the weighted Unifrac distances showed an apparent clustering pattern in the structure of intestinal microbiota between buffalo and cattle. We could attribute the structural difference to the genera of Sutterella, Coprococcus and Dorea. A further analysis of microbial functional features revealed that the biosynthesis of amino acids (including lysine, valine, leucine and isoleucine), lipopolysaccharide biosynthesis and cofactor/vitamin biosynthesis were enriched in the buffalo. In contrast, dairy cattle had higher levels of pyruvate metabolism and carbon fixation in photosynthetic organisms. A further correlation analysis based on different milk components and the typical microbiome uncovered a significant positive correlation between milk protein and the microbial biosynthesis of amino acids, which was also positively correlated in the genera of Parabacteroides, Dorea and Sutterella. This study will expand our understanding of the intestinal microbiome of buffalo and cattle as representative ruminants, as well as provide new views about how to improve the production and nutritional qualities of animal milk.

Polymorphisms in the ghrelin gene and their associations with milk yield and quality in water buffaloes.

PubMed

Gil, F M M; de Camargo, G M F; Pablos de Souza, F R; Cardoso, D F; Fonseca, P D S; Zetouni, L; Braz, C U; Aspilcueta-Borquis, R R; Tonhati, H

2013-05-01

Ghrelin is a gastrointestinal hormone that acts in releasing growth hormone and influences the body general metabolism. It has been proposed as a candidate gene for traits such as growth, carcass quality, and milk production of livestock because it influences feed intake. In this context, the aim of this study was to verify the existence of polymorphisms in the ghrelin gene and their associations with milk, fat and protein yield, and percentage in water buffaloes (Bubalus bubalis). A group of 240 animals was studied. Five primer pairs were used and 11 single nucleotide polymorphisms (SNP) were found in the ghrelin gene by sequencing. The animals were genotyped for 8 SNP by PCR-RFLP. The SNP g.960G>A and g.778C>T were associated with fat yield and the SNP g.905T>C was associated with fat yield and percentage and protein percentage. These SNP are located in intronic regions of DNA and may be in noncoding RNA sites or affect transcriptional efciency. The ghrelin gene in buffaloes influences milk fat and protein synthesis. The polymorphisms observed can be used as molecular markers to assist selection. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Muzzle secretion electrolytes as a possible indicator of sodium status in buffalo (Bubalus bubalis) calves: effects of sodium depletion and aldosterone administration.

PubMed

Kumar, S; Singh, S P

1981-01-01

In two separate experiments, the effects of sodium depletion and aldosterone administration on sodium and potassium concentrations in muzzle secretion, saliva and urine were studied in buffalo calves. Sodium deficiency in the animals was experimentally produced by unilateral parotid saliva deprivation for 18 days. During sodium depletion, the sodium levels in saliva and muzzle secretion gradually fell while the potassium level gradually rose. The concentrations of both of these cations in urine gradually fell during the course of sodium depletion. Aldosterone administration in normal (sodium-replete) animals simulated the effects of sodium depletion as far as cationic changes in saliva were concerned. However, aldosterone did not affect sodium and potassium concentration in the urine and in muzzle secretion in a manner similar to that caused by sodium depletion. Though the hormone decreased urinary sodium without affecting urinary potassium, it did not affect the muzzle sodium or potassium. Results suggest that aldosterone affects the composition of saliva and urine in buffaloes as it does in sheep and other ruminants. Similar changes in composition of muzzle secretion and saliva during sodium depletion indicate that the concentration of sodium in muzzle secretion could possibly be used to evaluate the sodium status of animals.

Phenotypic Characterization and Multivariate Analysis to Explain Body Conformation in Lesser Known Buffalo (Bubalus bubalis) from North India

PubMed Central

Vohra, V.; Niranjan, S. K.; Mishra, A. K.; Jamuna, V.; Chopra, A.; Sharma, Neelesh; Jeong, Dong Kee

2015-01-01

Phenotypic characterization and body biometric in 13 traits (height at withers, body length, chest girth, paunch girth, ear length, tail length, length of tail up to switch, face length, face width, horn length, circumference of horn at base, distances between pin bone and hip bone) were recorded in 233 adult Gojri buffaloes from Punjab and Himachal Pradesh states of India. Traits were analysed by using varimax rotated principal component analysis (PCA) with Kaiser Normalization to explain body conformation. PCA revealed four components which explained about 70.9% of the total variation. First component described the general body conformation and explained 31.5% of total variation. It was represented by significant positive high loading of height at wither, body length, heart girth, face length and face width. The communality ranged from 0.83 (hip bone distance) to 0.45 (horn length) and unique factors ranged from 0.16 to 0.55 for all these 13 different biometric traits. Present study suggests that first principal component can be used in the evaluation and comparison of body conformation in buffaloes and thus provides an opportunity to distinguish between early and late maturing to adult, based on a small group of biometric traits to explain body conformation in adult buffaloes. PMID:25656215

Effects of smallmouth buffalo, Ictiobus bubalus biomass on water transparency, nutrients, and productivity in shallow experimental ponds

USGS Publications Warehouse

Goetz, Daniel B.; Kroger, Robert; Miranda, Leandro E.

2014-01-01

The smallmouth buffalo Ictiobus bubalus is a native benthivore to floodplain lakes in the Yazoo River Basin, USA. Based on evidence from other benthivorous fish studies we hypothesized high biomasses of I. bubalus contribute to poor water quality conditions. We tested this hypothesis in shallow (< 1.5 m) 0.05 ha earthen ponds at three stocking biomasses over a 10-week period during the summer of 2012. The most notable results from the permutational multivariate analysis of variance suggest I. bubalus at high and moderate biomasses significantly (p < 0.05) enhanced turbidity and suspended solid levels while decreasing Secchi depth. Our results suggest that effects of I. bubalus on water clarity may have considerable ecological implications in natural habitats such as shallow floodplain lakes.

A review on breeding and genetic strategies in Iranian buffaloes (Bubalus bubalis).

PubMed

Safari, Abbas; Ghavi Hossein-Zadeh, Navid; Shadparvar, Abdol Ahad; Abdollahi Arpanahi, Rostam

2018-04-01

The aim of current study was to review breeding progress and update information on genetic strategies in Iranian buffaloes. Iranian buffalo is one of the vital domestic animals throughout north, north-west, south and south-west of Iran with measurable characteristics both in milk and meat production. The species plays an important role in rural economy of the country due to its unique characteristics such as resistance to diseases and parasites, having long productive lifespan and showing higher capability of consuming low-quality forage. In Iran, total production of milk and meat devoted to buffaloes are 293,000 and 24,700 tons, respectively. Selection activities and milk yield recording are carrying out by the central government through the Animal Breeding Centre of Iran. The main breeding activities of Iranian buffaloes included the estimation of genetic parameters and genetic trends for performance traits using different models and methods, estimation of economic values and selection criteria and analysis of population structure. Incorporating different aspects of dairy buffalo management together with improved housing, nutrition, breeding and milking, is known to produce significant improvements in buffalo production. Therefore, identifying genetic potential of Iranian buffaloes, selection of superior breeds, improving nutritional management and reproduction and developing the education and increasing the skills of practical breeders can be useful in order to enhance the performance and profitability of Iranian buffaloes.

A possible case of caprine-associated malignant catarrhal fever in a domestic water buffalo (Bubalus bubalis) in Switzerland

PubMed Central

2011-01-01

Background Malignant catarrhal fever (MCF) is a fatal herpesvirus infection, affecting various wild and domestic ruminants all over the world. Water buffaloes were reported to be particularly susceptible for the ovine herpesvirus-2 (OvHV-2) causing the sheep-associated form of MCF (SA-MCF). This report describes the first case of possibly caprine-associated malignant catarrhal fever symptoms in a domestic water buffalo in Switzerland. Case presentation The buffalo cow presented with persistent fever, dyspnoea, nasal bleeding and haematuria. Despite symptomatic therapy, the buffalo died and was submitted to post mortem examination. Major findings were an abomasal ulceration, a mild haemorrhagic cystitis and multifocal haemorrhages on the epicardium and on serosal and mucosal surfaces. Eyes and oral cavity were not affected. Histopathology revealed a mild to moderate lymphohistiocytic vasculitis limited to the brain and the urinary bladder. Although these findings are typical for MCF, OvHV-2 DNA was not detected in peripheral blood lymphocytes or in paraffin-embedded brain, using an OvHV-2 specific real time PCR. With the aid of a panherpesvirus PCR, a caprine herpesvirus-2 (CpHV-2) sequence could be amplified from both samples. Conclusions To our knowledge, this is the first report of malignant catarrhal fever in the subfamily Bovinae, where the presence of CpHV-2 could be demonstrated. The etiological context has yet to be evaluated. PMID:22132808

Differential actions of proteinases and neuraminidase on mammalian erythrocyte surface and its impact on erythrocyte agglutination by concanavalin A.

PubMed

Sharma, Savita; Gokhale, Sadashiv M

2012-12-01

Action of proteinases viz. trypsin and chymotrypsin, and neuraminidase on intact erythrocyte membrane proteins and glycophorins (sialoglycoproteins) exposed to cell surface and its impact on lectin (concanavalin A)-mediated agglutination were studied in Homo sapiens (human), Capra aegagrus hircus (goat) and Bubalus bubalis (buffalo). Membrane proteins and glycophorins analysis by SDS-PAGE as visualized by coomassie brilliant blue and periodic acid-schiff stains, respectively, and agglutination behaviour revealed marked differences: 1) there were prominent dissimilarities in the number and molecular weights of glycophorins in human, goat and buffalo erythrocyte membranes; 2) proteinase action(s) on human and buffalo erythrocyte surface membrane proteins and glycophorins showed similarity but was found different in goat; 3) significant differences in erythrocyte agglutinability with concanavalin A can be attributed to differences in membrane composition and alterations in the surface proteins after enzyme treatment; 4) a direct correlation was found between degradation of glycophorins and concanavalin A agglutinability; 5) action of neuraminidase specifically indicated the negative role of cell surface sialic acids in determining concanavalin A agglutinability of goat and buffalo erythrocytes, similar to human. Present studies clearly indicate that there are some basic differences in human, goat and buffalo erythrocyte membrane proteins, especially with respect to glycophorins, which determine the concanavalin A-mediated agglutination in enzyme treated erythrocytes.

Blood gas and serum biochemical RIs for healthy newborn Murrah buffaloes (Bubalus bubalis).

PubMed

Santana, André M; Silva, Daniela G; Clemente, Virna; Pizauro, Lucas J L; Bernardes, Priscila A; Santana, Clarissa H; Eckersall, Peter D; Fagliari, José J

2018-03-01

There is a lack of published work on RIs for newborn buffaloes. Establishing blood gas and serum biochemical RIs for newborn buffaloes is important for monitoring health. This study establishes blood gas and serum biochemical RIs of newborn buffaloes. Twenty-eight newborn buffaloes, 10-30 days old, were selected. Thirty blood biochemical variables were analyzed. The Anderson-Darling test was used to assess the normality of the distribution. The Dixon test and the Tukey test were used to identify outliers. The RI and 90% CI were determined using standard and robust methods and the Box-Cox transformation. A total of 30 RIs for healthy buffalo calves have been reported in this study. RIs for blood gas variables were reported for pH, partial pressure of oxygen (pO 2 ), partial pressure of carbon dioxide (pCO 2 ), saturation of O 2 (SO 2 ), bicarbonate (cHCO 3 - ), base excess (BE), total carbon dioxide (ctCO 2 ), and anion gap (AG). RIs for serum biochemical variables were reported for glucose (GLU), direct bilirubin (DB), total bilirubin (TB), AST, ALP, GGT, CK, LDH, creatinine (CREA), urea, cholesterol (CHOL), triglycerides (TG), Ca, P, Mg, Na, K, iCa, Cl, iron, total protein (TP), and albumin (ALB). This is the first reported study covering complete serum chemistry and blood gas RIs for healthy 1-month-old Murrah buffaloes. © 2018 American Society for Veterinary Clinical Pathology.

High prevalence of Schistosoma japonicum and Fasciola gigantica in bovines from Northern Samar, the Philippines.

PubMed

Gordon, Catherine A; Acosta, Luz P; Gobert, Geoffrey N; Jiz, Mario; Olveda, Remigio M; Ross, Allen G; Gray, Darren J; Williams, Gail M; Harn, Donald; Li, Yuesheng; McManus, Donald P

2015-02-01

The cause of zoonotic schistosomiasis in the Philippines is Schistosoma japonicum, which infects up to 46 mammalian hosts, including humans and bovines. In China, water buffaloes have been identified as major reservoir hosts for schistosomiasis japonica, contributing up to 75% of human transmission. In the Philippines, water buffaloes (carabao; Bubalus bubalis carabanesis) have, historically, been considered unimportant reservoirs. We therefore revisited the possible role of bovines in schistosome transmission in the Philippines, using the recently described formalin-ethyl acetate sedimentation (FEA-SD) technique and a qPCR assay to examine fecal samples from 153 bovines (both carabao and cattle) from six barangays in Northern Samar. A high prevalence of S. japonicum was found using qPCR and FEA-SD in both cattle (87.50% and 77.08%, respectively) and carabao (80.00% and 55.24%, respectively). The average daily egg output for each bovine was calculated at 195,000. High prevalence and infection intensity of F. gigantica was also found in the bovines by qPCR and FEA-SD (95.33% and 96.00%, respectively). The identification of bovines as major reservoir hosts for S. japonicum transmission suggests that bovine treatment and/or vaccination, as one becomes available, should be included in any future control program that aims to reduce the disease burden due to schistosomiasis in the Philippines.

Cross-cultural management of pest animal damage: a case study of feral buffalo control in Australia's Kakadu National Park.

PubMed

Robinson, Cathy J; Whitehead, Peter

2003-10-01

Government agencies responsible for pest animal management often assume that their views and assumptions about the benefits of control are widely shared, especially if these pests are exotics. This was certainly the case when tens of thousands of feral Asian water buffalo (Bubalus bubalis) were to be culled in Australia's Kakadu National Park as part of a national Brucellosis and Tuberculosis Eradication Campaign (BTEC). Implementation of the campaign sparked considerable dispute between officials and aboriginal and non-aboriginal interests about the risks posed by buffalo relative to their value as a potential resource. Drawing upon a variety of written and oral sources relating to the era of buffalo control in Kakadu, this paper critically analyzes the way in which detriment caused by buffalo was appraised and managed under the BTEC program. In particular, the paper focuses the ways in which the BTEC program affected aboriginal people in Kakadu, who view buffalo as a source of customary and economic benefit as well as a source of change on their lands. The paper then considers what lessons can be learned from the BTEC for the development of sensible feral management objectives and strategies. It is argued that effective management of feral animals such as buffalo will require environmental managers to engage with local people and involve them in the definition and management of pest animal damage and methods of control.

The influence of cumulus cells during in vitro fertilization of buffalo (Bubalus bubalis) denuded oocytes that have undergone vitrification.

PubMed

Attanasio, Laura; De Rosa, Anna; De Blasi, Marina; Neglia, Gianluca; Zicarelli, Luigi; Campanile, Giuseppe; Gasparrini, Bianca

2010-11-01

The aim of this work was to evaluate whether providing a support of cumulus cells during IVF of buffalo denuded oocytes submitted to vitrification-warming enhances their fertilizing ability. In vitro matured denuded oocytes were vitrified by Cryotop in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3M). Oocytes that survived vitrification were fertilized: 1) in the absence of a somatic support (DOs); 2) in the presence of bovine cumulus cells in suspension (DOs+susp); 3) on a bovine cumulus monolayer (DOs+monol); and 4) with intact bovine COCs in a 1:1 ratio (DOs+COCs). In vitro matured oocytes were fertilized and cultured to the blastocyst stage as a control. An increased cleavage rate was obtained from DOs+COCs (60.9%) compared to DOs, DOs+susp (43.6 and 38.4, respectively; P < 0.01) and DOs+monol (47.5%; P < 0.05). Interestingly, cleavage rate of DOs+COCs was similar to that of fresh control oocytes (67.8%). However, development to blastocysts significantly decreased in all vitrification groups compared to the control (P < 0.01). In conclusion the co-culture with intact COCs during IVF completely restores fertilizing capability of buffalo denuded vitrified oocytes, without improving blastocyst development. Copyright © 2010 Elsevier Inc. All rights reserved.

Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation.

PubMed

Naresh, S; Atreja, S K

2015-12-01

In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa. © 2015 Blackwell Verlag GmbH.

High Prevalence of Schistosoma japonicum and Fasciola gigantica in Bovines from Northern Samar, the Philippines

PubMed Central

Gordon, Catherine A.; Acosta, Luz P.; Gobert, Geoffrey N.; Jiz, Mario; Olveda, Remigio M.; Ross, Allen G.; Gray, Darren J.; Williams, Gail M.; Harn, Donald; Li, Yuesheng; McManus, Donald P.

2015-01-01

The cause of zoonotic schistosomiasis in the Philippines is Schistosoma japonicum, which infects up to 46 mammalian hosts, including humans and bovines. In China, water buffaloes have been identified as major reservoir hosts for schistosomiasis japonica, contributing up to 75% of human transmission. In the Philippines, water buffaloes (carabao; Bubalus bubalis carabanesis) have, historically, been considered unimportant reservoirs. We therefore revisited the possible role of bovines in schistosome transmission in the Philippines, using the recently described formalin-ethyl acetate sedimentation (FEA-SD) technique and a qPCR assay to examine fecal samples from 153 bovines (both carabao and cattle) from six barangays in Northern Samar. A high prevalence of S. japonicum was found using qPCR and FEA-SD in both cattle (87.50% and 77.08%, respectively) and carabao (80.00% and 55.24%, respectively). The average daily egg output for each bovine was calculated at 195,000. High prevalence and infection intensity of F. gigantica was also found in the bovines by qPCR and FEA-SD (95.33% and 96.00%, respectively). The identification of bovines as major reservoir hosts for S. japonicum transmission suggests that bovine treatment and/or vaccination, as one becomes available, should be included in any future control program that aims to reduce the disease burden due to schistosomiasis in the Philippines. PMID:25643317

In-gel and OFFGEL-based proteomic approach for authentication of meat species from minced meat and meat products.

PubMed

Naveena, Basappa M; Jagadeesh, Deepak S; Kamuni, Veeranna; Muthukumar, Muthupalani; Kulkarni, Vinayak V; Kiran, Mohan; Rapole, Srikanth

2018-02-01

Fraudulent mislabelling of processed meat products on a global scale that cannot be detected using conventional techniques necessitates sensitive, robust and accurate methods of meat authentication to ensure food safety and public health. In the present study, we developed an in-gel (two-dimensional gel electrophoresis, 2DE) and OFFGEL-based proteomic method for authenticating raw and cooked water buffalo (Bubalus bubalis), sheep (Ovis aries) and goat (Caprus hircus) meat and their mixes. The matrix-assisted liquid desorption/ionization time-of-flight mass spectrometric analysis of proteins separated using 2DE or OFFGEL electrophoresis delineated species-specific peptide biomarkers derived from myosin light chain 1 and 2 (MLC1 and MLC2) of buffalo-sheep-goat meat mix in definite proportions at 98:1:1, 99:0.5:0.5 and 99.8:0.1:0.1 that were found stable to resist thermal processing. In-gel and OFFGEL-based proteomic approaches are efficient in authenticating meat mixes spiked at minimum 1.0% and 0.1% levels, respectively, in triple meat mix for both raw and cooked samples. The study demonstrated that authentication of meat from a complex mix of three closely related species requires identification of more than one species-specific peptide due to close similarity between their amino acid sequences. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Dispositions of enrofloxacin and its major metabolite ciprofloxacin in Thai swamp buffaloes

PubMed Central

RUENNARONG, Nitwarat; WONGPANIT, Kannika; SAKULTHAEW, Chainarong; GIORGI, Mario; KUMAGAI, Susumu; POAPOLATHEP, Amnart; POAPOLATHEP, Saranya

2015-01-01

Given the limited information available in this species, the aim of this study was to investigate the pharmacokinetic characteristics of enrofloxacin (ER) and its major metabolite ciprofloxacin (CP) in buffaloes, Bubalus bubalis. ER was administered intravenously (i.v.) or subcutaneously (s.c.) to buffaloes at doses of 5.0 and 7.5 mg/kg BW, and plasma, urine and fecal samples were collected until 48 hr post-administration. The concentrations of ER and CP in the plasma, urine and feces were analyzed using high-performance liquid chromatography equipped with a fluorescence detector. The plasma concentrations of ER and CP could be determined up to 24 hr and 32 hr after i.v. and s.c. administrations at doses of 5.0 and 7.5 mg/kg BW, respectively. CP concentrations were always lower than those of parental drug. The s.c. bioavailability of ER was 52.36 ± 4.24% and 72.12 ± 5.39% at doses of 5.0 and 7.5 mg/kg BW, respectively. Both ER and CP were detectable in urine and feces up to 24 hr. ER and CP were mainly excreted via the urine. Based on the pharmacokinetic data and PK-PD indices, s.c. administration of ER at doses of 5.0 and 7.5 mg/kg BW might be appropriate for the treatment of susceptible bacterial diseases in Thai swamp buffaloes. PMID:26596287

Effects of smallmouth buffalo, Ictiobus bubalus biomass on water transparency, nutrients, and productivity in shallow experimental ponds.

PubMed

Goetz, D; Kröger, R; Miranda, L E

2014-05-01

The smallmouth buffalo Ictiobus bubalus is a native benthivore to floodplain lakes in the Yazoo River Basin, USA. Based on evidence from other benthivorous fish studies we hypothesized high biomasses of I. bubalus contribute to poor water quality conditions. We tested this hypothesis in shallow (<1.5 m) 0.05 ha earthen ponds at three stocking biomasses over a 10-week period during the summer of 2012. The most notable results from the permutational multivariate analysis of variance suggest I. bubalus at high and moderate biomasses significantly (p < 0.05) enhanced turbidity and suspended solid levels while decreasing Secchi depth. Our results suggest that effects of I. bubalus on water clarity may have considerable ecological implications in natural habitats such as shallow floodplain lakes.

A new extinct dwarfed buffalo from Sulawesi and the evolution of the subgenus Anoa: An interdisciplinary perspective

NASA Astrophysics Data System (ADS)

Rozzi, Roberto

2017-02-01

The fossil and extant faunas of Sulawesi, the largest island within the Wallacea biogeographic region, exhibit a high degree of endemism. The lowland anoa Bubalus depressicornis and the mountain anoa Bubalus quarlesi, two closely-related dwarfed buffaloes, are among the most peculiar endemic mammals of the region. Here, I describe a new species, Bubalus grovesi, from the Late Pleistocene/Holocene of South Sulawesi and I give a revised diagnosis of Anoa. Bubalus grovesi sp. nov. differs from all previously described Bubalus in both the size and proportions of the skeleton and in possessing a unique combination of discrete character states. Body mass estimates suggest an average mass of 117 kg for Bubalus grovesi sp. nov. and a body size reduction of about 90% with respect to a typical water buffalo. A comprehensive overview of body mass estimates of dwarfed buffaloes and differences in their dental and postcranial features is included. Finally, new evidence on the taxonomy and island dwarfing of the anoas and available data from different disciplines are used to discuss the timing and mode of their evolution. The representatives of the subgenus Anoa would be dwarfed forms of the Asian water buffalo that arose following dispersal to Sulawesi during the Middle/Late Pleistocene.

Differentiated evolutionary relationships among chordates from comparative alignments of multiple sequences of MyoD and MyoG myogenic regulatory factors.

PubMed

Oliani, L C; Lidani, K C F; Gabriel, J E

2015-10-16

MyoD and MyoG are transcription factors that have essential roles in myogenic lineage determination and muscle differentiation. The purpose of this study was to compare multiple amino acid sequences of myogenic regulatory proteins to infer evolutionary relationships among chordates. Protein sequences from Mus musculus (P10085 and P12979), human Homo sapiens (P15172 and P15173), bovine Bos taurus (Q7YS82 and Q7YS81), wild pig Sus scrofa (P49811 and P49812), quail Coturnix coturnix (P21572 and P34060), chicken Gallus gallus (P16075 and P17920), rat Rattus norvegicus (Q02346 and P20428), domestic water buffalo Bubalus bubalis (D2SP11 and A7L034), and sheep Ovis aries (Q90477 and D3YKV7) were searched from a non-redundant protein sequence database UniProtKB/Swiss-Prot, and subsequently analyzed using the Mega6.0 software. MyoD evolutionary analyses revealed the presence of three main clusters with all mammals branched in one cluster, members of the order Rodentia (mouse and rat) in a second branch linked to the first, and birds of the order Galliformes (chicken and quail) remaining isolated in a third. MyoG evolutionary analyses aligned sequences in two main clusters, all mammalian specimens grouped in different sub-branches, and birds clustered in a second branch. These analyses suggest that the evolution of MyoD and MyoG was driven by different pathways.

Optimization of culture conditions to support long-term self-renewal of buffalo (Bubalus bubalis) embryonic stem cell-like cells.

PubMed

Sharma, Ruchi; George, Aman; Kamble, Nitin Manchindra; Singh, Karn Pratap; Chauhan, Manmohan Singh; Singla, Suresh Kumar; Manik, Radhey Sham; Palta, Prabhat

2011-12-01

A culture system capable of sustaining self-renewal of buffalo embryonic stem (ES) cell-like cells in an undifferentiated state over a long period of time was developed. Inner cell masses were seeded on KO-DMEM+15% KO-serum replacer on buffalo fetal fibroblast feeder layer. Supplementation of culture medium with 5 ng/mL FGF-2 and 1000 IU/mL mLIF gave the highest (p<0.05) rate of primary colony formation. The ES cell-like cells' colony survival rate and increase in colony size were highest (p<0.05) following supplementation with FGF-2 and LIF compared to other groups examined. FGF-2 supplementation affected the quantitative expression of NANOG, SOX-2, ACTIVIN A, BMP 4, and TGFβ1, but not OCT4 and GREMLIN. Supplementation with SU5402, an FGFR inhibitor (≥20 μM) increased (p<0.05) the percentage of colonies that differentiated. FGFR1-3 and ERK1, K-RAS, E-RAS, and SHP-2, key signaling intermediates of FGF signaling, were detected in ES cell-like cells. Under culture conditions described, three ES cell lines were derived that, to date, have been maintained for 135, 95, and 85 passages for over 27, 19, and 17 months, respectively, whereas under other conditions examined, ES cell-like cells did not survive beyond passage 10. The ES cell-like cells were regularly monitored for expression of pluripotency markers and their potency to form embryoid bodies.

ROCK inhibitor Y-27632 enhances the survivability of dissociated buffalo (Bubalus bubalis) embryonic stem cell-like cells.

PubMed

Sharma, Ruchi; George, Aman; Chauhan, Manmohan S; Singla, Suresh; Manik, Radhey S; Palta, Prabhat

2013-01-01

This study investigated the effects of supplementation of culture medium with 10 μM Y-27632, a specific inhibitor of Rho kinase activity, for 6 days on self-renewal of buffalo embryonic stem (ES) cell-like cells at Passage 50-80. Y-27632 increased mean colony area (P<0.05) although it did not improve their survival. It decreased OCT4 expression (P<0.05), increased NANOG expression (P<0.05), but had no effect on SOX2 expression. It also increased expression of anti-apoptotic gene BCL-2 (P<0.05) and decreased that of pro-apoptotic genes BAX and BID (P<0.05). It increased plating efficiency of single-cell suspensions of ES cells (P<0.05). Following vitrification, the presence of Y-27632 in the vitrification solution or thawing medium or both did not improve ES cell colony survival. However, following seeding of clumps of ES cells transfected with pAcGFP1N1 carrying green fluorescent protein (GFP), Y-27632 increased colony formation rate (P<0.01). ES cell colonies that formed in all Y-27632-supplemented groups were confirmed for expression of pluripotency markers alkaline phosphatase, SSEA-4 and TRA-1-60, and for their ability to generate embryoid bodies containing cells that expressed markers of ectoderm, mesoderm and endoderm. In conclusion, Y-27632 improves survival of buffalo ES cells under unfavourable conditions such as enzymatic dissociation to single cells or antibiotic-assisted selection after transfection, without compromising their pluripotency.

Spermatozoa with high mitochondrial membrane potential and low tyrosine phosphorylation preferentially bind to oviduct explants in the water buffalo (Bubalus bubalis).

PubMed

Saraf, Kaustubh Kishor; Kumaresan, Arumugam; Chhillar, Shivani; Nayak, Samiksha; Lathika, Sreela; Datta, Tirtha Kumar; Gahlot, Subhash Chand; Karan, Prabha; Verma, Kiran; Mohanty, Tushar Kumar

2017-05-01

Although it is understood that spermatozoa are subjected to selection processes to form a functional sperm reservoir in the oviduct, the mechanism remains obscure. With the aim to understand the sperm selection process in the oviduct, in the present in vitro study, we analyzed mitochondrial membrane potential and tyrosine phosphorylation status in oviduct-explants bound and unbound spermatozoa. Frozen semen from Murrah buffalo bulls (n=10) used under progeny testing programme were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 4h. Mitochondrial membrane potential (MMP) and tyrosine phosphorylation status of bound and unbound spermatozoa were assessed at 1h and 4h of incubation. The proportion of spermatozoa with high MMP was significantly higher (P<0.001) among the bound spermatozoa (range 84.67-96.56%) compared to unbound (range 8.70-21.03%) spermatozoa. The proportion of tyrosine phosphorylated spermatozoa was significantly higher (P<0.001) among unbound population as compared to bound population. The proportion of spermatozoa displaying tyrosine phosphorylation at acrosomal area was significantly (P<0.05) lower in bound sperm population compared to unbound population. It was inferred that spermatozoa with high MMP and low tyrosine phosphorylation were preferred for oviduct-explants binding in the buffalo. Copyright © 2017 Elsevier B.V. All rights reserved.

l-Cysteine improves antioxidant enzyme activity, post-thaw quality and fertility of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa.

PubMed

Iqbal, S; Riaz, A; Andrabi, S M H; Shahzad, Q; Durrani, A Z; Ahmad, N

2016-11-01

The effects of l-cysteine in extender on antioxidant enzymes profile during cryopreservation, post-thaw quality parameters and in vivo fertility of Nili-Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of l-cysteine (0.0, 0.5, 1.0, 2.0 and 3.0 mm) and frozen in 0.5-ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P < 0.05) at pre-freezing and post-thawing in extender containing 2.0 mm l-cysteine as compared to other groups. Post-thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm s -1 ), straight line velocity (μm s -1 ), curvilinear velocity (μm s -1 ), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0 mm l-cysteine as compared to other groups (P < 0.05). The fertility rates (59 versus 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mm of l-cysteine than in the control. In conclusion, the addition of 2.0 mm l-cysteine in extender improved the antioxidant enzymes profile, post-thaw quality and in vivo fertility of Nili-Ravi buffalo bull spermatozoa. © 2016 Blackwell Verlag GmbH.

Bovine calves as ideal bio-indicators for fluoridated drinking water and endemic osteo-dental fluorosis.

PubMed

Choubisa, S L

2014-07-01

Relative susceptibility to fluoride (F) toxicosis in the form of osteo-dental fluorosis was observed in an observational survey of 2,747 mature and 887 immature domestic animals of diverse species living in areas with naturally fluoridated (>1.5 ppm F) drinking water. These animals included buffaloes (Bubalus bubalis), cattle (Bos taurus), camels (Camelus dromedarius), donkeys (Equus asinus), horses (Equus caballus), goats (Capra hircus), and sheep (Ovis aries). Of these mature and immature animals, 899 (32.7 %) and 322 (36.3 %) showed evidence of dental fluorosis with varying grades, respectively. Their incisor teeth were stained with light to deep brownish color. On clinical examination, 31.2 % mature and 10.7 % immature animals revealed periosteal exostoses, intermittent lameness, and stiffness of tendons in the legs as signs of skeletal fluorosis. The maximum susceptibility to fluoride toxicosis was found in bovines (buffaloes and cattle) followed by equines (donkeys and horses), flocks (goats and sheep), and camelids (camels). The bovine calves were found to be more sensitive and highly susceptible to F toxicosis and revealed the maximum prevalence (92.2 %) of dental fluorosis. This indicates that bovine calves are less tolerant and give early sign of F poisoning (dental fluorosis) and therefore, they can be considered as bio-indicators for fluoridated water as well as for endemicity of osteo-dental fluorosis. Causes for variation in susceptibility to F toxicosis (fluorosis) in various species of domestic animal are also discussed.

Gastrointestinal parasitic infection in diverse species of domestic ruminants inhabiting tribal rural areas of southern Rajasthan, India.

PubMed

Choubisa, S L; Jaroli, V J

2013-10-01

A total of 415 adult domesticated ruminants, 130 cattle (Bos taurus), 108 buffaloes (Bubalus bubalis), 94 goats (Capra hircus) and 83 sheep (Ovis aries) inhabiting tribal rural areas of southern Rajasthan, India were investigated for evidence of gastrointestinal protozoan and helminthic infections. In southern Rajasthan humid ecosystem is predominant and has number of perennial freshwater bodies. Fresh faecal samples of these animals were examined microscopically by direct wet smear with saline and 1 % Lugol's iodine and formalin ether concentration. Of these 296 (71.32 %) were found to be infected with different species of gastrointestinal parasites. The highest (93.84 %) prevalence of these parasitic infections was found in cattle followed by goats (82.97 %), sheep (55.42 %) and buffaloes (46.29 %). Except cattle no other ruminants revealed protozoan infection. A total 8 species of gastrointestinal parasites were encountered. Among these parasites Fasciola hepatica was the commonest (15.18 %) followed by Haemonchus contortus (11.32 %), Ancylostoma duodenale (10.36 %), Trichuris trichiura (9.15 %), Amphistome species (7.95 %), Moniezia expansa (6.98 %), Strongyloides stercoralis (4.57 %) and Balantidium coli (3.37 %). The prevalence rate of these parasitic infections also varied seasonally. The highest prevalence rate was found in rainy season (84.21 %) followed by winter (73.9 %) and summer (52.8 %). The possible causes for variation in prevalence of parasitic infections are also discussed.

Characterisation and In Silico Analysis of Interleukin-4 cDNA of Nilgai (Boselaphus tragocamelus) and Indian Buffalo (Bubalus bubalis)

PubMed Central

Saini, M.; Palai, T. K.; Das, D. K.; Hatle, K. M.; Gupta, P. K.

2013-01-01

Interleukin-4 (IL-4) produced from Th2 cells modulates both innate and adaptive immune responses. It is a common belief that wild animals possess better immunity against diseases than domestic and laboratory animals; however, the immune system of wild animals is not fully explored yet. Therefore, a comparative study was designed to explore the wildlife immunity through characterisation of IL-4 cDNA of nilgai, a wild ruminant, and Indian buffalo, a domestic ruminant. Total RNA was extracted from peripheral blood mononuclear cells of nilgai and Indian buffalo and reverse transcribed into cDNA. Respective cDNA was further cloned and sequenced. Sequences were analysed in silico and compared with their homologues available at GenBank. The deduced 135 amino acid protein of nilgai IL-4 is 95.6% similar to that of Indian buffalo. N-linked glycosylation sequence, leader sequence, Cysteine residues in the signal peptide region, and 3′ UTR of IL-4 were found to be conserved across species. Six nonsynonymous nucleotide substitutions were found in Indian buffalo compared to nilgai amino acid sequence. Tertiary structure of this protein in both species was modeled, and it was found that this protein falls under 4-helical cytokines superfamily and short chain cytokine family. Phylogenetic analysis revealed a single cluster of ruminants including both nilgai and Indian buffalo that was placed distinct from other nonruminant mammals. PMID:24348167

Seroprevalence and risk factors associated with exposure of water buffalo (Bubalus bubalis) to Neospora caninum in northeast Thailand.

PubMed

Kengradomkij, Chanya; Inpankaew, Tawin; Kamyingkird, Ketsarin; Wongpanit, Kannika; Wongnakphet, Sirichai; Mitchell, Thomas J; Xuan, Xuenan; Igarashi, Ikuo; Jittapalapong, Sathaporn; Stich, Roger W

2015-01-15

Water buffalo are important draft animals for agriculture in resource-restricted areas worldwide. Water buffalo were shown to be experimentally susceptible to infection with Neospora caninum, potentially affected by neosporosis, and naturally exposed to the parasite in Asia. Although enzootic to Thailand, the distribution of N. caninum among Thai water buffalo is unclear. The objectives of this study were to determine the seroprevalence of N. caninum among water buffalo of northeast Thailand and to identify risk factors associated with their exposure to N. caninum. Sera from 628 water buffalo from 288 farms were tested with an indirect fluorescent antibody test (IFAT). A total of 57 samples from 48 herds contained antibodies to N. caninum, indicating overall seroprevalence of 9.1% and 16.7% among individual animals and herds, respectively. The overall seroprevalence was highest in provinces located in the Khorat Basin in the southern part of the region tested. Host age was also associated with seroprevalence, with the greatest seroprevalence (16.1%) among buffalo over 10 years of age, followed by 5-10 years of age (13.4%), 3-5 years (9.2%), and less than 3 years (1.2%). These results collectively suggested that horizontal transmission from canine definitive hosts was an important route of water buffalo exposure to N. caninum. These results also verified the importance of risk factor analysis for effective bovine neosporosis control strategies at the local level. Copyright © 2014 Elsevier B.V. All rights reserved.

In-vitro indicators of natural resistance and milk-producing ability in dairy buffaloes (Bubalus bubalis).

PubMed

Miarelli, Maria; Signorelli, Federica

2015-01-01

The aim of this study was to explore the possibility of detecting novel phenotypes of natural resistance at the molecular level through the in-vitro stimulation of monocyte-derived macrophages (MDMs). This study was conducted with 16 healthy buffaloes who were reared for milk production and for whom data on milk-producing ability were available for several lactations. MDMs from circulating monocytes were activated with interferon-gamma and lipopolysaccharide. The response was evaluated using Western blotting to detect the presence of 2 types of proteins separated by electrophoresis: tyrosine-phosphorylated proteins, which are indicators of the dynamic control of biochemical pathways, and IkB-alpha (Kappa light polipeptide gene enhancer in B-cells Inhibitor, alpha) protein, which controls the activity of nuclear factor kappa-light-chain-enhancer of activated B cells-a transcription factor that is responsible for the expression of proinflammatory cytokines. The results showed that the buffaloes who were positive for IkB-alpha proteins had a significantly higher milk-producing ability than the buffaloes who did not express IkB-alpha. On the contrary, no significant difference was detected between the high and low milk-producing buffaloes with regard to the presence of tyrosine-phosphorylated proteins. This preliminary study indicated that it may be possible to identify the more disease-resistant nonhuman animals on a molecular level. The results, therefore, indicate that an intense selection toward the increase of milk yield could impair natural disease resistance in future dairy buffalo generations.

Molecular characterization and phylogenetic analysis of Fasciola gigantica from western Java, Indonesia.

PubMed

Hayashi, Kei; Ichikawa-Seki, Madoka; Allamanda, Puttik; Wibowo, Putut Eko; Mohanta, Uday Kumar; Sodirun; Guswanto, Azirwan; Nishikawa, Yoshifumi

2016-10-01

Fasciola gigantica and aspermic (hybrid) Fasciola flukes are thought to be distributed in Southeast Asian countries. The objectives of this study were to investigate the distribution of these flukes from unidentified ruminants in western Java, Indonesia, and to determine their distribution history into the area. Sixty Fasciola flukes from western Java were identified as F. gigantica based on the nucleotide sequences of the nuclear phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold) genes. The flukes were then analyzed phylogenetically based on the nucleotide sequence of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene, together with Fasciola flukes from other Asian countries. All but one F. gigantica fluke were classified in F. gigantica haplogroup C, which mainly contains nad1 haplotypes detected in flukes from Thailand, Vietnam, and China. A population genetic analysis suggested that haplogroup C spread from Thailand to the neighboring countries including Indonesia together with domestic ruminants, such as the swamp buffalo, Bubalus bubalis. The swamp buffalo is one of the important definitive hosts of Fasciola flukes in Indonesia, and is considered to have been domesticated in the north of Thailand. The remaining one fluke displayed a novel nad1 haplotype that has never been detected in the reference countries. Therefore, the origin of the fluke could not be established. No hybrid Fasciola flukes were detected in this study, in contrast to neighboring Asian countries. Copyright © 2016. Published by Elsevier Ireland Ltd.

Insights into resistome and stress responses genes in Bubalus bubalis rumen through metagenomic analysis.

PubMed

Reddy, Bhaskar; Singh, Krishna M; Patel, Amrutlal K; Antony, Ancy; Panchasara, Harshad J; Joshi, Chaitanya G

2014-10-01

Buffalo rumen microbiota experience variety of diets and represents a huge reservoir of mobilome, resistome and stress responses. However, knowledge of metagenomic responses to such conditions is still rudimentary. We analyzed the metagenomes of buffalo rumen in the liquid and solid phase of the rumen biomaterial from river buffalo adapted to varying proportion of concentrate to green or dry roughages, using high-throughput sequencing to know the occurrence of antibiotics resistance genes, genetic exchange between bacterial population and environmental reservoirs. A total of 3914.94 MB data were generated from all three treatments group. The data were analysed with Metagenome rapid annotation system tools. At phyla level, Bacteroidetes were dominant in all the treatments followed by Firmicutes. Genes coding for functional responses to stress (oxidative stress and heat shock proteins) and resistome genes (resistance to antibiotics and toxic compounds, phages, transposable elements and pathogenicity islands) were prevalent in similar proportion in liquid and solid fraction of rumen metagenomes. The fluoroquinolone resistance, MDR efflux pumps and Methicillin resistance genes were broadly distributed across 11, 9, and 14 bacterial classes, respectively. Bacteria responsible for phages replication and prophages and phage packaging and rlt-like streptococcal phage genes were mostly assigned to phyla Bacteroides, Firmicutes and proteaobacteria. Also, more reads matching the sigma B genes were identified in the buffalo rumen. This study underscores the presence of diverse mechanisms of adaptation to different diet, antibiotics and other stresses in buffalo rumen, reflecting the proportional representation of major bacterial groups.

Modelling lactation curve for milk fat to protein ratio in Iranian buffaloes (Bubalus bubalis) using non-linear mixed models.

PubMed

Hossein-Zadeh, Navid Ghavi

2016-08-01

The aim of this study was to compare seven non-linear mathematical models (Brody, Wood, Dhanoa, Sikka, Nelder, Rook and Dijkstra) to examine their efficiency in describing the lactation curves for milk fat to protein ratio (FPR) in Iranian buffaloes. Data were 43 818 test-day records for FPR from the first three lactations of Iranian buffaloes which were collected on 523 dairy herds in the period from 1996 to 2012 by the Animal Breeding Center of Iran. Each model was fitted to monthly FPR records of buffaloes using the non-linear mixed model procedure (PROC NLMIXED) in SAS and the parameters were estimated. The models were tested for goodness of fit using Akaike's information criterion (AIC), Bayesian information criterion (BIC) and log maximum likelihood (-2 Log L). The Nelder and Sikka mixed models provided the best fit of lactation curve for FPR in the first and second lactations of Iranian buffaloes, respectively. However, Wood, Dhanoa and Sikka mixed models provided the best fit of lactation curve for FPR in the third parity buffaloes. Evaluation of first, second and third lactation features showed that all models, except for Dijkstra model in the third lactation, under-predicted test time at which daily FPR was minimum. On the other hand, minimum FPR was over-predicted by all equations. Evaluation of the different models used in this study indicated that non-linear mixed models were sufficient for fitting test-day FPR records of Iranian buffaloes.

Effect of supplemental light on growth, prolactin, progesterone and luteinizing hormone in water buffalo ( Bubalus bubalis)

NASA Astrophysics Data System (ADS)

Perera, K. S.; Gwazdauskas, F. C.; Akers, R. M.; McGilliard, M. L.

1989-06-01

Fifty non-pregnant Surti buffalo heifers aged between 17 and 42 months ( n=24, <24 months; n=26, >24 months) were randomly assigned to groups subject to either natural daylight +4h supplemental light ( n=25) or natural day light ( n=25), to study changes in growth, serum prolactin (Prl), progesterone (P4) and luteinizing hormone (LH) to supplemental lighting. Ambient temperatures (T) and relative humidity (RH) generally were >27° C and <70% during the day-time, respectively. Light-supplemented heifers had 16.2 kg net body weight (BW) gain at 9 weeks compared to 20.8 kg for controls, but higher mean Prl after 6.5 weeks ( P<0.01), and higher P4 (0.41 vs 0.19 ng/ml; P<0.06) than control heifers. Older heifers had 39.7% greater BW ( P<0.01), but a net 4.3% BW gain compared to a 10.1% gain for younger heifers at 10 weeks. Older, light-supplemented heifers had higher mean P4 (0.63 vs 0.19 ng/ml; P<0.07) than the other groups. These weight and hormonal changes suggest that 4 h supplemental light can alter growth and endocrine function in buffaloes under similar planes of nutrition. While light supplementation did not have a positive effect on body wieght during the 10 week study, body weight and endocrine changes due to supplemental light may be important factors for initiation of reproductive cyclicity.

Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa.

PubMed

Akhter, S; Ansari, M S; Andrabi, S M H; Rakha, B A; Ullah, N; Khalid, M

2012-10-01

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen. © 2011 Blackwell Verlag GmbH.

Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls

PubMed Central

Jain, Varsha; Patel, Brijesh; Umar, Farhat Paul; Ajithakumar, H. M.; Gurjar, Suraj K.; Gupta, I. D.; Verma, Archana

2017-01-01

Aim: This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls. Materials and Methods: Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme. Results: The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study. Conclusion: A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible. PMID:28344410

Ketosis in buffalo (Bubalus bubalis): clinical findings and the associated oxidative stress level.

PubMed

Youssef, Mohamed A; El-Khodery, Sabry Ahmed; El-deeb, Wael M; Abou El-Amaiem, Waleed E E

2010-12-01

As little is known about the oxidant/antioxidant status in buffalo with ketosis, the present study was delineated to assess the oxidative stress level associated with clinical ketosis in water buffalo. A total of 91 parturient buffalo at smallholder farms were studied (61 suspected to be ketotic and 30 healthy). Clinical and biochemical investigations were carried out for each buffalo. Based on clinical findings and the level of beta-hydroxybutyrate (BHB), buffalo were allocated into ketotic (42), subclinical cases (19). Clinically, there was an association between clinical ketosis and anorexia (p<0.001), constipation (p<0.001), decreased milk yield (p<0.001), ruminal stasis (p<0.001), and loss of body condition (p<0.01). Biochemically, in clinical ketosis compared with subclinical and control cases, there was a significant increase (p<0.05) of BHB, malondialdehyde (MDA), nitric oxide (NO), aspartate aminotransferase (AST), L-alanine aminotransferase (ALT). However, there was a significant decrease of glucose, phosphorus, magnesium,total cholesterol and HDL-cholesterol. There was a positive correlation between BHB and MDA (r=0.433), BHB and NO (r=0.37), MDA and NO (r=0.515), and Glucose and phosphorus(r=0.521). However, there was a negative correlation between BHB and glucose (r= -0.341) and HDL and NO (r= -0.379). The result of the present study indicates that hyperketonemia in buffalo is associated with an increase of oxidative stress levels. Further studies need to be done on the efficacy of antioxidants as an ancillary treatment to relief the oxidative stress caused by ketosis.

Replacement of serum with ocular fluid for cryopreservation of immature testes.

PubMed

Pothana, Lavanya; Devi, Lalitha; Venna, Naresh Kumar; Pentakota, Niharika; Varma, Vivek Phani; Jose, Jedy; Goel, Sandeep

2016-12-01

Cryopreservation of immature testis is a feasible approach for germplasm preservation of male animals. Combinations of dimethyl sulfoxide (DMSO) and foetal bovine serum (FBS) are used for testis cryopreservation. However, an alternative to FBS is needed, because FBS is expensive. Buffalo ocular fluid (BuOF), a slaughter house by-product, could be an economical option. The objective of the present study was to assess whether BuOF can replace FBS for cryopreservation of immature mouse (Mus musculus), rat (Rattus norvegicus), and buffalo (Bubalus bubalis) testes. Results showed that rodent and buffalo testes frozen in DMSO (10% for rodents and 20% for buffalo) with 20% FBS or BuOF had similar numbers of viable and DNA-damaged cells (P > 0.05). The expression of cell proliferation- (PCNA) and apoptosis-specific proteins (Annexin V and BAX/BCL2 ratio) were also comparable in mouse and buffalo testes frozen in DMSO with FBS or BuOF (P > 0.05). Interestingly, rat testis frozen in DMSO with BuOF had lower expression of Annexin V protein than testis frozen in DMSO with FBS (P < 0.05). The percentage of meiotic germ cells (pachytene-stage spermatocytes) in xenografts from testis frozen either in DMSO with BuOF or FBS did not significantly differ in rats or buffalo (P > 0.05). These findings provide evidence that BuOF has potential to replace FBS for cryopreservation of immature rodent and buffalo testis. Further investigation is needed to explore whether BuOF can replace FBS for testis cryopreservation of other species. Copyright © 2016 Elsevier Inc. All rights reserved.

The effects of high temperature and roof modification on physiological responses of swamp buffalo ( Bubalus bubalis) in the tropics

NASA Astrophysics Data System (ADS)

Khongdee, Titaporn; Sripoon, S.; Vajrabukka, C.

2013-05-01

The objective of the experiments reported here was to measure the effects of cooling techniques (Modified roof vs Normal roof) on the performance and physiology of 12 young male buffaloes with a similar live weight of 160 kg. The study was conducted at Chainat Agriculture and Technology College, Chainat Province, Thailand. The animals were divided randomly into two groups, each group comprising six buffaloes, and the two groups were studied to evaluate the effects of modified roofing (normal roof fitted with woven polypropylene shade cloth) on the subjects' physiological responses to heat stress under hot humid conditions. The modified roof resulted in lowered heat stress in buffaloes compared to those under a standard roof. The difference was shown by the buffaloes having a significantly lower mean rectal temperature (39.14 ± 0.07 vs 40.00 ± 0.10°C) and plasma cortisol (2.14 ± 0.24 vs 3.38 ± 0.37 ng/ml). The average daily water consumption was significantly lower in the MR group (MR, 29.71 ± 0.86 vs NR, 34.14 ± 1.06 L head -1 day-1), while there was a tendency for the roughage intake to be higher in the MR group compared to that of the NR group (MR, 5.88 ± 0.18 vs NR, 6.44 ± 0.19 kg head-1 -1 day-1; P = 0.0508). It was concluded that roof modification facilitated a reduction in heat load from roof re-radiation, and was an effective means of alleviating thermal stress in young buffaloes.

A novel selection signature in stearoyl-coenzyme A desaturase (SCD) gene for enhanced milk fat content in Bubalus bubalis.

PubMed

Maryam, J; Babar, M E; Bao, Zhang; Nadeem, A

2016-10-01

Modern molecular interventions are dynamic gears for breeding animals with superior genetic make-up. These scientific efforts lead us toward sustainable dairy herds with improved milk production in terms of yield and quality. Many of candidate genes have been dissected at molecular level, and suitable genetic markers have been identified in cattle, but this work has not been validated in buffaloes so far. Stearoyl-coenzyme A desaturase (SCD) has been a potential candidate gene for fat content of milk. Genomic analysis of SCD revealed a total of six variations that were identified through DNA sequencing of animals with lower and higher butter fat %age. After statistical analysis, genotype AB of p.K158I could be associated (P value <0.0001) with higher milk fat %age (10.5 ± 0.5464). This SNP was validated on larger data set by cleaved amplified polymorphic sequences (CAPS) by using DdeI. To scrutinize the functional consequences of p.K158I, 3D protein structure of SCD was predicted by homology modeling and this variation was found located in the vicinity of functional domain and a part of transmembrane helix of this membrane integrated protein. This is a first report toward genetic screening of SCD gene at molecular level in buffalo. This report illustrates the implication of SCD gene and in particular p.K158I variation, in imparting its effect on milk fat %age, which can be targeted in selection of superior dairy buffaloes.

The first serological evidence for Rift Valley fever infection in the camel, goitered gazelle and Anatolian water buffaloes in Turkey.

PubMed

Gür, Sibel; Kale, Mehmet; Erol, Nural; Yapici, Orhan; Mamak, Nuri; Yavru, Sibel

2017-10-01

Rift valley fever (RVF), a vector-borne zoonotic disease, is caused by a phlebovirus (family Bunyaviridae). The virus was initially characterized approximately 80 years ago in Kenya and disseminated to many countries in the continental Africa, Saudi Arabia, and Yemen. The infection has not been reported in Turkey. In this study, blood serum samples collected from camel (Camelus dromedairus), goitered gazelle (Gazella subgutturosa subgutturosa), and buffaloes (Bubalus bubalis linneaus) from 2000 to 2006 were investigated for RVF using C-ELISA. Camel samples (n = 72) were obtained from private small enterprises in Aydın province in theAegean region. Gazella samples (82) were taken from the biggest captive gazelle herd in Şanlıurfa province in the southeast Anatolia. Buffalo samples were collected mostly from small private family type farms in Afyon (168), Amasya (80), Samsun (69), Ankara (35), Sivas (21), Tokat (19), Konya (10), and Elazığ (8) provinces in the central, north, west, and east Anatolia. All of the gazella samples were negative; whereas, one of the 71 camel samples (1.3%) was positive for RVF-specific antibodies. Buffalos from Sivas, Tokat, Konya, and Elazığ provinces were negative. However, 35 of the 410 samples (8.5%) from rural areas in the following four provinces were positive: Amasya (12/80, 15%), Ankara (5/35, 14.2%), Samsun (8/69, 11.5%), and Afyon (10/168, 5.9%). To our knowledge, this is the first report of presence of RVF infection in Turkey.

Myxobolus ictiobus n. sp. and Myxobolus minutus n. sp. (Cnidaria: Myxobolidae) from the gills of the smallmouth buffalo Ictiobus bubalus Rafinesque (Cypriniformes: Castostomidae)

USDA-ARS?s Scientific Manuscript database

The smallmouth buffalo Ictiobus bubalus Rafinesque (Catostomidae) is native to North American waterways and occasionally grown in pond aquaculture. Species of Myxobolus Bütschli, 1882 have been reported from the gills, integument, and intestinal tract of buffalo fish, although there is ambiguity in ...

Molecular characterisation of Theileria orientalis in imported and native bovines from Pakistan.

PubMed

Gebrekidan, Hagos; Abbas, Tariq; Wajid, Muhammad; Ali, Aamir; Gasser, Robin B; Jabbar, Abdul

2017-01-01

The epidemiological aspects of Theileria orientalis in Pakistan are unknown; therefore, investigations using sensitive and precise molecular techniques are required. This study reports the first molecular characterisation of T. orientalis detected from imported (Bos taurus) and native cattle (Bos indicus×Bos taurus) and buffaloes (Bubalus bubalis) selected from four districts of Punjab, Pakistan. DNA samples from blood (n=246) were extracted and tested using conventional PCR utilising the major piroplasm surface protein (MPSP) gene and multiplexed tandem PCR (MT-PCR). Theileria orientalis DNA was detected (15%; 22/147) only in imported cattle by conventional PCR, whereas 24.5% (36/147), 6% (3/50) and 6.1% (3/49) of the imported cattle and native Pakistani cattle and buffaloes, respectively were test-positive for T. orientalis using MT-PCR. Using MT-PCR, the prevalence of T. orientalis was significantly higher (P<0.0001) in imported cattle compared to that of detected in native Pakistani bovines. The prevalence of T. orientalis and DNA copies of chitose and ikeda were significantly higher (P<0.05) in imported cattle than those detected in native Pakistani bovines. DNA sequencing of amplicons of the conventional PCR revealed the presence of buffeli, chitose and ikeda genotypes of T. orientalis. Phylogenetic analysis revealed that the MPSP sequences of buffeli, chitose and ikeda from imported cattle were closely related to those sequences reported previously from Australia and other regions. This study provides the first survey of T. orientalis infection in imported and native bovines in Pakistan, and highlights the need for future studies to understand the spread of transboundary animal diseases. Copyright © 2016. Published by Elsevier B.V.

Biocomputational identification and validation of novel microRNAs predicted from bubaline whole genome shotgun sequences.

PubMed

Manku, H K; Dhanoa, J K; Kaur, S; Arora, J S; Mukhopadhyay, C S

2017-10-01

MicroRNAs (miRNAs) are small (19-25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc. To date, not a single miRNA has been identified in bubaline (Bubalus bubalis), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis).

PubMed

Ashish, Shende; Bhure, S K; Harikrishna, Pillai; Ramteke, S S; Muhammed Kutty, V H; Shruthi, N; Ravi Kumar, G V P P S; Manish, Mahawar; Ghosh, S K; Mihir, Sarkar

2017-04-01

The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines. Copyright © 2017 Elsevier Inc. All rights reserved.

Peripheral blood mononuclear cells: a potential cellular system to understand differential heat shock response across native cattle (Bos indicus), exotic cattle (Bos taurus), and riverine buffaloes (Bubalus bubalis) of India.

PubMed

Kishore, Amit; Sodhi, Monika; Kumari, Parvesh; Mohanty, A K; Sadana, D K; Kapila, Neha; Khate, K; Shandilya, Umesh; Kataria, R S; Mukesh, M

2014-09-01

Circulating leukocytes can be used as an effective model to understand the heat stress response of different cattle types and buffaloes. This investigation aimed to determine the temporal profile of HSPs (HSP40, HSP60, HSP70, and HSP90) expression in circulating peripheral blood mononuclear cells (PBMCs) of Murrah buffaloes, Holstein-Friesian (HF), and Sahiwal cows in response to sublethal heat shock at 42 °C. The viability data indicated HF PBMCs to be the most affected to the heat shock, whereas Sahiwal PBMCs were least affected, indicating its better survivability during the heat stress condition. The qRT-PCR expression data showed significant increase in mRNA expression of the analyzed HSPs genes after heat stimuli to the PBMCs under in vitro condition. In each case, the HSPs were most upregulated at 2 h after the heat stress. Among the HSPs, HSP70 was relatively more expressed followed by HSP60 indicating the action of molecular chaperones to stabilize the native conformation of proteins. However, PBMCs from different cattle types and buffaloes showed difference in the extent of transcriptional response. The level of expression of HSPs throughout the time period of heat stress was highest in buffaloes, followed by HF and Sahiwal cows. The higher abundance of HSP70 mRNA at each time point after heat stress showed prolonged effect of heat stress in HF PBMCs. The data presented here provided initial evidence of transcriptional differences in PBMCs of different cattle types and buffaloes and warrant further research.

Molecular characterization of HOXC8 gene and methylation status analysis of its exon 1 associated with the length of cashmere fiber in Liaoning cashmere goat.

PubMed

Bai, Wen L; Wang, Jiao J; Yin, Rong H; Dang, Yun L; Wang, Ze Y; Zhu, Yu B; Cong, Yu Y; Deng, Liang; Guo, Dan; Wang, Shi Q; Yang, Shu H; Xue, Hui L

2017-02-01

Homeobox protein Hox-C8 (HOXC8) is a member of Hox family. It is expressed in the dermal papilla of the skin and is thought to be associated with the hair inductive capacity of dermal papilla cells. In the present study, we isolated and characterized a full-length open reading frame of HOXC8 cDNA from the skin tissue of Liaoning cashmere goat, as well as, established a phylogenetic relationship of goat HOXC8 with that of other species. Also, we investigated the effect of methylation status of HOXC8 exon 1 at anagen secondary hair follicle on the cashmere fiber traits in Liaoning cashmere goat. The sequence analysis indicated that the obtained cDNA was 1134-bp in length containing a complete ORF of 729-bp. It encoded a peptide of 242 amino acid residues in length. The structural analysis indicated that goat HOXC8 contained a typical homeobox domain. The phylogenetic analysis revealed that Capra hircus HOXC8 had a closer genetic relationship with that of Ovis aries, followed by Bos Taurus and Bubalus bubalis. The methylation analysis suggested that the methylation degree of HOXC8 exon 1 in anagen secondary hair follicle might be involved in regulating the growth of cashmere fiber in Liaoning cashmere goat. Our results provide new evidence for understanding the molecular structural and evolutionary characteristics of HOXC8 in Liaoning cashmere goat, as well as, for further insight into the role of methylation degree of HOXC8 exon 1 regulates the growth of cashmere fiber in goat.

Effect of feeding of blend of essential oils on methane production, growth, and nutrient utilization in growing buffaloes

PubMed Central

2018-01-01

Objective An experiment was conducted to study the effect of a blend of essential oils (BEO) on enteric methane emission and growth performance of buffaloes (Bubalus bubalis). Methods Twenty one growing male buffaloes (average body weight of 279±9.3 kg) were divided in to three groups. The animals of all the three groups were fed on a ration consisting of wheat straw and concentrate mixture targeting 500 g daily live weight gain. The three dietary groups were; Group 1, control without additive; Group 2 and 3, supplemented with BEO at 0.15 and 0.30 mL/kg of dry matter intake (DMI), respectively. Results During six months feeding trial, the intake and digestibility of dry matter and nutrients (organic matter, crude protein, ether extract, neutral detergent fibre, and acid detergent fibre) were similar in all the groups. The average body weight gain was tended to improve (p = 0.084) in Group 2 and Group 3 as compared to control animals. Feeding of BEO did not affect feed conversion efficiency of the animals. The calves of all the three groups were in positive nitrogen balance with no difference in nitrogen metabolism. During respiration chamber studies the methane production (L/kg DMI and L/kg digestible dry matter intake was significantly (p<0.001) lower in Group 2 and Group 3 as compared to control animals. Conclusion The results indicated that the BEO tested in the present study have shown potential to reduce enteric methane production without compromising the nutrient utilization and animal performance and could be further explored for its use as feed additive to mitigate enteric methane production in livestock. PMID:28231698

Heat stress and antioxidant enzyme activity in bubaline ( Bubalus bubalis) oocytes during in vitro maturation

NASA Astrophysics Data System (ADS)

Waiz, Syma Ashraf; Raies-ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T. Sridhar; Chauhan, M. S.; Upadhyay, R. C.

2016-09-01

In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly ( P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly ( P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

Programmable fast-freezing method improves the post-thaw motion dynamics, integrities of plasmalemma, mitochondrial transmembrane, DNA and, acrosome, and in vivo fertility of water buffalo (Bubalus bubalis) spermatozoa.

PubMed

Khalil Ur Rehman, H; Andrabi, S M H; Ahmed, H; Shah, S A H

2017-10-01

The effects of freezing methods (FR1, nonprogrammable/static, 5 cm above liquid nitrogen [LN 2 ] for 10 min, plunging in LN 2 ; FR2, programmable medium, +4°C to -15°C at 3°C min -1 , from -15 to -80°C at 10°C min -1 and final holding for 1 min at -80°C, plunging in LN 2 ; FR3, programmable fast, from initial holding at +4°C for 2 min, from +4°C to -20°C at 10°C min -1 , from -20°C to -100°C at 30°C min -1 , final holding for 1 min at -100°C and plunging in LN 2 ) were assessed on post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. Mean sperm progressive motility (%), rapid velocity (%), average path velocity (μm s -1 ), straight line velocity (μm s -1 ), curved line velocity (μm s -1 ), integrities (%) of plasmalemma, mitochondrial transmembrane, DNA and acrosome were higher (p < .05) in samples cryopreserved with FR3 compared to FR1 and FR2. Similarly, in vivo fertility (%) of buffalo spermatozoa was higher (p < .05) with FR3 than FR1 (%; 68.0 versus 50.0). We concluded that programmable fast-freezing method (FR3) improves the post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. © 2016 Blackwell Verlag GmbH.

Detomidine-diazepam-ketamine anaesthesia in buffalo (Bubalus bubalis) calves.

PubMed

Pawde, A M; Amarpal; Kinjavdekar, P; Aithal, H P; Pratap, K; Bisht, G S

2000-04-01

Eight buffalo calves (8-12 months, 70-100 kg) were randomly assigned to two groups of four animals each. Animals of group I were given detomidine (100 micrograms/kg), whereas animals of group II received a mixture of detomidine (100 micrograms/kg), diazepam (100 micrograms/kg) and ketamine (3 mg/kg) (DDK) intravenously. Various clinical parameters, such as weak time, down time, pedal and pinprick reflexes, muscle relaxation and extent of sedation, as well as heart and respiratory rates and electrocardiograms were measured before (time 0) and 15, 30, 45, 60, 75 and 90 min after treatment. In all the animals of group II (DDK), the pedal reflex was completely abolished (score: 3.00 +/- 0.00) within 5 min, the pinprick response was either very weak or it was completely abolished at this interval. Muscle relaxation and sedation were excellent within 5 min of DDK administration. The depth of sedation and analgesia was maximum from 5 to 15 min postinjection. Detomidine alone, however, failed to produce appropriate depression of the pedal and pinprick reflexes, sedation was mild and muscle relaxation was inadequate. Heart rate showed a significant (P < 0.05) decrease in group I, but the decrease was non-significant in group II. A more pronounced increase in respiratory rate was observed in group I as compared to group II. Animals of both groups recovered within 90 min without any complication. Minimal changes in the cardiovascular system in the group given the DDK combination were an advantage over the group given detomidine. The results indicated that DDK combination is safe and suitable for 15 min of anaesthesia with excellent muscle relaxation and has only limited cardiorespiratory effects in buffaloes.

Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

PubMed

Guha, Anirban; Gera, Sandeep; Sharma, Anshu

2012-03-01

Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (p<0.01) increase in SCC, Fe, Zn, Co and LDH occurred in SCM milk containing gram positive bacterial agents only. ALP was found to be elevated in milk infected by both gram positive and negative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×10(5) cells/ml of milk as the benchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology.

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices.

PubMed

Sharma, Manjinder; Dubey, Pawan K; Kumar, Rajesh; Nath, Amar; Kumar, G Sai; Sharma, G Taru

2013-05-01

Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

Ethnomedicinal and cultural practices of mammals and birds in the vicinity of river Chenab, Punjab-Pakistan.

PubMed

Altaf, Muhammad; Javid, Arshad; Umair, Muhammad; Iqbal, Khalid Javed; Rasheed, Zahid; Abbasi, Arshad Mehmood

2017-07-12

Although, use of animal species in disease treatment and culture practices is as ancient as that of plant species; however ethnomedicinal uses and cultural values of animal species have rarely been reported. Present study is the first report on the medicinal uses of mammals and bird species in Pakistan. Questionnaires and semi-structured interviews were applied to collect qualitative and quantitative data from local informants (N = 109). Relative frequency of mention (RFM), fidelity level (FL), relative popularity level (RPL), similarity index (SI) and rank order priority (ROP) indices were used to analyzed the data. One hundred and eight species of animals, which include: 83% birds and 17% mammals were documented. In total 30 mammalian and 28 birds' species were used to treat various diseases such as rheumatic disorders, skin infections and sexual weakness among several others. Fats, flesh, blood, milk and eggs were the most commonly utilized body parts. Bos taurus, Bubalus bubalis, Capra aegagrus hircus, Felis domesticus, Lepus nigricollis dayanus and Ovis aries (mammals) and Anas platyrhynchos domesticus, Columba livia, Coturnix coturnix, Gallus gallus and Passer domesticus (birds) were the highly utilized species. Medicinal and cultural uses of 30% mammals and 46% birds were reported for the first time, whereas 33% mammals and 79% birds depicted zero similarity with previous reports. Present study exhibits significant ethnozoological knowledge of local inhabitants and their strong association with animal species, which could be helpful in sustainable use of biodiversity of the region. Additionally, in vitro and in vivo evaluation of biological activities in the mammalian and birds' species with maximum fidelity level and frequency of mention could be important to discover animal based novel drugs. Some commonly used mammals and birds species of the study area.

Evaluation of thermal comfort, physiological, hematological, and seminal features of buffalo bulls in an artificial insemination station in a tropical environment.

PubMed

Barros, Daniel Vale; Silva, Lilian Kátia Ximenes; de Brito Lourenço, José; da Silva, Aluizio Otávio Almeida; E Silva, André Guimarães Maciel; Franco, Irving Montanar; Oliveira, Carlos Magno Chaves; Tholon, Patrícia; Martorano, Lucieta Guerreiro; Garcia, Alexandre Rossetto

2015-06-01

This study aimed to assess the variation over time in thermal comfort indices and the behavior of physiological parameters related to thermolysis, blood parameters, and semen in natura of buffalo bulls reared in tropical climate. The study was carried out in an artificial insemination station under a humid tropical climate (Afi according to Köppen). Ten water buffalo bulls (Bubalus bubalis) were used during the 5 months (April to August) of study. The environmental Temperature Humidity Index (THId) and the pen microclimate Temperature Humidity Index (THIp) were calculated. Every 25 days, respiratory rate (RR), heart rate (HR), rectal temperature (RT), and Benezra's thermal comfort index (BTCI) were assessed in the morning and in the afternoon. A blood assay was performed every month, while semen was collected weekly. THIp did not vary over the months (P > 0.05) and was higher in the afternoon than in the morning (77.7 ± 2.6 versus 81.8 ± 2.1, P < 0.05). RR, HR, and BTCI significantly increased over the months and were different between the periods of the day (P > 0.05) but within the physiological limits. RT varied between the periods of the day and decreased over the months, being the lowest in August (37.8 ± 0.7 °C), time-impacted hematocrit, mean corpuscular volume, hemoglobin levels, and spermatic gross motility and vigor (P < 0.05). Thus, buffalo bulls reared under a humid tropical climate may have variations in thermal comfort during the hotter periods but are able to efficiently activate thermoregulatory mechanisms and maintain homeothermy, hence preserving their physiological and seminal parameters at normal levels.

Sequential Cross-Species Chromosome Painting among River Buffalo, Cattle, Sheep and Goat: A Useful Tool for Chromosome Abnormalities Diagnosis within the Family Bovidae

PubMed Central

Pauciullo, Alfredo; Perucatti, Angela; Cosenza, Gianfranco; Iannuzzi, Alessandra; Incarnato, Domenico; Genualdo, Viviana; Di Berardino, Dino; Iannuzzi, Leopoldo

2014-01-01

The main goal of this study was to develop a comparative multi-colour Zoo-FISH on domestic ruminants metaphases using a combination of whole chromosome and sub-chromosomal painting probes obtained from the river buffalo species (Bubalus bubalis, 2n = 50,XY). A total of 13 DNA probes were obtained through chromosome microdissection and DOP-PCR amplification, labelled with two fluorochromes and sequentially hybridized on river buffalo, cattle (Bos taurus, 2n = 60,XY), sheep (Ovis aries, 2n = 54,XY) and goat (Capra hircus, 2n = 60,XY) metaphases. The same set of paintings were then hybridized on bovine secondary oocytes to test their potential use for aneuploidy detection during in vitro maturation. FISH showed excellent specificity on metaphases and interphase nuclei of all the investigated species. Eight pairs of chromosomes were simultaneously identified in buffalo, whereas the same set of probes covered 13 out 30 chromosome pairs in the bovine and goat karyotypes and 40% of the sheep karyotype (11 out of 27 chromosome pairs). This result allowed development of the first comparative M-FISH karyotype within the domestic ruminants. The molecular resolution of complex karyotypes by FISH is particularly useful for the small chromosomes, whose similarity in the banding patterns makes their identification very difficult. The M-FISH karyotype also represents a practical tool for structural and numerical chromosome abnormalities diagnosis. In this regard, the successful hybridization on bovine secondary oocytes confirmed the potential use of this set of probes for the simultaneous identification on the same germ cell of 12 chromosome aneuploidies. This is a fundamental result for monitoring the reproductive health of the domestic animals in relation to management errors and/or environmental hazards. PMID:25330006

Genetic Evaluation of Dual-Purpose Buffaloes (Bubalus bubalis) in Colombia Using Principal Component Analysis

PubMed Central

Agudelo-Gómez, Divier; Pineda-Sierra, Sebastian; Cerón-Muñoz, Mario Fernando

2015-01-01

Genealogy and productive information of 48621 dual-purpose buffaloes born in Colombia between years 1996 and 2014 was used. The following traits were assessed using one-trait models: milk yield at 270 days (MY270), age at first calving (AFC), weaning weight (WW), and weights at the following ages: first year (W12), 18 months (W18), and 2 years (W24). Direct additive genetic and residual random effects were included in all the traits. Maternal permanent environmental and maternal additive genetic effects were included for WW and W12. The fixed effects were: contemporary group (for all traits), sex (for WW, W12, W18, and W24), parity (for WW, W12, and MY270). Age was included as covariate for WW, W12, W18 and W24. Principal component analysis (PCA) was conducted using the genetic values of 133 breeding males whose breeding-value reliability was higher than 50% for all the traits in order to define the number of principal components (PC) which would explain most of the variation. The highest heritabilities were for W18 and MY270, and the lowest for AFC; with 0.53, 0.23, and 0.17, respectively. The first three PCs represented 66% of the total variance. Correlation of the first PC with meat production traits was higher than 0.73, and it was -0.38 with AFC. Correlations of the second PC with maternal genetic component traits for WW and W12 were above 0.75. The third PC had 0.84 correlation with MY270. PCA is an alternative approach for analyzing traits in dual-purpose buffaloes and reduces the dimension of the traits. PMID:26230093

Freezability of water buffalo bull (Bubalus bubalis) spermatozoa is improved with the addition of curcumin (diferuoyl methane) in semen extender.

PubMed

Shah, S A H; Andrabi, S M H; Qureshi, I Z

2017-10-01

Effects of curcumin as antioxidant in extender were evaluated on freezability of buffalo spermatozoa. Semen from each of the five bulls (n = 3 replicates, six ejaculates/bull, a total of 30 ejaculates) was diluted in Tris-citric acid extender containing curcumin (0.5, 1.0, 1.5 or 2.0 mM) or control. At pre-freezing and post-thawing, total antioxidant contents (μM/L) and lipid peroxidation levels (μM/ml) were higher (p < .05) and lower (p < .05) respectively, with 1.5 and 2.0 mM compared to 0.5 and 1.0 mM curcumin and control. At post-thawing, progressive motility (PM, %) and rapid velocity (RV, %) were higher (p < .05) with 1.5 mM compared to other doses of curcumin and control (except in case of RV, 1.5 was similar with 1.0 mM). Kinematics (average path velocity, μm/s; straight-line velocity, μm/s; curved-line velocity, μm/s; straightness, %; linearity, %), in vitro longevity (%, PM and RV) and DNA integrity (%) at post-thawing were higher (p < .05) with 1.5 mM compared to control. At post-thawing, supravital plasma membrane integrity (%) and viable spermatozoa with intact acrosome (%) were higher with 1.5 compared to 2.0 mM curcumin and control. We concluded that freezability of water buffalo spermatozoa is improved with the addition of 1.5 mM curcumin in extender. © 2016 Blackwell Verlag GmbH.

Spatial-temporal and phylogeographic characterization of Trypanosoma spp. in cattle (Bos taurus) and buffaloes (Bubalus bubalis) reveals transmission dynamics of these parasites in Colombia.

PubMed

Jaimes-Dueñez, Jeiczon; Triana-Chávez, Omar; Mejía-Jaramillo, Ana M

2018-01-15

Animal Trypanosomiasis (AT) is one of the most important problems in the Colombian livestock industry reducing its production around 30%. Caribbean and Orinoquia regions play a significant role in the development of this industry, having about 6.9 million cattle and 113,000 buffaloes. Considering the paucity in studies to understand the epidemiological features and control of AT in Colombia, the present study reports the seasonal transmission patterns and phylogeographic traits of the causal agents of AT in cattle and buffaloes from these regions. Between 2014 and 2016, a three-point longitudinal survey was designed to evaluate the mentioned characteristics. Molecular analysis in cattle showed an AT prevalence of 39.2% (T. theileri 38.6%, T. evansi 6.7% and T. vivax 0.2%), with higher values during wet and late wet seasons, while in buffaloes the prevalence was 28.2% (T. theileri 28.2% and T. evansi 1.3%), with higher values during the dry season. Additionally, variables such as tabanid abundance, vector control, breeding system, age and anemia signs were significantly associated with AT prevalence (P<0.05). Only T. theileri infection was higher in cattle with anemia signs than those with normal packed cell volume. Finally, phylogeographic analysis revealed that Colombian T. theileri isolates were associated to specific host genotypes IA and IIB, described worldwide; T. vivax isolates were related to the genotype from West Africa; while T. evansi isolates are related to the South American genotypes and to new genotypes. This is the first longitudinal survey that evaluates through molecular methods, the infection of Trypanosoma spp. in two important livestock regions from Colombia, showing that the clinical effects and prevalence of these trypanosomes in cattle and buffaloes are modulated by seasonal variations, host factors, and parasite traits. The results suggest that these factors have to be taken into account to successfully control AT in these regions. Copyright © 2017 Elsevier B.V. All rights reserved.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taru Sharma, G., E-mail: gts553@gmail.com; Dubey, Pawan K.; Verma, Om Prakash

Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBsmore » from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.« less

Effect of consecutive re-synchronization protocols on pregnancy rate in buffalo (Bubalus bubalis) heifers out of the breeding season.

PubMed

Neglia, Gianluca; Capuano, Massimo; Balestrieri, Anna; Cimmino, Roberta; Iannaccone, Francesco; Palumbo, Francesco; Presicce, Giorgio A; Campanile, Giuseppe

2018-06-01

The combined effect of six consecutive timed artificial inseminations (TAIs) on pregnancy rates, following two different synchronization protocols on buffalo heifers, over a period of seven months typically characterized by low breeding performances, were investigated in this study. A total of 2189 TAIs were performed on 1463 buffalo heifers within a large buffalo farm in the south of Italy. Individual animals were allowed to undergo synchronization protocol (either a slightly modified Ovsynch or Progesterone treatment) and TAI until establishment of pregnancy or else for not more than six consecutive times. Semen of seven proven bulls was used throughout the study, which was carried out from March to September of the same year. Therefore, other than the effect given by consecutive TAIs over time, a monthly and a seasonal effect could also be tested, once the entire period was split into a Low Breeding Season (LBS) from March to June, and a Transition to Breeding Season (TBS) from July to September. From the data recorded in this study and the statistical analysis performed, it can be stated that the two protocols for the synchronization of ovulation were similar in efficiency in determining pregnancies with an overall fertility rate of 89.4% when the comparison was run both on a monthly basis or when months were grouped into two different seasons. In addition, an average of 1.83 AI/pregnancy was reported, slightly higher for the Ovsynch when compared to the Progesterone protocol: 1.91 vs 1.70, respectively. Finally, when considering the number of progressive synchronization treatments implemented over time as covariate, neither Ovsynch nor Progesterone treatment significantly affected pregnancy rates following the first of the six synchronization sessions. However, repeating the synchronization procedure, the progesterone based protocol resulted in significantly higher probability of success in terms of established pregnancies during the second and third re-synchronization sessions. Copyright © 2018 Elsevier Inc. All rights reserved.

Incubation of spermatozoa with Anandamide prior to cryopreservation reduces cryocapacitation and improves post-thaw sperm quality in the water buffalo (Bubalus bubalis).

PubMed

Kumar, Puneeth; Mohanty, Tushar Kumar; Kumaresan, Arumugam; Nag, Pradeep; Saraf, Kaustubh Kishor; Kumar, Vimlesh; Lathika, Sreela; Nayak, Samiksha; Bhakat, Mukesh

2018-02-01

Anandamide (AEA), an endocannabinoid, has been shown to reduce capacitation and acrosomal exocytosis in human spermatozoa. Because buffalo spermatozoa are highly susceptible to cryopreservation induced damage, AEA was assessed as to whether it could protect spermatozoa from cryo-damage. Six ejaculates from six Murrah buffalo bulls (total 36 ejaculates) were utilized for the study. Each ejaculate was divided into four aliquots; spermatozoa in Aliquot 1 were extended in Tris-Citrate-Egg Yolk and frozen as per the standard protocol. Spermatozoa in Aliquots 2, 3 and 4 were incubated with AEA at 1 nM, 1 μM and 10 μM, respectively in Tris-Citrate extender for 15 min at 37 °C before cryopreservation. Cryopreserved spermatozoa were thawed at 37 °C for 30 s before assessment of sperm motility, membrane integrity, capacitation, acrosome reaction, mitochondrial membrane potential (MMP) and lipid peroxidation status. The proportion of motile and membrane intact spermatozoa were greater (P < 0.05) with use of 1 μM AEA incorporated group compared with other groups. The proportion of un-capacitated and acrosome intact spermatozoa was greater (P < 0.05) with use of 1 or 10 μM of AEA compared with the other groups. When compared to the control group, use of 1 μM AEA resulted in a greater proportion of spermatozoa with high MMP (P < 0.05). There was no significant difference in the lipid peroxidation status of spermatozoa among any of the four groups. It was inferred that the protective role of AEA during cryopreservation of buffalo spermatozoa was dose dependent and incubation of spermatozoa with AEA at 1 μM concentration prior to cryopreservation reduced cryo-capacitation and improved post-thaw sperm quality in buffalo. Copyright © 2017 Elsevier B.V. All rights reserved.

Chicken egg yolk plasma in tris-citric acid extender improves the quality and fertility of cryopreserved water buffalo (Bubalus bubalis) spermatozoa.

PubMed

Hussain Shah, S Aftab; Hassan Andrabi, S Murtaza; Ahmed, Hussain; Qureshi, Irfan Zia

2017-02-01

This study was primarily designed to evaluate the effect of different concentrations of ultraviolet (UV)-C-irradiated chicken egg yolk plasma (EYP; v:v; 10%, P1; 15%, P2; 20%, P3) or 20% (v:v) of whole chicken egg yolk (WCEY) in tris-citric acid (TCA) extender on water buffalo sperm quality during cryopreservation (postdilution, PD; postequilibration, PE; post-thawing, PT). Also the effect of best evolved concentration of UV-C-irradiated EYP in extender on in vivo fertility of buffalo spermatozoa was evaluated. At PE and PT, computer-assisted sperm analysis progressive motility (PM, %) was significantly higher in P3 compared with P1 and WCEY. Rapid velocity (RV, %) was higher (P < 0.05) in P3 compared with P1 and WCEY during cryopreservation (PD, PE, and PT). Average path velocity (μm/s) and straight line velocity (μm/s) were higher (P < 0.05) in P2 and P3 than WCEY at PE and PT. The decline percentage (%, longevity) in PM and RV was lower (P < 0.05) in P3 compared with WCEY during 2 hours incubation under in vitro condition at PT. Supravital plasma membrane integrity (%) was higher (P < 0.05) in P2 and P3 compared with control at different stages (PE and PT). Mitochondrial transmembrane potential (%) was higher (P < 0.05) in P2 and P3 compared with P1 and WCEY at different stages (PD and PT). Percentage of viable sperm with intact acrosome, and sperm DNA integrity (%) were higher (P < 0.05) in P2 and P3 compared with WCEY at PT. The in vivo fertility rate (%) was significantly higher with P3 compared with WCEY (76.61 vs. 64.49). In conclusion, WCEY (20%) can be replaced with UV-C-irradiated chicken EYP (20%) in TCA extender for cryopreservation of water buffalo spermatozoa. Copyright © 2016 Elsevier Inc. All rights reserved.

Controlled breeding and reproductive management in water buffaloes (Bubalus bubalis) using Eazi Breed controlled internal drug release.

PubMed

Hiremath, Shivayogi; Ramesha, Kerekoppa P

2015-06-04

Buffalo reproduction is considerably affected by late maturity, poor oestrus symptoms and long postpartum periods. This study was undertaken to evaluate the efficiency of Eazi Breed controlled internal drug release (CIDR), an intravaginal progesterone-releasing device, in relation to oestrus and fertility. Five hundred true anoestrus buffalo cows, in the age group 4-6 years in 10 villages of Dharwad district in Karnataka state in India, were randomly selected and treated with CIDR for 9 days. Two mL of Cidirol (1 mg oestradiol benzoate) was administered intramuscularly to all animals on day 10. Forty-two buffaloes (8.4%) that failed to show oestrus signs (1.6%) or showed weak signs of oestrus (6.8%) after the first treatment were treated again 72 h after the Cidriol injection with a new device, and inseminated after the expression of oestrus. After the second treatment all the animals showed oestrus signs. The percentage of buffaloes showing intense oestrus was 67.40%, intermediate oestrus was shown by 25.80%, whilst 6.80% buffaloes showed weak oestrus even after the second treatment. The buffaloes showing oestrus signs were inseminated twice with an interval of 12 h, starting 12 h after the start of the oestrus signs. In 86 buffaloes showing prolonged oestrus signs a third insemination was done. The conception rates were 85.16%, 60.47% and 44.11% respectively in buffaloes showing intense, intermediate and weak oestrus. Transrectal palpation of the genital tract was performed 45-60 days post-insemination to diagnose pregnancy status, and in doubtful cases pregnancy was reconfirmed at 90 days after insemination. Out of 500 buffaloes treated in this way 380 animals became pregnant and the pregnancy rate was 76%. This study revealed the usefulness of Eazi Breed CIDR along with Cidirol treatment in buffaloes to improve their reproductive performance.

Improving smallholder food security through investigations of carcass composition and beef marketing of buffalo and cattle in northern Lao PDR.

PubMed

Nampanya, Sonevilay; Khounsy, Syseng; Phonvisay, Aloun; Bush, Russell David; Windsor, Peter Andrew

2015-04-01

This study determined the carcass composition of Lao indigenous buffalo (Bubalus bubalis) and cattle (Bos indicus), then examined trends in bovine meat marketing following review of records of beef production and prices in the two major cities of Luang Prabang (LPB) and Xieng Khoung (XK) provinces in northern Laos. Samples from 41 buffalo and 81 cattle (n = 122) were collected from animals slaughtered in May-June 2014, with live weights, carcass weights and other carcass-related variables collected. The animals were classified into four age cohort groups (<2, 2-<4, 4-6 and >6 years) with quantitative and dichotomous qualitative traits determined. There were significant differences in buffalo and cattle predicted mean carcass weights between age classification categories (p = 0.003 and 0.001) but not in dressing percentages (p = 0.1 and 0.1). The carcass weight of buffalo was 104 (±23.1)-176 (±12.0) kg compared to 65 (±8.7)-84 (±6.5) kg of cattle, with dressing percentages of 37-40 and 39-42 %, respectively. Despite an average bovine meat price increase of 42-48 % between 2011 and 2013, there was a reduction in the numbers of large ruminants slaughtered in the surveyed cities of LPB (11 %) and XK (7 %), with bovine meat availability per person of 5.2-6.6 kg (LPB) and 3.0-3.8 kg (XK). Improving the sustainability of the bovine meat supply in Laos requires a systems approach involving improvements to animal health and production, livestock marketing, plus the critical development of improved slaughterhouse facilities enabling a meat-processing sector to emerge. This development pathway is of particular importance for building the capacity of Laos to reduce food insecurity and alleviate the poverty of its largely rural smallholder community.

Significance of clinical observations and biochemical alterations in buffalo calves with dietary abomasal impaction.

PubMed

El-Ashker, Maged R; Salama, Mohamed F; El-Boshy, Mohamed E; Abo El-Fadle, Eman A

2018-01-02

The present study aimed to throw light on the clinical characteristics of abomasal impaction in buffalo calves and its associated biochemical alterations. For this reason, a total of 20 male buffalo calves (Bubalus bubalis) with abomasal impaction were studied. The investigated calves were at 6 to 12 months of age and were belonged to three private farms in Dakahlia Governorate besides sporadic cases admitted to the Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Mansoura University, Egypt. Ten apparently healthy buffalo calves were also included as controls. According to the clinical outcome, the diseased calves were categorized into survivors (n = 11) and non-survivors (n = 9). Blood samples were collected from all animals to estimate blood gases besides a panel of selected biochemical parameters. The definitive diagnosis of dietary abomasal impaction was achieved by either left flank exploratory laparotomy or by necropsy. Both survivors and non-survivors demonstrated common clinical findings including distension of ventro-lateral aspect of the right abdomen, and varying degrees of dehydration. The great majority of survivors (81%) and 100% of non-survivors were anorexic and had rumen stasis as well as hard texture upon ballottement of the left flank. Approximately 45% of non-survivors had frothy salivation, expiratory grunting and were being tender when strong percussion was applied on the right flank. Diseased calves had metabolic alkalosis, while plasma potassium and chloride were significantly lower in non-survivors than those of survivors (P < 0.05). Serum malondialdehyde, superoxide dismutase and uric acid were significantly higher in diseased buffalo than controls and in non-survivors than survivors (P < 0.05). Serum total protein, albumin, creatinine, urea, aspartate aminotransferase, gamma-glutamyl transferase, and total bilirubin levels were also higher in non-survivors than those of survivors (P < 0.05). Buffalo calves with dietary abomasal impaction were associated with marked clinical and biochemical alterations that could be helpful for an accurate diagnosis of the disease.

IGF-1 attenuates LPS induced pro-inflammatory cytokines expression in buffalo (Bubalus bubalis) granulosa cells.

PubMed

Onnureddy, K; Ravinder; Onteru, Suneel Kumar; Singh, Dheer

2015-03-01

Interaction between immune and endocrine system is a diverse process influencing cellular function and homeostasis in animals. Negative energy balance (NEB) during postpartum period in dairy animals usually suppresses these systems resulting in reproductive tract infection and infertility. These negative effects could be due to competition among endocrine and immune signaling pathways for common signaling molecules. The present work studied the effect of IGF-1 (50 ng/ml) on LPS (1 μg/ml) mediated pro-inflammatory cytokine expression (IL-1β, TNF-α, IL-6) and aromatase (CYP19A1) genes' expressions as well as proliferation of buffalo granulosa cells. The crosstalk between LPS and IGF-1 was also demonstrated through studying the activities of downstream signaling molecules (ERK1/2, Akt, NF-κB) by western blot and immunostaining. Gene expression analysis showed that IGF-1 significantly reduced the LPS induced expression of IL-1β, TNF-α and IL-6. LPS alone inhibited the CYP19A1 expression. However, co-treatment with IGF-1 reversed the inhibitory effect of LPS on CYP19A1 expression. LPS alone did not affect granulosa cell proliferation, but co-treatment with IGF-1, and IGF-1 alone enhanced the proliferation. Western blot results demonstrated that LPS caused the nuclear translocation of the NF-κB and increased the phosphorylation of ERK1/2 and Akt maximum at 15 min and 60 min, respectively. Nonetheless, co-treatment with IGF-1 delayed LPS induced phosphorylation of ERK1/2 (peak at 120 min), while promoting early Akt phosphorylation (peak at 5 min) with no effect on NF-κB translocation. Overall, IGF-1 delayed and reversed the effects of LPS, suggesting that high IGF-1 levels may combat infection during critical periods like NEB in postpartum dairy animals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of roughage on rumen microbiota composition in the efficient feed converter and sturdy Indian Jaffrabadi buffalo (Bubalus bubalis).

PubMed

Nathani, Neelam M; Patel, Amrutlal K; Mootapally, Chandra Shekar; Reddy, Bhaskar; Shah, Shailesh V; Lunagaria, Pravin M; Kothari, Ramesh K; Joshi, Chaitanya G

2015-12-29

The rumen microbiota functions as an effective system for conversion of dietary feed to microbial proteins and volatile fatty acids. In the present study, metagenomic approach was applied to elucidate the buffalo rumen microbiome of Jaffrabadi buffalo adapted to varied dietary treatments with the hypothesis that the microbial diversity and subsequent in the functional capacity will alter with diet change and enhance our knowledge of effect of microbe on host physiology. Eight adult animals were gradually adapted to an increasing roughage diet (4 animals each with green and dry roughage) containing 50:50 (J1), 75:25 (J2) and 100:0 (J3) roughage to concentrate proportion for 6 weeks. Metagenomic sequences of solid (fiber adherent microbiota) and liquid (fiber free microbiota) fractions obtained using Ion Torrent PGM platform were analyzed using MG-RAST server and CAZymes approach. Taxonomic analysis revealed that Bacteroidetes was the most abundant phylum followed by Firmicutes, Fibrobacter and Proteobacteria. Functional analysis revealed protein (25-30 %) and carbohydrate (15-20 %) metabolism as the dominant categories. Principal component analysis demonstrated that roughage proportion, fraction of rumen and type of forage affected rumen microbiome at taxonomic as well as functional level. Rumen metabolite study revealed that rumen fluid nitrogen content reduced in high roughage diet fed animals and pathway analysis showed reduction in the genes coding enzymes involved in methanogenesis pathway. CAZyme annotation revealed the abundance of genes encoding glycoside hydrolases (GH), with the GH3 family most abundant followed by GH2 and GH13 in all samples. Results reveals that high roughage diet feed improved microbial protein synthesis and reduces methane emission. CAZyme analysis indicated the importance of microbiome in feed component digestion for fulfilling energy requirements of the host. The findings help determine the role of rumen microbes in plant polysaccharide breakdown and in developing strategies to maximize productivity in ruminants.

Clinical efficacy and pharmacokinetics of meloxicam in Mediterranean buffalo calves (Bubalus bubalis)

PubMed Central

Cagnardi, Petra; Villa, Roberto; D’Andrea, Luigi; Di Loria, Antonio; Ferrante, Maria Carmela; Borriello, Giuliano; Zicarelli, Luigi; Ciaramella, Paolo

2017-01-01

The aims of the investigation were to establish for the first time (i) clinical efficacy and (ii) pharmacokinetic profile of meloxicam intravenously (IV) administered in male Mediterranean buffalo calves after surgical orchiectomy. The study was performed on 10 healthy buffalo calves, between 4 and 5 months old and between 127 and 135 kg of body weight (b.w.). An IV injection of 0.5 mg/kg b.w. of meloxicam was administered in six calves (treated group, TG) immediately after surgery; the other four animals were used as untreated control group (CG). The clinical efficacy of meloxicam was evaluated pre- and post-surgery by monitoring respiratory rate (RR), heart rate (HR), rectal temperature (T°C), serum cortisol levels (SCL) and pain score (PS). Significant inter-groups differences were detected at sampling times (T): 4 hour (h) for RR (P<0.05), at T1-4-6-8 h for PS (P<0.05) and at T4-6-8 h for SCL (P < 0.0001). Regarding the mean intra-group values observed pre (T0) and post-surgery (from T15 min to T72 h), significant difference between the groups were found for RR (P<0.01), PS and SCL (P<0.05). The pharmacokinetic profile was best fitted by a two-compartmental model and characterized by a fast distribution half-life and slow elimination half-life (0.09 ± 0.06 h and 21.51 ± 6.4 h, respectively) and meloxicam mean concentrations at 96 h was of 0.18 ± 0.14 μg/mL. The volume of distribution and clearance values were quite low, but reasonably homogenous among individuals (Vdss 142.31 ± 55.08 mL/kg and ClB 4.38 ± 0.95 mL/kg/h, respectively). The IV administration of meloxicam in buffalo calves shows encouraging effects represented by significant and prolonged analgesic effects, significant reduction of SCL as well as similar pharmacokinetic profile to bovine calves. PMID:29077759

Anthelmintic Effect of Biocompatible Zinc Oxide Nanoparticles (ZnO NPs) on Gigantocotyle explanatum, a Neglected Parasite of Indian Water Buffalo

PubMed Central

Khan, Yasir Akhtar; Singh, Braj Raj; Ullah, Rizwan; Shoeb, Mohd; Naqvi, Alim H.; Abidi, Syed M. A.

2015-01-01

Helminth parasites of veterinary importance cause huge revenue losses to agrarian economy worldwide. With the emergence of drug resistance against the current formulations, there is a need to focus on the alternative approaches in order to control this menace. In the present study, biocompatible zinc oxide nanoparticles (ZnO NPs) were used to see their in vitro effect on the biliary amphistomes, Gigantocotyle explanatum, infecting Bubalus bubalis because these nanoparticles are involved in generation of free radicals that induce oxidative stress, resulting in disruption of cellular machinery. The ZnO NPs were synthesized by using egg albumin as a biotemplate and subsequently characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction and Spectrophotometrical, which showed that ZnO NPs were highly purified wurtzite type polycrystals, with a mean size of 16.7 nm. When the parasites were treated with lower concentrations (0.004% and 0.008%) of the ZnO NPs, the worms mounted a protective response by stimulating the antioxidant system but the treatment of G. explanatum with 0.012% ZnO NPs produced significant inhibition of the antioxidant enzymes like superoxide dismutase (SOD) (p< 0.05) and glutathione S- transferase (GST) (p<0.01), while the level of malondialdehyde (MDA), a lipid peroxidation marker, was significantly (p< 0.01) elevated. SEM and histopathology revealed pronounced tegumental damage showing the disruption of surface papillae and the annulations, particularly in the posterior region near acetabulum. The under expression of a number of polypeptides, loss of worm motility in a time dependent manner, further reflect strong anthelmintic potential of ZnO NPs. It can be concluded that the anthelmintic effect might be due to the production of reactive oxygen species that target a variety of macromolecules such as nucleic acid, protein and lipids which are involved in different cellular processes. PMID:26177503

Anthelmintic Effect of Biocompatible Zinc Oxide Nanoparticles (ZnO NPs) on Gigantocotyle explanatum, a Neglected Parasite of Indian Water Buffalo.

PubMed

Khan, Yasir Akhtar; Singh, Braj Raj; Ullah, Rizwan; Shoeb, Mohd; Naqvi, Alim H; Abidi, Syed M A

2015-01-01

Helminth parasites of veterinary importance cause huge revenue losses to agrarian economy worldwide. With the emergence of drug resistance against the current formulations, there is a need to focus on the alternative approaches in order to control this menace. In the present study, biocompatible zinc oxide nanoparticles (ZnO NPs) were used to see their in vitro effect on the biliary amphistomes, Gigantocotyle explanatum, infecting Bubalus bubalis because these nanoparticles are involved in generation of free radicals that induce oxidative stress, resulting in disruption of cellular machinery. The ZnO NPs were synthesized by using egg albumin as a biotemplate and subsequently characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction and Spectrophotometrical, which showed that ZnO NPs were highly purified wurtzite type polycrystals, with a mean size of 16.7 nm. When the parasites were treated with lower concentrations (0.004% and 0.008%) of the ZnO NPs, the worms mounted a protective response by stimulating the antioxidant system but the treatment of G. explanatum with 0.012% ZnO NPs produced significant inhibition of the antioxidant enzymes like superoxide dismutase (SOD) (p< 0.05) and glutathione S- transferase (GST) (p<0.01), while the level of malondialdehyde (MDA), a lipid peroxidation marker, was significantly (p< 0.01) elevated. SEM and histopathology revealed pronounced tegumental damage showing the disruption of surface papillae and the annulations, particularly in the posterior region near acetabulum. The under expression of a number of polypeptides, loss of worm motility in a time dependent manner, further reflect strong anthelmintic potential of ZnO NPs. It can be concluded that the anthelmintic effect might be due to the production of reactive oxygen species that target a variety of macromolecules such as nucleic acid, protein and lipids which are involved in different cellular processes.

In the elephant's seed shadow: the prospects of domestic bovids as replacement dispersers of three tropical Asian trees.

PubMed

Sekar, Nitin; Lee, Chia-lo; Sukumar, Raman

2015-08-01

As populations of the world's largest animal species decline, it is unclear how ecosystems will react to their local extirpation. Due to the unique ecological characteristics of megaherbivores such as elephants, seed dispersal is one ecosystem process that may be affected as populations of large animals are decimated. In typically disturbed South Asian ecosystems, domestic bovids (cattle, Bosprimigenius, and buffalo, Bubalus bubalis) may often be the species most available to replace Asian elephants (Elephas maximus) as endozoochorous dispersers of large-fruited mammal-dispersed species. We use feeding trials, germination trials, and movement data from the tropical moist forests of Buxa Tiger Reserve (India) to examine whether domestic bovids are viable replacements for elephants in the dispersal of three large- fruited species: Dillenia indica, Artocarpus chaplasha, and Careya arborea. We find that (1) once consumed, seeds are between 2.5 (C. arborea) and 26.5 (D. indica) times more likely to pass undigested into elephant dung than domestic bovid dung; and (2) seeds from elephant dung germinated as well as or better than seeds taken from bovid dung for all plant species, with D. indica seeds from elephant dung 1.5 times more likely to germinate. Furthermore, since wild elephants have less constrained movements than even free-roaming domestic bovids, we calculate that maximum dispersal by elephants is between 9.5 and 11.2 times farther than that of domestic bovids, with about 20% of elephant-dispersed seeds being moved farther than the maximum distance seeds are moved by bovids. Our findings suggest that, while bovids are able to disperse substantial numbers of seeds over moderate distances for two of the three study species, domestic bovids will be unable to routinely emulate the reliable, long-distance dispersal of seeds executed by elephants in this tropical moist forest. Thus while domestic bovids can attenuate the effects of losing elephants as dispersers, they may not be able to prevent the decline of various mammal-dispersed fruiting species in the face of overhunting, habitat fragmentation, and climate change.

Effect of equilibration times, freezing, and thawing rates on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa.

PubMed

Shah, S A H; Andrabi, S M H; Qureshi, I Z

2016-09-01

The effects of equilibration times (E1, 2 h; E2, 4 h; E3, 6 h), freezing rates (FR1, manual, 5 cm above liquid nitrogen (LN2 ) for 10 min, plunging in LN2 ; FR2, programmable ultra-fast, holding at +4 °C for 2 min, from 4 to -10 °C at -10 °C/min, from -10 to -20 °C at -15 °C/min, from -20 to -120 °C at -60 °C/min, holding at -120 °C for 30 sec, plunging in LN2 ), and thawing rates (T1, 37 °C for 30 sec; T2, 50 °C for 15 sec; T3, 70 °C for 7 sec) were evaluated on quality of buffalo bull spermatozoa. Progressive motility (%), rapid velocity (%), average path velocity (VAP, μm/s), straight line velocity (VSL, μm/s), and mitochondrial transmembrane potential (%) were higher (p < 0.05) with E2, FR2, and T3 compared to other groups. Sperm curved line velocity (VCL, μm/s) was higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm straightness (%) and linearity (LIN, %) were higher (p < 0.05) with E2 compared to other groups. Sperm LIN was affected (p < 0.05) with T3 compared to other groups. Supravital-plasma membrane integrity (%), viability and acrosome integrity (%) of spermatozoa were higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm DNA integrity (%) was higher (p < 0.05) with FR2 and T1 compared to other groups. We concluded that inclusion of 4 h-equilibration time, programmable ultra-fast freezing rate, and rapid thawing at 70 °C for 7 sec in cryopreservation protocol improves the post-thaw quality of buffalo bull spermatozoa. © 2016 American Society of Andrology and European Academy of Andrology.

A brief and critical review on hydrofluorosis in diverse species of domestic animals in India.

PubMed

Choubisa, Shanti Lal

2018-02-01

India is one of the fluoride-endemic countries where the maximum numbers of ground or drinking water sources are naturally fluoridated. In India, a total of 23, out of 36 states and union territories have drinking water contaminated with fluoride in varying concentration. In the present scenario, especially in rural India, besides the surface waters (perennial ponds, dams, rivers, etc.), bore wells and hand pumps are the principal drinking water sources for domestic animals such as cattle (Bos taurus), water buffaloes (Bubalus bubalis), sheep (Ovis aries), goats (Capra hircus), horses (Equus caballus), donkeys (Equus asinus) and dromedary camels (Camelus dromedarius). Out of 23 states, 17 states, namely Andhra Pradesh, Assam, Bihar, Chhattisgarh, Gujarat, Haryana, Jharkhand, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Odisha (Orissa), Punjab, Rajasthan, Telangana, Uttar Pradesh and West Bengal, have fluoride beyond the maximum permissible limit of 1.0 or 1.5 ppm in drinking water. This situation is a great concern for the animal health because fluoride is a slow toxicant and causes chronic diverse serious health hazards or toxic effects. Despite the fact that domestic animals are the basic income sources in rural areas and possess a significant contributory role not only in the agriculture sector but also in the strengthening of economy as well as in sustainable development of the country, research work on chronic fluoride intoxication (hydrofluorosis) due to drinking of fluoridated water in domestic animals rearing in various fluoride-endemic states is not enough as compared to work done in humans. However, some interesting and excellent research works conducted on different aspects of hydrofluorosis in domesticated animals rearing in different states are briefly and critically reviewed in the present communication. Author believes that this review paper not only will be more useful for researchers to do some more advance research work on fluoride-induced toxicosis in different species of animals but will also be helpful in the making of health policy for domestic animals at state and national level for the mitigation of hydrofluorosis in India.

Foot-and-mouth disease in the Americas: epidemiology and ecologic changes affecting distribution.

PubMed

Saraiva, Victor

2004-10-01

Foot-and-mouth disease(FMD) was first recorded in South America (SA) circa 1870, in Buenos Aires, Argentina, in Uruguay, and in southern Brazil as a result of the introduction of cattle from Europe during the early days of colonization. Livestock production to trade with neighboring countries was established in the La Plata Region, and the trade of livestock and products with Chile, northeastern and central western states of Brazil, to Peru, Bolivia, and Paraguay spread FMD, which reached Venezuela and Colombia in the 1950s and finally Ecuador in 1961. The traditional forms of livestock husbandry influence the diffusion and maintenance of the FMD virus (FMDV) in different areas. Cattle production in SA depends mainly on a strong relation between cattle-calf operations and fattening operations in a complementary cycle, revealing the vulnerability and susceptibility of these areas to FMDV. Understanding the relationship between time-space behavior of the disease and the forms of production defines the FMD ecosystems, a key concept to elaborating the control/eradication strategies of national FMD eradication programs, which must be modified when trade opportunities between zones of differing sanitary status change. The role of other susceptible species besides bovines, including wildlife, in maintaining and spreading FMDV has been the subject of several studies, but in SA, bovines are so far considered to determine disease presentation. Buffalo (Bubalus bubalis) have been implicated in the spread of the disease between farms in at least one case in Brazil. Sheep are almost on a par with bovine in terms of number, especially in the Southern Cone, but their role in the maintenance of infection is not considered important, possibly owing to rearing practices. Camelid populations in the Andean region do not play an important role in the maintenance of FMD, because of short persistence of infection and low population densities in these species. The importance of wildlife is not clear, but it is accepted that animals are mostly affected as a spinoff during outbreaks in domestic species. Experimentally infected capybaras (Hydrochoerus hydrochoeris hydrochoeris) showed clinical signs and infected other susceptible species, but their role in the maintenance of infection in nature is so far not clear.

Molecular assays reveal the presence of Theileria spp. and Babesia spp. in Asian water buffaloes (Bubalus bubalis, Linnaeus, 1758) in the Amazon region of Brazil.

PubMed

Silveira, Júlia A G; de Oliveira, Cairo H S; Silvestre, Bruna T; Albernaz, Tatiana T; Leite, Rômulo C; Barbosa, José D; Oliveira, Carlos M C; Ribeiro, Múcio F B

2016-07-01

Approximately 50% of buffalo herds in Brazil are located in Pará state in northern Brazil. There are several properties where cattle and buffalo live and graze together, and thus, buffalo pathogens may threaten the health of cattle and vice versa. Therefore, knowledge of infectious agents of buffalo is essential for maintaining healthy livestock. Clinical disease caused by Theileria and Babesia parasites in the Asian water buffalo is not common, although these animals may act as reservoir hosts, and the detection of these hemoparasites in buffaloes is as important as it is in cattle. Studies of the infection of buffaloes by hemoparasites in Brazil are scarce. The objective of the present study was to investigate the occurrence of Piroplasmida parasites in Asian water buffaloes in the state of Pará in the Amazon region of Brazil using nested PCR assays and phylogenetic analysis. The 18S rRNA gene and ITS complete region were amplified from DNA extracted from blood samples collected from 308 apparently healthy buffaloes bred on six properties in the state of Pará, Brazil. The prevalence of positive buffalo samples was 4.2% (13/308) for Theileria spp., 3.6% (11/308) for Babesia bovis and 1% (3/308) for Babesia bigemina. Animals infected with Theileria were detected in 50% (3/6) of the assessed properties. Phylogenetic analyses indicated that the Theileria species detected in this study were closely related to Theileria buffeli, Theileria orientalis and Theileria sinensis. To our knowledge, this is the first report of Theileria in Asian water buffaloes in the Americas. The majority of Theileria-positive buffaloes (11/13) belong to a property that has a history of animals presenting lymphoproliferative disease of unknown etiology. Therefore, the present research suggests that this disorder can be associated with Theileria infection in this property. Our results provide new insights on the distribution and biological aspects of hemoparasites transmissible from buffaloes to cattle. Copyright © 2016 Elsevier GmbH. All rights reserved.

Lessons for livestock genomics from genome and transcriptome sequencing in cattle and other mammals.

PubMed

Taylor, Jeremy F; Whitacre, Lynsey K; Hoff, Jesse L; Tizioto, Polyana C; Kim, JaeWoo; Decker, Jared E; Schnabel, Robert D

2016-08-17

Decreasing sequencing costs and development of new protocols for characterizing global methylation, gene expression patterns and regulatory regions have stimulated the generation of large livestock datasets. Here, we discuss experiences in the analysis of whole-genome and transcriptome sequence data. We analyzed whole-genome sequence (WGS) data from 132 individuals from five canid species (Canis familiaris, C. latrans, C. dingo, C. aureus and C. lupus) and 61 breeds, three bison (Bison bison), 64 water buffalo (Bubalus bubalis) and 297 bovines from 17 breeds. By individual, data vary in extent of reference genome depth of coverage from 4.9X to 64.0X. We have also analyzed RNA-seq data for 580 samples representing 159 Bos taurus and Rattus norvegicus animals and 98 tissues. By aligning reads to a reference assembly and calling variants, we assessed effects of average depth of coverage on the actual coverage and on the number of called variants. We examined the identity of unmapped reads by assembling them and querying produced contigs against the non-redundant nucleic acids database. By imputing high-density single nucleotide polymorphism data on 4010 US registered Angus animals to WGS using Run4 of the 1000 Bull Genomes Project and assessing the accuracy of imputation, we identified misassembled reference sequence regions. We estimate that a 24X depth of coverage is required to achieve 99.5 % coverage of the reference assembly and identify 95 % of the variants within an individual's genome. Genomes sequenced to low average coverage (e.g., <10X) may fail to cover 10 % of the reference genome and identify <75 % of variants. About 10 % of genomic DNA or transcriptome sequence reads fail to align to the reference assembly. These reads include loci missing from the reference assembly and misassembled genes and interesting symbionts, commensal and pathogenic organisms. Assembly errors and a lack of annotation of functional elements significantly limit the utility of the current draft livestock reference assemblies. The Functional Annotation of Animal Genomes initiative seeks to annotate functional elements, while a 70X Pac-Bio assembly for cow is underway and may result in a significantly improved reference assembly.

Impact of livestock hygiene education programs on mastitis in smallholder water buffalo (Bubalus bubalis) in Chitwan, Nepal.

PubMed

Ng, Linda; Jost, Christine; Robyn, Misha; Dhakal, I P; Bett, Bernard; Dhakal, Pramod; Khadka, Rupak

2010-09-01

A project implemented from 2003 to 2005 trained women in Chitwan District, Nepal, in hygienic dairy production using a process of social mobilization. The aim of this research was to assess if the prevalence of mastitis in water buffalo in the households of women who were trained was lower one year after training than in untrained households, if the training influenced knowledge and practices for the prevention or control of mastitis, and if these practices and knowledge were associated with a lower prevalence of mastitis. A total of 202 households from Eastern and Western Chitwan District were included in the study. Of these, 60 households had participated in the project and 142 had not. Milk samples were collected from 129 households (33 project households and 96 non-project households). Clinical mastitis was determined using visual inspection of udders and detection of macroscopic clots and flakes in milk. The California Mastitis Test was used to diagnose sub-clinical mastitis from milk samples, and the IDEXX SNAP test to identify the presence of tetracycline residues. The prevalence of mastitis in trained households (39.4%) was 43.78% of that in untrained households (60.4%), lower but not significantly so (p=0.08, 95% CI 0.17-1.12). Thirteen indicators of knowledge or practice for the control or prevention of mastitis were more likely to occur in trained households, four significantly so (not consuming milk from sick buffalo (p=0.001), using soap to wash hands before milking (p=0.001), discarding milk after antibiotic usage (p=0.01), and choosing appropriate flooring for their livestock (p=0.03)). Trained households that discarded milk from sick buffalo were 2.96 times more likely to have at least one animal with mastitis in the household (p=0.03, 95% CI 1.15-7.65). Trained households that knew to wash buffalos' teats after milking were less likely (OR 0.25) to have mastitis in their herd (p=0.02, 95% CI 0.08-0.80). Of the 138 buffalos tested, only one tested positive for tetracycline residues. 2010 Elsevier B.V. All rights reserved.

Expression and localization of insulin-like growth factor system in corpus luteum during different stages of estrous cycle in water buffaloes (Bubalus bubalis) and the effect of insulin-like growth factor I on production of vascular endothelial growth factor and progesterone in luteal cells cultured in vitro.

PubMed

Uniyal, S; Panda, R P; Chouhan, V S; Yadav, V P; Hyder, I; Dangi, S S; Gupta, M; Khan, F A; Sharma, G T; Bag, S; Sarkar, M

2015-01-01

This study investigated the expression and localization of insulin-like growth factor (IGF) system at different stages of buffalo CL and the role of IGF-I in stimulating vascular endothelial growth factor (VEGF) and progesterone (P4) production in cultured luteal cells. The mRNA expression of IGF system, VEGF, steroidogenic acute regulatory protein, P450scc, and hydroxysteroid dehydrogenase (HSD) was investigated by quantitative real-time polymerase chain reaction (PCR). Protein expression of IGF was demonstrated by Western blot and localization by immunohistochemistry. Progesterone and VEGF production was assayed using RIA and ELISA. A relatively high mRNA expression of IGF-I and IGF-II in early, mid- and late luteal phases with immunoreactivity mostly restricted to cytoplasm of large luteal cells indicates their autocrine role, whereas very weak immunoreactivity in endothelial cells during the mid-luteal phase indicates their paracrine role. Insulin-like growth factor receptors, IGF-IR and IGF-IIR, were restricted to large luteal cells with high mRNA and protein expressions in the mid-luteal phase. The significantly higher expression of insulin-like growth factor binding protein (IGFBP)-1, -3, -5, and -6 in the early or mid-luteal phase suggested their stimulatory role, whereas that of IGFBP-2 and -4 in mid-, late, and regressive luteal stages implied their inhibitory role. The mRNA expressions of key steroidogenic factors and VEGF were significantly higher (P < 0.05) when the culture medium was supplemented with 100 ng/mL of IGF-I for 72 hours. Moreover, IGF-I at a dose of 100 ng/mL increased P4 and VEGF production (P < 0.05). It can be concluded that IGF family members via their autocrine and paracrine effect play significant roles in promoting angiogenesis through the production of VEGF in luteal cells and steroid synthesis through the production of key steroidogenic factors. Copyright © 2015 Elsevier Inc. All rights reserved.

DETOMIDINE AND BUTORPHANOL FOR STANDING SEDATION IN A RANGE OF ZOO-KEPT UNGULATE SPECIES.

PubMed

Bouts, Tim; Dodds, Joanne; Berry, Karla; Arif, Abdi; Taylor, Polly; Routh, Andrew; Gasthuys, Frank

2017-09-01

General anesthesia poses risks for larger zoo species, like cardiorespiratory depression, myopathy, and hyperthermia. In ruminants, ruminal bloat and regurgitation of rumen contents with potential aspiration pneumonia are added risks. Thus, the use of sedation to perform minor procedures is justified in zoo animals. A combination of detomidine and butorphanol has been routinely used in domestic animals. This drug combination, administered by remote intramuscular injection, can also be applied for standing sedation in a range of zoo animals, allowing a number of minor procedures. The combination was successfully administered in five species of nondomesticated equids (Przewalski horse [ Equus ferus przewalskii; n = 1], onager [ Equus hemionus onager; n = 4], kiang [ Equus kiang ; n = 3], Grevy's zebra [ Equus grevyi ; n = 4], and Somali wild ass [ Equus africanus somaliensis; n = 7]), with a mean dose range of 0.10-0.17 mg/kg detomidine and 0.07-0.13 mg/kg butorphanol; the white ( Ceratotherium simum simum; n = 12) and greater one-horned rhinoceros ( Rhinoceros unicornis ; n = 4), with a mean dose of 0.015 mg/kg of both detomidine and butorphanol; and Asiatic elephant bulls ( Elephas maximus ; n = 2), with a mean dose of 0.018 mg/kg of both detomidine and butorphanol. In addition, the combination was successfully used for standing sedation in six species of artiodactylids: giraffe ( Giraffa camelopardalis reticulata; n = 3), western bongo ( Tragelaphus eurycerus eurycerus; n = 2), wisent ( Bison bonasus ; n = 5), yak ( Bos grunniens ; n = 1), water buffalo ( Bubalus bubalis ; n = 4) and Bactrian camel ( Camelus bactrianus ; n = 5). The mean dose range for artiodactylid species except bongo was 0.04-0.06 mg/kg detomidine and 0.03-0.06 mg/kg butorphanol. The dose in bongo, 0.15-0.20 mg/kg detomidine and 0.13-0.15 mg/kg butorphanol, was considerably higher. Times to first effect, approach, and recovery after antidote were short. The use of detomidine and butorphanol has been demonstrated to be a reliable, safe alternative to general anesthesia for a number of large ungulate species.

Effect of sexual excitation on testosterone and nitric oxide levels of water buffalo bulls (Bubalus bubalis) with different categories of sexual behavior and their correlation with each other.

PubMed

Swelum, Ayman Abdel-Aziz; Saadeldin, Islam M; Zaher, Hany A; Alsharifi, Sawsan A M; Alowaimer, Abdullah N

2017-06-01

We studied the effect of sexual excitation on serum testosterone and nitric oxide (NO) levels in water buffalo bulls with different categories of sexual behavior and their correlation with each other. Buffalo bulls were classified according to their sexual behavior (including reaction time, sexual aggressiveness and mating ability): acceptable (good to excellent) (n=5), fair (n=5), and unacceptable (poor) (n=5) sexual behavior. Blood samples were collected from all animals immediately before and after sexual teasing and/or mounting to estimate the testosterone and NO levels using a commercial radioimmunoassay kit and Griess reaction test, respectively. Comparisons among groups were evaluated using a mixed-design analysis of variance. Pearson's correlation coefficients were calculated to determine the relationship between testosterone and NO levels before and after sexual excitation besides sexual behavior. The level of testosterone before sexual excitation was higher (p≤0.05) in bulls with acceptable and fair sexual behavior than in bulls with unacceptable sexual behavior (0.86±0.01, 0.69±0.02, and 0.29±0.02ng/mL, respectively). The level of NO was higher (p≤0.05) in bulls with acceptable and fair sexual behavior than in bulls with unacceptable sexual behavior (8.00±0.03, 7.66±0.19, and 6.29±0.33μM, respectively). Sexual excitation significantly (p<0.05) increase testosterone and NO levels in bulls with acceptable (1.45±0.01ng/mL and 19.04±0.32μM, respectively) or fair (0.92±0.02ng/mL and 14.95±0.34μM, respectively) sexual behavior, but not in bulls with unacceptable sexual behavior. The unacceptable sexual behavior bulls had significantly lower testosterone and NO levels than the other bulls. There was a strong correlation and association between serum testosterone and NO levels besides sexual behavior of buffalo bulls. In conclusion, the alteration in the testosterone and NO levels after sexual excitation depends on the sexual behavior category of buffalo-bull. Testosterone and NO can be used to create a sexual behavior score. The testosterone and NO levels of can be predicted via evaluation of sexual behavior of buffalo bull. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation and characterization of EG-like cells from Chinese swamp buffalo (Bubalus bubalis).

PubMed

Huang, Ben; Xie, Ti-San; Shi, De-Shun; Li, Tong; Wang, Xiao-Li; Mo, Yi; Wang, Zhi-Qiang; Li, Meng-Mei

2007-10-01

There have been few studies done on the isolation and characterization of Chinese swamp buffalo embryonic germ cells (EG cells). Here, we first report on EG-like cells isolated from Chinese swamp buffalo fetuses. The results showed the cells grew in large, multilayered colonies, which were densely packed with an obvious border resembling mouse embryonic stem cells (ES cells) and EG cells. The buffalo EG-like cells expressed AP, SSEA-1, SSEA-3, SSEA-4 and OCT-4. By RT-PCR, we found that undifferentiated swamp buffalo EG-like cells expressed the OCT-4, NANOG, SOX2, FOXD3, GP130, STAT3, and HEB gene mRNA, but not Fgf4. When these cells were cultured for more than 2weeks without passage, they could differentiate into several types of cells including fibroblast-like, neuron-like, smooth muscle-like, and epithelial-like cells. Some cells formed simple embryoid bodies (EBs) and cystic EBs by suspension culture. By RT-PCR, we found cystic EBs expressed FOXD3, GP130, STAT3 and HEB gene mRNA, but not OCT-4, NANOG, and SOX2 gene mRNA, which could be detected in undifferentiated buffalo EG-like cells. At the same time, the expression of KERATIN-14 (Endoderm), GATA4, ACTA2 (Mesoderm) and TUBB3 (Ectoderm) gene mRNA were also detected in cystic EBs. The results suggested that these cells were capable of forming three germ layers in in vitro differentiation. The expression of OCT-4, NANOG and SOX2 might be essential for Chinese swamp buffalo EG-like cells in a pluripotent state. During the isolation and culture of Chinese swamp buffalo EG-like cells, we found the fetuses that were at 30-80days post-coitus were more efficient than others; and the mechanical method was better than trypsin digestion. The maximal passage of the mechanical method was eight, but the trypsin digestion was just three passages. So it seemed like that the buffalo EG-like cells were sensitive to trypsin. In summary, we were the first to isolate and characterize Chinese swamp buffalo EG-like cells that had morphology and characterization similar to those of established EG/EG-like cells in mouse and human.

In silico mining of putative microsatellite markers from whole genome sequence of water buffalo (Bubalus bubalis) and development of first BuffSatDB

PubMed Central

2013-01-01

Background Though India has sequenced water buffalo genome but its draft assembly is based on cattle genome BTau 4.0, thus de novo chromosome wise assembly is a major pending issue for global community. The existing radiation hybrid of buffalo and these reported STR can be used further in final gap plugging and “finishing” expected in de novo genome assembly. QTL and gene mapping needs mining of putative STR from buffalo genome at equal interval on each and every chromosome. Such markers have potential role in improvement of desirable characteristics, such as high milk yields, resistance to diseases, high growth rate. The STR mining from whole genome and development of user friendly database is yet to be done to reap the benefit of whole genome sequence. Description By in silico microsatellite mining of whole genome, we have developed first STR database of water buffalo, BuffSatDb (Buffalo MicroSatellite Database (http://cabindb.iasri.res.in/buffsatdb/) which is a web based relational database of 910529 microsatellite markers, developed using PHP and MySQL database. Microsatellite markers have been generated using MIcroSAtellite tool. It is simple and systematic web based search for customised retrieval of chromosome wise and genome-wide microsatellites. Search has been enabled based on chromosomes, motif type (mono-hexa), repeat motif and repeat kind (simple and composite). The search may be customised by limiting location of STR on chromosome as well as number of markers in that range. This is a novel approach and not been implemented in any of the existing marker database. This database has been further appended with Primer3 for primer designing of the selected markers enabling researcher to select markers of choice at desired interval over the chromosome. The unique add-on of degenerate bases further helps in resolving presence of degenerate bases in current buffalo assembly. Conclusion Being first buffalo STR database in the world , this would not only pave the way in resolving current assembly problem but shall be of immense use for global community in QTL/gene mapping critically required to increase knowledge in the endeavour to increase buffalo productivity, especially for third world country where rural economy is significantly dependent on buffalo productivity. PMID:23336431

[Polymorphisms of inhibin α gene exon 1 in buffalo (Bubalus bubalis), gayal (Bos frontalis) and yak (Bos grunniens)].

PubMed

Miao, Yong-Wang; Ha, Fu; Gao, Hua-Shan; Yuan, Feng; Li, Da-Lin; Yuan, Yue-Yun

2012-08-01

To elucidate the genetic characteristics of the bovine Inhibin α subunit (INHA) gene, the polymorphisms in exon 1 of INHA and its bilateral sequences were assayed using PCR with direct sequencing in buffalo, gayal and yak. A comparative analysis was conducted by pooled the results in this study with the published data of INHA on some mammals including some bovine species together. A synonymous substitution c.73C>A was identified in exon 1 of INHA for buffalo, which results in identical encoding product in river and swamp buffalo. In gayal, two non-synonymous but same property substitutions in exon 1 of INHA, viz. c.62 C>T and c.187 G>A, were detected, which lead to p. P21L, p. V63M changes in INHA, respectively. In yak, nucleotide substitution c.62C> T, c.129A>G were found in exon 1 of INHA, the former still causes p. P21L substitution and the latter is synonymous. For the sequence of the 5'-flanking region of INHA examined, no SNPs were found within the species, but a substitution, c. -6T>G, was found. The nucleotide in this site in gayal, yak and cattle was c. -6G, whereas in buffalo it was c. -6T. Meanwhile, a 6-bp deletion, namely c. 262+31_262+36delTCTGAC, was found in the intron of buffalo INHA gene. For this deletion, wild types (+/+) account for main part in river buffalo while mutant types (-/-) are predominant in swamp buffalo. This deletion was not found in gayal, yak and cattle, though these all have another deletion in the intron of INHA, c. 262+78_262+79delTG. The results of sequence alignment showed that the substitutions c. 43A and c. 67G in exon 1 of INHA are specific to buffalo, whereas the substitutions c. 173A and c. 255G are exclusive to gayal, yak and cattle, and c. 24C, c. 47G, c. 174T and c. 206T are specific to goat. Furthermore, there are few differences among gayal, yak and cattle, but there relatively great differences between buffalo, goat and other bovine species regarding the sequences of INHA exon 1.

In silico mining of putative microsatellite markers from whole genome sequence of water buffalo (Bubalus bubalis) and development of first BuffSatDB.

PubMed

Sarika; Arora, Vasu; Iquebal, Mir Asif; Rai, Anil; Kumar, Dinesh

2013-01-19

Though India has sequenced water buffalo genome but its draft assembly is based on cattle genome BTau 4.0, thus de novo chromosome wise assembly is a major pending issue for global community. The existing radiation hybrid of buffalo and these reported STR can be used further in final gap plugging and "finishing" expected in de novo genome assembly. QTL and gene mapping needs mining of putative STR from buffalo genome at equal interval on each and every chromosome. Such markers have potential role in improvement of desirable characteristics, such as high milk yields, resistance to diseases, high growth rate. The STR mining from whole genome and development of user friendly database is yet to be done to reap the benefit of whole genome sequence. By in silico microsatellite mining of whole genome, we have developed first STR database of water buffalo, BuffSatDb (Buffalo MicroSatellite Database (http://cabindb.iasri.res.in/buffsatdb/) which is a web based relational database of 910529 microsatellite markers, developed using PHP and MySQL database. Microsatellite markers have been generated using MIcroSAtellite tool. It is simple and systematic web based search for customised retrieval of chromosome wise and genome-wide microsatellites. Search has been enabled based on chromosomes, motif type (mono-hexa), repeat motif and repeat kind (simple and composite). The search may be customised by limiting location of STR on chromosome as well as number of markers in that range. This is a novel approach and not been implemented in any of the existing marker database. This database has been further appended with Primer3 for primer designing of the selected markers enabling researcher to select markers of choice at desired interval over the chromosome. The unique add-on of degenerate bases further helps in resolving presence of degenerate bases in current buffalo assembly. Being first buffalo STR database in the world , this would not only pave the way in resolving current assembly problem but shall be of immense use for global community in QTL/gene mapping critically required to increase knowledge in the endeavour to increase buffalo productivity, especially for third world country where rural economy is significantly dependent on buffalo productivity.

The relationship between rumen bacterial growth, intake of dry matter, digestible organic matter and volatile fatty acid production in buffalo (Bos bubalis) calves.

PubMed

Singh, U B; Verma, D N; Varma, A; Ranjhan, S K

1977-11-01

1. The production rates of bacteria in the rumen of buffalo (Bos bubalis) calves were estimated using an isotope-dilution technique. A series of fifteen experiments was done with animals given green maize and nine experiments with animals given cowpea (Vigna unguiculata). 2. The turnover time ranged from 205 to 567 min in the group given green maize and from 330 to 648 min in animals offered cowpea. The production rates of bacteria were (mean +/- SE; g/d) 145.77 +/- 7.240 and 237.09 +/- 11.847 in animals given green maize and cowpea respectively. 3. There was a significant correlation between bacterial production rates and dry matter intake, digestible organic matter and total volatile fatty acids formed in the rumen. 4. Regression equations obtained for the two foodstuffs were different suggesting that the bacterial growth rate may vary depending upon the quantity and quality of foodstuff digested and possibly the ratio nitrogen:energy of the foodstuff.

PHYLOGEOGRAPHIC PATTERNS IN LARGE RIVER ECOSYSTEMS: GENETIC STRUCTURE OF SMALLMOUTH BUFFALO (ICTIOBUS BUBALUS) IN THE OHIO RIVER

EPA Science Inventory

Genetic studies on populations of large river fishes provide a potentially useful but underutilized research and assessment tool. Population genetic research on freshwater systems has provided meaningful insight into stock structure, hybridization issues, and gene flow/migration...

77 FR 3493 - Endangered Species Receipt of Applications for Permit

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-24

... (Bubalus depressicornis), removed from the wild and captive bred in zoos for the purpose of scientific... the applicant over a 5-year period. Applicant: Kansas City Zoo, Kansas City, MO; PRT-681588. The... activities to be conducted by the applicant over a 5-year period. [[Page 3495

Effect of dietary protein on intake, nutrients utilization, nitrogen balance, blood metabolites, growth and puberty in growing Bhadawari buffalo (Bubalus bubalis) heifers.

PubMed

Singh, Sultan; Kushwaha, Badri Prasad; Maity, Subendu Bikas; Singh, Krishan Kunwar; Das, Nityanand

2015-01-01

Fifteen Bhadawari buffalo heifers of 207 ± 9.78 kg mean body weight were randomly distributed into three dietary groups to evaluate the effect of protein level on nutrient utilization, nitrogen (N) balance, growth rate, blood metabolites, and puberty. All animals were offered wheat straw-berseem diets supplemented with concentrate mixtures of similar energy (2.7 Mcal/kg) and different protein levels (14.3-22%). Animals of standard-protein group (SPG) were offered protein and energy as per requirement, while animals of low-protein group (LPG) and high-protein group (HPG) were fed 20% less and 20% more protein, respectively, than SPG. Feed dry matter (DM) and metabolizable energy (ME) intake (% body wt. and g/kg w(0.75)) were similar for all three diets; however, the crude protein (CP) and digestible crude protein (DCP) intake on percent body weight and per kilogram metabolic weight was higher (P < 0.05) in HPG than in SPG or LPG. Digestibility of CP, cellulose, and hemicellulose was higher (P < 0.05) in HPG versus LPG. Fecal N excretion was similar, while urinary N excretion was highest (P < 0.05) in HPG (74.83 g/day) compared with SPG (50.03 g/day) and LPG (47.88 g/day), which resulted in lower N retention in HPG than in the other dietary groups. Level of dietary N had no effect on blood metabolites viz. glucose, urea, and N. Digestible energy (DE) and ME contents of diets were identical, while DCP contents were higher (P < 0.05) in HPG than in LPG. Feed and nutrient (CP and ME) conversion efficiency to produce a unit kilogram weight gain was identical among the dietary groups. Dietary protein level had no effect on the heifer's weight and age at puberty. The mean growth rate of heifers at 240 days was higher (P > 0.05) in SPG (330.8 g/day) than in LPG (296.7 g/day), while the animals gained more weight in January to March months and the lowest weight in May to July months. Protein level had no effect on conception rate of heifers. Results revealed that 20% higher or less protein than the ICAR requirement had no significant (P > 0.05) on feed intake, nutrient conversion efficiency for weight gain, heifer growth, and puberty; however, 20% more protein increased urinary N loss.

Influence of season, age and management on scrotal thermal profile in Murrah bulls using scrotal infrared digital thermography

NASA Astrophysics Data System (ADS)

Ahirwar, Maneesh Kumar; Kataktalware, Mukund Amritrao; Ramesha, Kerekoppa Puttaiah; Pushpadass, Heartwin Amaladhas; Jeyakumar, Sakthivel; Revanasiddu, Deginal; Kour, Reen Jagish; Nath, Sapna; Nagaleekar, Anand Kumar; Nazar, Sayyad

2017-12-01

The aim of the present study was to examine the effects of non-genetic factors on scrotal thermographic profile viz., proximal pole temperature (PPT °C), mid pole temperature (MPT °C), distal pole temperature (DPT °C) and ocular temperature (OcT) of Murrah ( Bubalus bubalis) breeding bulls. A total of 109 buffalo bulls, maintained at three semen stations (SS), were monitored for scrotal surface and ocular temperatures using infrared thermography twice daily during rainy, winter and summer seasons using an FLIR i5 infrared camera and temperatures were measured. Thermograms were analysed by FLIR QuickReport v.1.2 SP2 software. Statistical analysis revealed that semen station, season, temperature humidity index (THI), housing system and timing of observations had significant ( P < 0.05) effect on scrotal surface temperature (SST) and OcT. In SS-I, the PPT and MPT were significantly ( P < 0.05) higher as compared to SS-II and SS-III. THI had significant ( P < 0.05) effect on SST and OcT, whereas PPT (°C), MPT (°C), DPT (°C) and OcT (°C) values during high THI (>80.88; <0.05) period were higher as compared to medium THI period (70.06-80.88) and during low THI period (<70.06). Temperature gradient (TG) of the testes was significantly ( P < 0.05) higher during low THI period (4.50 ± 0.06 °C) as compared to medium THI (2.38 ± 0.03 °C) and high THI (1.61 ± 0.05 °C). Season of the year had a significant effect ( P < 0.05) on the SST and OcT. During the rainy season, PPT (34.50 ± 0.09 °C), MPT (33.44 ± 0.12 °C) and DPT (32.11 ± 0.15 °C) were significantly ( P < 0.05) higher as compared to winter and summer seasons. Age of the bulls had non-significant effect on SST and OcT but had a marked influence on thermal profile of scrotum. It could be concluded semen station, season, temperature humidity index, housing system and timing of observations had a significant influence on scrotal surface temperature. The monitoring of scrotal surface temperature by infrared thermography was found to be useful in evaluating the effects of thermal stress on physiology and health of buffalo bulls.

Minimum number of spermatozoa per dose in Mediterranean Italian buffalo (Bubalus bubalis) using sexed frozen semen and conventional artificial insemination.

PubMed

Gaviraghi, A; Puglisi, R; Balduzzi, D; Severgnini, A; Bornaghi, V; Bongioni, G; Frana, A; Gandini, L M; Lukaj, A; Bonacina, C; Galli, A

2013-05-01

In buffaloes, AI with sexed semen is not fully optimized, and the procedure has only been performed using the approach currently in use for cattle. The objective of the present work was to compare the pregnancy rates in Mediterranean Italian buffalo cows inseminated with sexed frozen-thawed semen at 2, 4, 6, and 8 million sperm per dose, using the Ovsynch protocol and conventional AI at a fixed time. Fresh ejaculates from three buffalo bulls were processed according to Beltsville sperm sorting technology, and packaged in 0.25-mL straws with two total concentrations of 2 and 4 million live sorted sperm per straw. After thawing, semen was evaluated for total motility, forward motility, average path velocity, membrane and DNA integrity, and membrane fluidity. Sorting efficiency was estimated using a real time polymerase chain reaction method developed and validated in our laboratory. The artificial inseminations were conducted during the breeding season on 849 Italian Mediterranean buffalo heifers and cows distributed in 13 farms in northern and central Italy. No significant difference in quality parameters was reported between nonsexed and sexed straws produced with 2 and 4 million sperm. Lower pregnancy rate (P < 0.001) was reported when inseminating doses of sexed semen at 2 million were used (53/170; 31.2%), with respect to conventional nonsexed (78/142; 54.9%), and sexed doses at 4, 6, and 8 million spermatozoa (102/205, 49.8%; 84/175, 48.0%; and 74/157, 47.1%, respectively). No differences were evident using conventional doses and sexed semen with sperm numbers equal or higher than 4 million per dose. Pregnancies were not affected by the sire; 39/82 (47.6%), 120/270 (44.4%), and 151/355 (42.5%), respectively, for the three bulls. Variability in pregnancy rates observed in different herds was not significant. Furthermore, no significant difference was reported between pregnancies obtained with sexed semen in heifers and multiparous, respectively, 179/407 (44.0%) and 131/300 (43.7%). The results of the present work indicate that in Mediterranean Italian buffalo the dose of 4 million represents an optimal compromise when using sexed semen with conventional technologies of insemination, together with estrus synchronization, and the minimum number of spermatozoa per dose. In addition, the real time polymerase chain reaction method was optimized and is now available for estimating sorting efficiency in buffalo. Copyright © 2013 Elsevier Inc. All rights reserved.

Influence of season, age and management on scrotal thermal profile in Murrah bulls using scrotal infrared digital thermography.

PubMed

Ahirwar, Maneesh Kumar; Kataktalware, Mukund Amritrao; Ramesha, Kerekoppa Puttaiah; Pushpadass, Heartwin Amaladhas; Jeyakumar, Sakthivel; Revanasiddu, Deginal; Kour, Reen Jagish; Nath, Sapna; Nagaleekar, Anand Kumar; Nazar, Sayyad

2017-12-01

The aim of the present study was to examine the effects of non-genetic factors on scrotal thermographic profile viz., proximal pole temperature (PPT °C), mid pole temperature (MPT °C), distal pole temperature (DPT °C) and ocular temperature (OcT) of Murrah (Bubalus bubalis) breeding bulls. A total of 109 buffalo bulls, maintained at three semen stations (SS), were monitored for scrotal surface and ocular temperatures using infrared thermography twice daily during rainy, winter and summer seasons using an FLIR i5 infrared camera and temperatures were measured. Thermograms were analysed by FLIR QuickReport v.1.2 SP2 software. Statistical analysis revealed that semen station, season, temperature humidity index (THI), housing system and timing of observations had significant (P < 0.05) effect on scrotal surface temperature (SST) and OcT. In SS-I, the PPT and MPT were significantly (P < 0.05) higher as compared to SS-II and SS-III. THI had significant (P < 0.05) effect on SST and OcT, whereas PPT (°C), MPT (°C), DPT (°C) and OcT (°C) values during high THI (>80.88; <0.05) period were higher as compared to medium THI period (70.06-80.88) and during low THI period (<70.06). Temperature gradient (TG) of the testes was significantly (P < 0.05) higher during low THI period (4.50 ± 0.06 °C) as compared to medium THI (2.38 ± 0.03 °C) and high THI (1.61 ± 0.05 °C). Season of the year had a significant effect (P < 0.05) on the SST and OcT. During the rainy season, PPT (34.50 ± 0.09 °C), MPT (33.44 ± 0.12 °C) and DPT (32.11 ± 0.15 °C) were significantly (P < 0.05) higher as compared to winter and summer seasons. Age of the bulls had non-significant effect on SST and OcT but had a marked influence on thermal profile of scrotum. It could be concluded semen station, season, temperature humidity index, housing system and timing of observations had a significant influence on scrotal surface temperature. The monitoring of scrotal surface temperature by infrared thermography was found to be useful in evaluating the effects of thermal stress on physiology and health of buffalo bulls.

Cellular and functional characterization of buffalo (Bubalus bubalis) corpus luteum during the estrous cycle and pregnancy.

PubMed

Baithalu, Rubina Kumari; Singh, S K; Gupta, Chhavi; Raja, Anuj K; Saxena, Abhishake; Kumar, Yogendra; Singh, R; Agarwal, S K

2013-08-01

In the present paper, cellular composition of buffalo corpus luteum (CL) with its functional characterization based on 3β-HSD and progesterone secretory ability at different stages of estrous cycle and pregnancy was studied. Buffalo uteri along with ovaries bearing CL were collected from the local slaughter house. These were classified into different stages of estrous cycle (Stage I, II, III and IV) and pregnancy (Stage I, II and III) based on morphological appearance of CL, surface follicles on the ovary and crown rump length of conceptus. Luteal cell population, progesterone content and steroidogenic properties were studied by dispersion of luteal cells using collagenase type I enzyme, RIA and 3β-HSD activity, respectively. Large luteal cells (LLC) appeared as polyhedral or spherical in shape with a centrally placed large round nucleus and an abundance of cytoplasmic lipid droplets. However, small luteal cells (SLC) appeared to be spindle shaped with an eccentrically placed irregular nucleus and there was paucity of cytoplasmic lipid droplets. The size of SLC (range 12-23μm) and LLC (range 25-55μm) increased (P<0.01) with the advancement of stage of estrous cycle and pregnancy. The mean progesterone concentration per gram and per CL increased (P<0.01) from Stage I to III of estrous cycle with maximum concentration at Stage III of estrous cycle and pregnancy. The progesterone concentration decreased at Stage IV (day 17-20) of estrous cycle coinciding with CL regression. Total luteal cell number (LLC and SLC) also increased (P<0.01) from Stage I to III of estrous cycle and decreased (P<0.05), thereafter, at Stage IV indicating degeneration of luteal cells and regression of the CL. Total luteal cell population during pregnancy also increased (P<0.01) from Stage I to II and thereafter decreased (P>0.05) indicating cessation of mitosis. Increased (P<0.05) large luteal cell numbers from Stage I to III of estrous cycle and pregnancy coincided with the increased progesterone secretion and 3β-HSD activity of CL. Thus, proportionate increases of large compared with small luteal cells were primarily responsible for increased progesterone secretion during the advanced stages of the estrous cycle and pregnancy. Total luteal cells and progesterone content per CL during the mid-luteal stage in buffalo as observed in the present study seem to be less than with cattle suggesting inherent luteal deficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators.

PubMed

Fukuda, Tomokazu; Iino, Yuuka; Eitsuka, Takahiro; Onuma, Manabu; Katayama, Masafumi; Murata, Koichi; Inoue-Murayama, Miho; Hara, Kumiko; Isogai, Emiko; Kiyono, Tohru

2016-10-01

Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.

Effect of environmental contaminants in the Mississippi River Basin on carboxylesterases from four aquatic species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaiswal, R.; Huang, T.; Obih, P.

1995-12-31

The objectives of this study are to investigate the sensitivity of different classes of esterases in various aquatic species to environmental contaminants and the possible use of these enzymes as biomarkers for monitoring the effects of pollutants. Acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and the non-specific carboxylesterases (CaE) were analyzed in three fish species, Ictiobus bubalus (small mouth buffalo), Ictiobus cyprinellus (big mouth buffalo) and Lepisosteus oculatus (spotted gar) and the green tree frog, Hyla cinerea. These samples were collected from the Devil`s Swamp Site (DSS), an industrial site known to be highly contaminated at the Mississippi River Basin, and Lake Tunica,more » a nonindustrial site. ACHE and BuChE activities in the subcellular fractions of liver and brain were significantly lower in fishes and frogs obtained from DSS when compared to the same species obtained from Tunica swamp site. The greatest decrease was observed with ACHE activity in the liver and brain of Ictiobus bubalus from DSS. CaE activity analyzed with p-nitrophenyl acetate was found to be significantly lower in the liver of all three fish species collected from DSS when compared to the same fish species obtained from the Tunica swamp site.« less

Loss of heat shock protein 70 from apical region of buffalo (Bubalus bubalis) sperm head after freezing and thawing.

PubMed

Varghese, Tincy; Divyashree, Bannur C; Roy, Sudhir C; Roy, Kajal S

2016-03-15

The post-thaw fertility of frozen-thawed mammalian spermatozoa is substantially low as compared with that of fresh sperm. Furthermore, the post-thaw fertility of the cryopreserved buffalo sperm has been reported to be poor as compared with that of cattle sperm. Recently, heat shock protein 70 (HSP70) has been found to play a critical role in mammalian fertilization and early embryonic development in boar and cattle. However, the presence of such fertility-related HSP70 in buffalo sperm and its status after cryopreservation has not been reported so far. Thus, a study was conducted to determine the effect of cryopreservation on the level and distribution pattern of HSP70 molecule in buffalo sperm after cryopreservation. Buffalo semen samples, after dilution in semen extender, were aliquoted in straws and divided into two groups. One group was not cryopreserved, and the other group was cryopreserved for 60 days. Sperm proteins were extracted from both non-cryopreserved (NC) and cryopreserved (C) sperm and subjected to Western blot analysis for detection of HSP70 using a monoclonal anti-HSP70 antibody. The distribution pattern of these proteins in buffalo sperm was also monitored before and after cryopreservation using indirect immunofluorescence technique. A prominent 70-kDa protein band of HSP70 protein was detected in protein extracts of both NC and C buffalo sperm. Densitometry analysis revealed that the intensity of 70-kDa HSP70 protein band of cryopreserved sperm decreased significantly (P < 0.05) compared with that of NC sperm. However, the level of HSP70 in cryopreserved extended seminal plasma (ESP) did not change as compared with that of NC samples indicating a possible degradation of HSP70 in the spermatozoa itself rather than leakage of the protein into the ESP. Furthermore, Western blot also confirmed that several HSP70 immunoreactive protein bands detected in the ESP were contributed by the egg yolk that was added to the extender. Immunocytochemistry revealed that HSP70 proteins were distributed over the apical region of sperm head and/or acrosome, post-acrosomal, and middle piece regions of NC buffalo spermatozoa. However, the fluorescence signal of apical region of sperm head was lost significantly (P < 0.05) after a cycle of freezing and thawing. Thus, the present study confirmed that there was loss of HSP70 from buffalo sperm head after freezing and thawing of buffalo spermatozoa, and this may be one of the causes of the reduced post-thaw fertility of sperm in this species. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficiency to reach age of puberty and behaviour of buffalo heifers (Bubalus bubalis) kept on pasture or in confinement.

PubMed

Sabia, E; Napolitano, F; De Rosa, G; Terzano, G M; Barile, V L; Braghieri, A; Pacelli, C

2014-11-01

In order to evaluate the influence of rearing system (free-ranging (FR) v. confinement (C)) on buffalo heifer efficiency to reach age of puberty and on behavioural and immune functions, two experiments were conducted from September 2010 to October 2011. In Experiment I, 32 subjects aged 8 to 9 months at the start of experiment were used. A total of 16 animals (group C) were group housed in an indoor slatted floor pen (4 m2/animal) with an outdoor paddock (4 m2/animal); 16 others grazed on a Mediterranean natural pasture of 40 ha (group FR). Behavioural data were collected and organic matter digestibility, blood metabolites and progesterone were determined. At the end of the experiment, a novel object test and a skin test were conducted, and the avoidance distance (AD) at the manger was measured. Free-ranging animals were able to express natural behaviours such as wallowing and grazing. C animals devoted more time to the novel object than FR animals, whereas AD at manger was lower in group FR than in group C (P<0.01). Cellular immune response was higher in FR heifers than in C animals (P<0.01). FR animals also showed a higher digestibility of organic matter (P<0.01). Heifers from group FR had higher plasma concentrations of non-esterified fatty acids (P<0.001) and lower concentrations of glucose than heifers from group C (P<0.001). C animals showed higher daily weight gains (P<0.01) and weight at the puberty (P<0.05), but there were no differences in terms of age of puberty between the two groups. The intakes of dry matter (DM), CP and energy to reach the age of puberty were similar in both groups. In order to verify whether the results obtained in Experiment I could be replicated in different rearing conditions (reduced pasture availability, different location and altitude), a second experiment was conducted on 26 animals, where only onset of age of puberty and metabolic profile were monitored. In Experiment II, 13 heifers grazed on a natural pasture of 5 ha, other 13 received the same space as indicated for Experiment I. Results from Experiment II generally confirmed those of Experiment I. Only the intakes of DM and energy to reach the age of puberty were higher in group C than in FR (P<0.001). A lower competition with human nutrition, reproductive performances similar to those shown by confined animals and the indications given by immune and behavioural variables, suggest that a free-range-based system may be conveniently used for buffalo heifer farming purposes.

Ultrasonographic and macroscopic anatomy of the enucleated eyes of the buffalo (Bos bubalis) and the one-humped camel (Camelus dromedarius) of different ages.

PubMed

Kassab, A

2012-02-01

The ultrasonographic appearance and measurements of the normal buffalo and camel eye globes were described in 60 buffaloes (Bos bubalis) aged 1 year (28 eyes) and 10 years (32 eyes), and in 51 humped camels (Camelus dromedarius) aged 1 year (26 eyes) and 10 years (24 eyes). Ocular measurements were recorded by A- and B-scan ultrasonographic examination of 40 buffalo eyes (18 young and 22 adult eyes) and 34 camel eyes (14 young and 20 adult eyes) using a KANGH ultrasound scanner equipped with 10 MHz probe. For gross measurements, 20 buffalo and 16 camel eye globes were frozen and dissected and the same measurements were made using fine callipers macroscopically. The aqueous and vitreous humour of the buffalo and camel eyes appeared anechoic. The cornea, anterior and posterior lens capsule and iris appeared hyperechoic. The ocular measurements for the axial length, vitreous chamber depth (VCD), corneal thickness, lens thickness and scleroretinal rim thickness increase with the advance of age in both buffaloes and camels. Except for the anterior chamber depth, VCD and lens thickness, which were larger in adult camels than in adult buffaloes, no other differences between ocular dimensions were observed in both species. The results of this study are valuable for comparative ocular anatomy and will be useful for ultrasonographic evaluation of ocular diseases in buffaloes and camels. © 2011 Blackwell Verlag GmbH.

The commercial fishery in Lake Oahe, North and South Dakota, 1964-70

USGS Publications Warehouse

Higham, Joseph H.

1974-01-01

Ten species were commercially harvested in Lake Oahe, among which bigmouth buffalo (Ictiobus cyprinellus) predominated (63.8% of the total weight), smallmouth buffalo (Ictiobus bubalus) and goldeye (Hiodon alosoides) ranked second, and third. Variations in the seasonal and annual production of buffalo were governed by market conditions and availability. Production of minor species depended on the amount of fishing effort directed toward the harvest of buffalo. Gill and hoop nets were the principal fishing gears. The fishing season usually extended from April through December; most fishing was in May through August.

Effect of pour-on alphacypermethrin on feed intake, body condition score, milk yield, pregnancy rates, and calving-to-conception interval in buffaloes.

PubMed

Bifulco, G; Veneziano, V; Cimmino, R; Esposito, L; Auletta, L; Varricchio, E; Balestrieri, A; Claps, S; Campanile, G; Neglia, G

2015-04-01

The aims of this study were to assess the efficacy of alphacypermethrin (ACYP) on pediculosis due to Haematopinus tuberculatus and to evaluate the influence of the treatment on productive and reproductive performance in buffaloes (Bubalus bubalis) reared in an intensive system. The trial was performed on 56 pluriparous buffaloes at 86.8 ± 8.1 d in milk. The animals underwent individual louse count and were divided into 2 homogenous groups according to louse count, age, number of lactations, days in milk, live BW, BCS, pregnancy status, and milk yield. Group A (n = 28) was treated by a pour-on formulation of ACYP, and Group S (n = 28) was treated by pour-on saline solution. Individual louse counts were performed weekly on 10 buffaloes in each group. Feed intake was recorded daily and the total mixed ration, individual ingredients, and orts were analyzed to calculate DM ingestion. Individual milk yield was recorded daily and milk samples were analyzed at the beginning of the trial, after 4 wk, and at the end of the trial to assess milk composition. Individual BCS was also evaluated simultaneously. Finally, the animals underwent synchronization of ovulation starting 4 wk after treatment and the pregnancy rate and the calving-conception interval were evaluated. Data were analyzed by the Mann-Whitney test and ANOVA for repeated measures. The infestation was constant in Group S, whereas no lice were present in Group A throughout the study. Daily DMI was similar in the 2 groups (16.7 ± 0.4 vs. 16.3 ± 0.3 kg/d in Group A vs. Group S, respectively), although buffaloes in Group A showed higher (P < 0.05) BCS score at the end of the trial (7.39 ± 0.1 vs. 7.14 ± 0.1 in Group A vs. Group S, respectively). The average milk yield/buffalo was higher (P < 0.05) in Group A compared to Group S (10.58 ± 0.1 vs. 10.39 ± 0.1 kg in Group A vs. Group S, respectively) and this was mainly due to the higher milk production recorded in buffaloes at less than 75 d in milk (11.81 ± 0.1 vs. 11.45 ± 0.1 kg in Group A vs. Group S, respectively). Despite of a similar fertility rate (90.5 vs. 80.9% in Group A vs. Group S, respectively), a lower (P < 0.05) calving-conception interval was recorded in Group A compared to Group S (118 ± 16 vs. 177 ± 16 d in Group A vs. Group S, respectively). In addition to the pour-on treatment against pediculosis, productive and reproductive performance were also improved. This represents a significant improvement in dairy buffalo herd management.

Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (Bubalus Bubalis)

PubMed Central

Kapila, Neha; Sharma, Ankita; Kishore, Amit; Sodhi, Monika; Tripathi, Pawan K.; Mohanty, Ashok K.

2016-01-01

The present study aims to identify the heat responsive genes and biological pathways in heat stressed buffalo mammary epithelial cells (MECs). The primary mammary epithelial cells of riverine buffalo were exposed to thermal stress at 42°C for one hour. The cells were subsequently allowed to recover at 37°C and harvested at different time intervals (30 min to 48 h) along with control samples (un-stressed). In order to assess the impact of heat stress in buffalo MECs, several in-vitro cellular parameters (lactate dehydrogenase activity, cell proliferation assay, cellular viability, cell death and apoptosis) and transcriptional studies were conducted. The heat stress resulted in overall decrease in cell viability and cell proliferation of MECs while induction of cellular apoptosis and necrosis. The transcriptomic profile of heat stressed MECs was generated using Agilent 44 K bovine oligonucleotide array and at cutoff criteria of ≥3-or ≤3 fold change, a total of 153 genes were observed to be upregulated while 8 genes were down regulated across all time points post heat stress. The genes that were specifically up-regulated or down-regulated were identified as heat responsive genes. The upregulated genes in heat stressed MECs belonged to heat shock family viz., HSPA6, HSPB8, DNAJB2, HSPA1A. Along with HSPs, genes like BOLA, MRPL55, PFKFB3, PSMC2, ENDODD1, ARID5A, and SENP3 were also upregulated. Microarray data revealed that the heat responsive genes belonged to different functional classes viz., chaperons; immune responsive; cell proliferation and metabolism related. Gene ontology analysis revealed enrichment of several biological processes like; cellular process, metabolic process, response to stimulus, biological regulation, immune system processes and signaling. The transcriptome analysis data was further validated by RT-qPCR studies. Several HSP (HSP40, HSP60, HSP70, HSP90, and HSPB1), apoptotic (Bax and Bcl2), immune (IL6, TNFα and NF-kβ) and oxidative stress (GPX1 and DUSP1) related genes showed differential expression profile at different time points post heat stress. The transcriptional data strongly indicated the induction of survival/apoptotic mechanism in heat stressed buffalo MECs. The overrepresented pathways across all time points were; electron transport chain, cytochrome P450, apoptosis, MAPK, FAS and stress induction of HSP regulation, delta Notch signaling, apoptosis modulation by HSP70, EGFR1 signaling, cytokines and inflammatory response, oxidative stress, TNF-alpha and NF- kB signaling pathway. The study thus identified several genes from different functional classes and biological pathways that could be termed as heat responsive in buffalo MEC. The responsiveness of buffalo MECs to heat stress in the present study clearly suggested its suitability as a model to understand the modulation of buffalo mammary gland expression signature in response to environmental heat load. PMID:27682256

Effect of long term feeding of ammoniated wheat straw treated with or without HCl on blood biochemical parameters in growing male buffalo (Bubalus bubalis) calves.

PubMed

Mehra, Usha Rani; Sahu, Dev Sharan; Naik, Prafulla Kumar; Dass, Ram Sharan; Verma, Ashok Kumar

2005-01-01

Twenty-four growing male buffalo calves (one year of age; 88.54 +/- 3.81 kg average body weight) were divided into three comparable groups (I, II and III) on the basis of their body weight (BW) in a completely randomised design to study the effect of long term feeding of ammoniated wheat straw (AWS) and hydrochloric acid treated ammoniated wheat straw (HCl-AWS) on blood biochemical changes. The animals were offered a concentrate mixture (CM) along with wheat straw (WS), ammoniated wheat straw (AWS) (4% urea at a 50% moisture level) and hydrochloric acid treated ammoniated wheat straw (HCI-AWS) (4% urea at a 50% moisture level and HCI added to trap 30% of NH3 evolved) in groups I, II and III, respectively for an average daily gain (ADG) of 500 g. All the diets were made iso-nitrogenous by preparing three types of concentrate mixtures of different CP levels. The blood was collected from the jugular vein randomly from three animals of each group initially after 8 months post feeding and subsequently after two months interval up to 14 months of experimental feeding. Due to urea ammoniation, the CP content of WS increased from 3.66 to 8.51 and was further increased to 11.35 due to the addition of HCl during urea-ammoniation of wheat straw. The cumulative period mean plasma glucose values (mg %), in group II (53.13) were significantly (P < 0.001) higher than those in groups I (48.44) and III (50.60). The cumulative period mean values of serum albumin and globulin (g %) were not significantly different and were comparable among the groups I (3.33 and 3.06), II (3.53 and 2.97) and III (3.49 and 2.94). The cumulative period mean values of serum albumin: globulin ratio and total protein values were not significantly different among the different groups. Serum urea and creatinine values were significantly (P < 0.001) higher in group III (58.66 and 2.24) as compared to groups I and II. The cumulative period mean values of serum alkaline phosphatase (ALP) (KA units) did not differ significantly, but serum glutamate pyruvate transaminase (SGPT) and glutamate oxaloacetate transaminase (SGOT) values (units x mL(-1) were significantly (P < 0.001) higher in groups II and III than in group I. The cumulative period mean values of T3 (ng x mL(-1)) did not differ significantly among the groups, but T4 values were significantly (P < 0.001) higher in group III (22.74) than in groups 1 (21.41) and II (20.89), respectively. Since the mean values of all the blood parameters were within the normal range, it may be concluded that feeding of ammoniated wheat straw treated with and without HCl to growing male buffalo calves for fourteen months has no adverse effect on the blood biochemical parameters.

Mastitis outcomes on pre-ovulatory follicle diameter, estradiol concentrations, subsequent luteal profiles and conception rate in Buffaloes.

PubMed

Mansour, Mohamed Mohsen; Zeitoun, Moustafa M; Hussein, Fekry M

2017-06-01

The objectives of this study was to investigate the outcome of mastitis, in its clinical or subclinical forms, on the mean diameter of pre-ovulatory follicle (POF), plasma estradiol concentration on the day of estrus, subsequent luteal profile and subsequent conception rate in buffaloes. Sixty dairy buffalo (Bubalus bubalus) conducted in this study were divided into three groups {healthy (H), n=20; subclinical mastitis (SCM), n=18; and clinical mastitis (CM), n=22}. Ultrasonography of ovaries revealed that mean diameter of POF was larger (P<0.05) in H buffalo (14.35mm) compared to SCM (12.40mm) and CM (10.25mm). Also, plasma estradiol concentration on the day of estrus was higher (P<0.05) in H buffalo compared to SCM and CM counterparts; 34.95 vs. 32.87 and 27.50pg/ml, respectively. Besides, positive correlation was observed between the POF diameter with plasma estradiol concentration in H, SCM and CM buffaloes (r=0.64, 0.74, 0.72 respectively, P<0.05). Moreover, positive correlations (P<0.01) were found on days 9, 12, 16, and 21 post-ovulation between POF diameter and luteal profile. Thus, the conception rate in H buffalo was higher (P<0.05) compared with SCM and CM counterparts; 55% vs. 38.89 and 18.18%, respectively. In conclusion, mastitis in its clinical or subclinical forms disrupts the functioning of the pre-ovulatory follicle on the day of estrus, associated with low follicular estradiol production, resulting in suppression to subsequent luteal profile leading to substantial decrease in pregnancy consequence of buffaloes. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of season, late embryonic mortality and progesterone production on pregnancy rates in pluriparous buffaloes (Bubalus bubalis) after artificial insemination with sexed semen.

PubMed

Campanile, Giuseppe; Vecchio, Domenico; Neglia, Gianluca; Bella, Antonino; Prandi, Alberto; Senatore, Elena M; Gasparrini, Bianca; Presicce, Giorgio A

2013-03-01

The use of sexed semen technology in buffaloes is nowadays becoming more and more accepted by farmers, to overcome the burden of unwanted male calves with related costs and to more efficiently improve production and genetic gain. The aim of this study was to verify the coupling of some variables on the efficiency of pregnancy outcome after deposition of sexed semen through AI. Pluriparous buffaloes from two different farms (N = 152) were screened, selected, and subjected to Ovsynch protocol for AI using nonsexed and sexed semen from four tested bulls. AI was performed in two distinct periods of the year: September to October and January to February. Neither farms nor bulls had a significant effect on pregnancy rates pooled from the two periods. The process for sexing sperm cells did not affect pregnancy rates at 28 days after AI, for nonsexed and sexed semen, respectively 44/73 (60.2%) and 50/79 (63.2%), P = 0.70, and at 45 days after AI, for nonsexed and sexed semen, respectively 33/73 (45.2%) and 33/79 (49.3%), P = 0.60. Pregnancy rate at 28 days after AI during the transitional period of January to February was higher when compared with September to October, respectively 47/67 (70.1%) versus 47/85 (55.2%), P = 0.06. When the same pregnant animals were checked at Day 45 after AI, the difference disappeared between the two periods, because of a higher embryonic mortality, respectively 32/67 (47.7%) versus 40/85 (47.0%), P = 0.93. Hematic progesterone concentration at Day 10 after AI did not distinguish animals pregnant at Day 28 that would or would not maintain pregnancy until Day 45 (P = 0.21). On the contrary, when blood samples were taken at Day 20 after AI, the difference in progesterone concentration between pregnant animals that would maintain their pregnancy until Day 45 was significant for both pooled (P = 0.00) and nonsexed (P = 0.00) and sexed semen (P = 0.09). A similar trend was reported when blood samples were taken at Day 25, being highly significant for pooled, nonsexed, and sexed semen (P = 0.00). Hematic progesterone concentration between the two periods of the year was highly significant for pregnant animals at 28 days from AI when blood samples were taken at Day 20 after AI for pooled, nonsexed, and sexed semen, respectively P = 0.00, 0.00, and 0.06, and for pregnant animals at Day 45 for pooled, nonsexed, and sexed semen, respectively P = 0.00, 0.00, and 0.01. From these results, it can be stated that hematic progesterone concentration measurement since Day 20 after AI can be predictive of possible pregnancy maintenance until Day 45. Furthermore, the transitional period of January to February, although characterized by a higher pregnancy outcome when compared with September to October, suffers from a higher late embryonic mortality as evidenced by a significant different hematic progesterone concentration between the two periods at Day 20 after AI. Copyright © 2013 Elsevier Inc. All rights reserved.

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos.

PubMed

Niemann, H; Brem, G; Sacher, B; Smidt, D; Kräusslich, H

1986-04-01

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.

Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

PubMed

Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

2016-09-01

The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes from domestic animals tested in our study, the feline ooplasm might be the most appropriate recipient to partially allow preimplantation embryo development of iSCNT equine embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Embryo density and medium volume effects on early murine embryo development.

PubMed

Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

1992-10-01

One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

How do laboratory embryo transfer techniques affect IVF outcomes? A review of current literature.

PubMed

Sigalos, George; Triantafyllidou, Olga; Vlahos, Nikos

2017-04-01

Over the last few years, many studies have focused on embryo selection methods, whereas little attention has been given to the standardization of the procedure of embryo transfer. In this review, several parameters of the embryo transfer procedure are examined, such as the: (i) culture medium volume and loading technique; (ii) syringe and catheters used for embryo transfer; (iii) viscosity and composition of the embryo transfer medium; (iv) environment of embryo culture; (v) timing of embryo transfer; (vi) and standardization of the embryo transfer techniques. The aim of this manuscript is to review these factors and compare the existing embryo transfer techniques and highlight the need for better embryo transfer standardization.

Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo

PubMed Central

Jusof, Wan-Hafizah Wan; Khan, Nor-Ashikin Mohamed Noor; Rajikin, Mohd Hamim; Satar, Nuraliza Abdul; Mustafa, Mohd-Fazirul; Jusoh, Norhazlin; Dasiman, Razif

2015-01-01

Background Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos. Materials and Methods In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test. Results A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001). Conclusion Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification. PMID:26246881

Arrested human embryos are more likely to have abnormal chromosomes than developing embryos from women of advanced maternal age.

PubMed

Qi, Shu-Tao; Liang, Li-Feng; Xian, Ye-Xing; Liu, Jian-Qiao; Wang, Weihua

2014-01-01

Aneuploidy is one of the major factors that result in low efficiency in human infertility treatment by in vitro fertilization (IVF). The development of DNA microarray technology allows for aneuploidy screening by analyzing all 23 pairs of chromosomes in human embryos. All chromosome screening for aneuploidy is more accurate than partial chromosome screening, as errors can occur in any chromosome. Currently, chromosome screening for aneuploidy is performed in developing embryos, mainly blastocysts. It has not been performed in arrested embryos and/or compared between developing embryos and arrested embryos from the same IVF cycle. The present study was designed to examine all chromosomes in blastocysts and arrested embryos from the same cycle in patients of advanced maternal ages. Embryos were produced by routine IVF procedures. A total of 90 embryos (45 blastocysts and 45 arrested embryos) from 17 patients were biopsied and analyzed by the Agilent DNA array platform. It was found that 50% of the embryos developed to blastocyst stage; however, only 15.6% of the embryos (both blastocyst and arrested) were euploid, and most (84.4%) of the embryos had chromosomal abnormalities. Further analysis indicated that 28.9% of blastocysts were euploid and 71.1% were aneuploid. By contrast, only one (2.2%) arrested embryo was euploid while others (97.8%) were aneuploid. The prevalence of multiple chromosomal abnormalities in the aneuploid embryos was also higher in the arrested embryos than in the blastocysts. These results indicate that high proportions of human embryos from patients of advanced maternal age are aneuploid, and the arrested embryos are more likely to have abnormal chromosomes than developing embryos.

Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish.

PubMed

Lehner, Adam; Kaszas, Zita; Murber, Akos; Rigo, Janos; Urbancsek, Janos; Fancsovits, Peter

2017-08-01

Culturing embryos in groups is a common practice in mammalian embryology. Since the introduction of different microwell dishes, it is possible to identify oocytes or embryos individually. As embryo density (embryo-to-volume ratio) may affect the development and viability of the embryos, the purpose of this study was to assess the effect of different embryo densities on embryo quality. Data of 1337 embryos from 228 in vitro fertilization treatment cycles were retrospectively analyzed. Embryos were cultured in a 25 μl microdrop in a microwell group culture dish containing 9 microwells. Three density groups were defined: Group 1 with 2-4 (6.3-12.5 μl/embryo), Group 2 with 5-6 (4.2-5.0 μl/embryo), and Group 3 with 7-9 (2.8-3.6 μl/embryo) embryos. Proportion of good quality embryos was higher in Group 2 on both days (D2: 18.9 vs. 31.5 vs. 24.7%; p < 0.001; D3: 19.7 vs. 27.1 vs. 21.2%; p = 0.029; Group 1. vs. Group 2. vs. Group 3). Cell number on Day 3 differed between Groups 1 and 2 (6.8 ± 2.2; 7.3 ± 2.1; p = 0.004) and Groups 2 and 3 (7.3 ± 2.1 vs. 7.0 ± 2.0; p = 0.014). Culturing 5-6 embryos together in a culture volume of 25 μl may benefit embryo quality. As low egg number, position, and distance of the embryos may influence embryo quality, results should be interpreted with caution.

Frozen-Thawed Embryo Transfer Cycles Have a Lower Incidence of Ectopic Pregnancy Compared With Fresh Embryo Transfer Cycles.

PubMed

Zhang, Xinyu; Ma, Caihong; Wu, Zhangxin; Tao, Liyuan; Li, Rong; Liu, Ping; Qiao, Jie

2017-01-01

To evaluate the risk of ectopic pregnancy of embryo transfer. A retrospective cohort study on the incidence of ectopic pregnancy in fresh and frozen-thawed embryo transfer cycles from January 1 st , 2010, to January 1 st , 2015. Infertile women undergoing frozen-thawed transfer cycles or fresh transfer cycles. In-vitro fertilization, fresh embryo transfer, frozen-thawed embryo transfer, ectopic pregnancy. Ectopic pregnancy rate and clinical pregnancy rate. A total of 69 756 in vitro fertilization-embryo transfer cycles from 2010 to 2015 were analyzed, including 45 960 (65.9%) fresh and 23 796 (34.1%) frozen-thawed embryo transfer cycles. The clinical pregnancy rate per embryo transfer was slightly lower in fresh embryo transfer cycles compared with frozen-thawed embryo transfer cycles (40.8% vs 43.1%, P < .001). Frozen-thawed embryo transfer is associated with a lower incidence of ectopic pregnancy per clinical pregnancy, compared with fresh embryo transfers (odds ratio = 0.31; 95% confidence interval = 0.24-0.39). Female age and body mass index have no influence on ectopic pregnancy. In the frozen-thawed embryo transfer cycles, blastocyst transfer shows a significantly lower incidence of ectopic pregnancy (0.8% vs 1.8%, P = .002) in comparison with day 3 cleavage embryo transfer. The risk of ectopic pregnancy is lower in frozen-thawed embryo transfer cycles than fresh embryo transfer cycles, and blastocyst transfer could further decrease the ectopic pregnancy rate in frozen-thawed embryo transfer cycles.

Transition of cell numbers in bovine preimplantation embryos: in vivo collected and in vitro produced embryos.

PubMed

Ushijima, Hitoshi; Akiyama, Kiyoshi; Tajima, Toshio

2008-08-01

The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0.16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos.

Patients' Attitudes towards the Surplus Frozen Embryos in China

PubMed Central

Jin, Xuan; Wang, GongXian; Liu, SiSun; Liu, Ming; Zhang, Jing; Shi, YuFa

2013-01-01

Background. Assisted reproductive techniques have been used in China for more than 20 years. This study investigates the attitudes of surplus embryo holders towards embryos storage and donation for medical research. Methods. A total of 363 couples who had completed in vitro fertilization (IVF) treatment and had already had biological children but who still had frozen embryos in storage were invited to participate. Interviews were conducted by clinics in a narrative style. Results. Family size was the major reason for participants' (dis)continuation of embryo storage; moreover, the moral status of embryos was an important factor for couples choosing embryo storage, while the storage fee was an important factor for couples choosing embryo disposal. Most couples discontinued the storage of their embryos once their children were older than 3 years. In our study, 58.8% of the couples preferred to dispose of surplus embryos rather than donate them to research, citing a lack of information and distrust in science as significant reasons for their decision. Conclusions. Interviews regarding frozen embryos, including patients' expectations for embryo storage and information to assist them with decisions regarding embryo disposal, are beneficial for policies addressing embryo disposition and embryo donation in China. PMID:23509811

9 CFR 98.9 - Embryos refused entry.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...

9 CFR 98.9 - Embryos refused entry.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...

9 CFR 98.9 - Embryos refused entry.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...

9 CFR 98.9 - Embryos refused entry.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...

9 CFR 98.9 - Embryos refused entry.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Embryos refused entry. 98.9 Section 98... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.9 Embryos refused entry. Any embryo refused entry into the...

A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate.

PubMed

de los Santos, Maria José; Arroyo, Gemma; Busquet, Ana; Calderón, Gloria; Cuadros, Jorge; Hurtado de Mendoza, Maria Victoria; Moragas, Marta; Herrer, Raquel; Ortiz, Agueda; Pons, Carme; Ten, Jorge; Vilches, Miguel Angel; Figueroa, Maria José

2014-04-01

To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. Prospective cross-sectional study. Multicenter study. Seven hundred embryo transfers and 1,028 early-stage human embryos. None. Implantation according to the presence of EC and embryo quality. The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Timing of first embryonic cleavage is a positive indicator of the in vitro developmental potential of porcine embryos derived from in vitro fertilization, somatic cell nuclear transfer and parthenogenesis.

PubMed

Isom, S Clay; Li, Rong Feng; Whitworth, Kristin M; Prather, Randall S

2012-03-01

Evidence in many species has suggested that those embryos that cleave earliest after fertilization are more developmentally competent than those that cleave relatively later after fertilization. Herein we document this phenomenon in porcine in vitro-fertilized (IVF), somatic cell nuclear transfer (SCNT), and parthenogenetic (PA) embryos. In vitro-matured pig oocytes were used to generate IVF, SCNT, and PA embryos. At 24 hr post-activation (or insemination; hpa/hpi), embryos were visually assessed, and cleaved embryos were moved into a new culture well. This process was repeated at 30 and 48 hpa/hpi. All embryos were allowed to develop 7 days in culture. For IVF embryos, 39.9%, 24.6%, and 10.5% of fast-, intermediate-, or slow-cleaving embryos, respectively, developed into blastocysts by day 7. For SCNT embryos, 31.8% of fast-, 5.7% of intermediate-, and 2.9% of late-cleaving embryos achieved the blastocyst stage of development. For PA embryos, the percentages of those cleaved embryos that developed to blastocyst were 59.3%, 36.7%, and 7.5% for early-, intermediate-, and late-cleaving embryos, respectively. Using RNA collected from early-, intermediate-, and late-cleaving embryos, real-time PCR was performed to assess the transcript levels of 14 different genes of widely varied function. The qPCR results suggest that maternal mRNA degradation may not proceed in an appropriate pattern in slow-cleaving embryos. These findings (1) confirm that, as observed in other species, earlier-cleaving porcine embryos are more successful at developing in culture than are slower-cleaving embryos, and (2) implicate mechanisms of maternal transcript destruction as potential determinants of oocyte/embryo quality. Copyright © 2011 Wiley Periodicals, Inc.

Transfer of bovine demi-embryos with and without the zona pellucida.

PubMed

Warfield, S J; Seidel, G E; Elsden, R P

1987-09-01

Bisected bovine embryos with or without the zona pellucida were transferred to recipients nonsurgically in five field trials. Embryos were collected from superovulated donors 6.5 to 7.5 d after estrus; only embryos of good and excellent quality were bisected. Demi-embryos were transferred either within a zona pellucida, without a zona pellucida, without a zona pellucida, or in the third and fourth trials, without a zona but embedded in 7% gelatin. Pregnancies were diagnosed at 44 to 68 d of gestation. In a preliminary trial, 9/29 zona pellucida-intact demi-embryos developed into fetuses compared with 1/10 zona pellucida-free demi-embryos (P greater than .1). The proportion of zona-free demi-embryos developing to fetuses was not significantly different from the zona-intact group in the second trial either, 24/49 and 5/19, respectively. In trial 3, the proportion of zona pellucida-free demi-embryos developing was 8/25; of zona-enclosed embryos, 29/88; and of zona-free demi-embryos embedded in gelatin, 8/22 (P greater than .1). Similarly, in the fourth trial the rate of development of zona-free demi-embryos to fetuses was 5/12, that of zona-enclosed embryos was 32/81, and that of zona-free demi-embryos embedded in gelatin was 3/12 (P greater than .1). In trial 5, survival of zona-enclosed demi-embryos to fetuses was 40/105, and of zona-free demi-embryos, 46/109 (P greater than .1). Except for trial 2, half of the demi-embryos were twinned, one to each uterine horn; twinning did not significantly affect the proportion developing to fetuses for any of the demi-embryo groups. It is concluded that placing post-compaction demi-embryos into the zona pellucida for transfer does not improve pregnancy rates significantly.

The effect of flurbiprofen on the development of anencephaly in early stage chicken embryos.

PubMed

Özeren, Ersin; Er, Uygur; Güvenç, Yahya; Demirci, Adnan; Arıkök, Ata Türker; Şenveli, Engin; Ergün, Rüçhan Behzat

2015-04-01

The study investigated the effect of flurbiprofen on the development of anencephaly in early stage chicken embryos. We looked at four groups with a total of 36 embryos. There was a control group, a normal saline group, a normal-dose group and a high-dose group with ten, ten, eight and eight eggs with embryo respectively. Two embryos in the control group, studied with light microscopy at 48 h, were consistent with 28-29 hours' incubation in the Hamburger-Hamilton System. They had open neural tubes. The other embryos in this group were considered normal. One embryo in the normal saline group was on the occlusion stage at 48 h. One embryo showed an open neural tube. They were compatible with 28-29 hours' incubation in the Hamburger-Hamilton system. The remaining eight embryos showed normal development. In the normal dose group, one embryo showed underdevelopment of the embryonic disc and the embryo was dead. In four embryos, the neural tubes were open. One cranial malformation was found that was complicated with anencephaly in one embryo. In two embryos the neural tubes were closed, as they showed normal development, and they reached their expected stages according to the Hamburger-Hamilton classification. There was no malformation or growth retardation. Four experimental embryos were anencephalic in the high dose group, and three embryos had open neural tubes. One embryo exhibited both anencephaly and a neural tube closure defect. None of the embryos in this group showed normal development. Even the usual therapeutic doses of flurbiprofen increased the risk of neural tube defect. Flurbiprofen was found to significantly increase the risk of anencephaly. The provision of improved technical materials and studies with larger sample sizes will reveal the stage of morphological disruption during the development of embryos.

[Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

PubMed

Zhao, H; Teng, X M; Li, Y F

2017-11-25

Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all P< 0.05]. The copy number of mtDNA and the mitochondrial membrane potential in classⅠ frozen embryo group were significantly higher than those in classⅡ frozen embryo group (both P< 0.05). Conclusion: The mtDNA copy number and the mitochondrial membrane potential of embryos of the better quality embryo are higher.

Surgical manipulation of mammalian embryos in vitro.

PubMed

Naruse, I; Keino, H; Taniguchi, M

1997-04-01

Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.

Equine cloning: in vitro and in vivo development of aggregated embryos.

PubMed

Gambini, Andrés; Jarazo, Javier; Olivera, Ramiro; Salamone, Daniel F

2012-07-01

The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.

Characteristics of yak platelet derived growth factors-alpha gene and expression in brain tissues.

PubMed

Huang, Zhenhua; Pan, Yangyang; Liu, Penggang; Yu, Sijiu; Cui, Yan

2017-05-29

Platelet derived growth factors (PDGFs) are key components of autocrine and paracrine signaling, both of which play important roles in mammalian developmental processes. PDGF expression levels also relate to oxygen levels. The characteristics of yak PDGFs, which are indigenous to hypoxic environments, have not been clearly described until the current study. We amplified the open reading frame encoding yak (Bos grunniens) platelet derived growth factor-a (PDGFA) from a yak skin tissue cDNA library by reverse transcriptase polymerase chain reaction (PCR) using specific primers and Sanger dideoxy sequencing. Expression of PDGFA mRNA in different portions of yak brain tissue (cerebrum, cerebellum, hippocampus, and spinal cord) was detected by quantitative real-time PCR (qRT-PCR). PDGFA protein expression levels and its location in different portions of the yak brain were evaluated by western blot and immunohistochemistry. We obtained a yak PDGFA 755 bp cDNA gene fragment containing a 636 bp open reading frame, encoding 211 amino acids (GenBank: KU851801). Phylogenetic analysis shows yak PDGFA to be well conserved, having 98.1% DNA sequence identity to homologous Bubalus bubalus and Bos taurus PDGFA genes. However, eight nucleotides in the yak DNA sequence and four amino acids in the yak protein sequence differ from the other two species. PDGFA is widely expressed in yak brain tissue, and furthermore, PDGFA expression in the cerebrum and cerebellum are higher than in the hippocampus and spinal cord (p > 0.05). PDGFA was observed by immunohistochemistry in glial cells of the cerebrum, cerebellum, and hippocampus, as well as in pyramidal cells of the cerebrum, and Purkinje cell bodies of the hippocampus, but not in glial cells of the spinal cord. The PDGFA gene is well conserved in the animal kingdom; however, the yak PDGFA gene has unique characteristics and brain expression patterns specific to this high elevation species.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

PubMed

Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

2016-01-01

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

Comparison of effects of albendazole sulfoxide on in vitro produced bovine embryos and rat embryos.

PubMed

Piscopo, S E; Smoak, I W

1997-09-01

To evaluate and compare effects of albendazole sulfoxide (ABZSO) on rat embryos and bovine embryos produced in vitro. In vitro produced bovine embryos. Rat embryos recovered from naturally bred Sprague-Dawley rats. 4- and 8-cell bovine embryos were randomly allocated to ABZSO or vehicle control groups. After 48 hours, embryos were evaluated for cell number and blastomere morphology. Rat embryos of similar stages, flushed from the uterine tube on gestational day 2-5, were randomly allocated to treatment or control groups. After 24 hours, embryos were evaluated as described previously. 44% of control bovine embryos divided in culture (> or = 16-cell stage). Fifteen percent of the controls had morphologic abnormalities, including disparity in blastomere size and cytoplasmic vacuoles and stippling. Treated (> or = 1 microgram of ABZSO/ml) bovine embryos differed (P < 0.0001) from controls, with 4% development and 93% abnormal morphology. Forty-five percent of control rat embryos divided in culture. Treated (> or = 500 ng of ABZSO/ml) rat embryos differed (P < 0.0003) from controls with regard to ability to divide. There were no consistent morphologic abnormalities in rat embryos. In vitro produced bovine embryos were susceptible to ABZSO at a concentration > or = 1 microgram/ ml, resulting in decreased ability to divide and presence of gross morphologic abnormalities. Rat embryos produced in vivo and exposed in vitro to ABZSO at a concentration > or = 500 ng/ml had decreased ability to divide in culture. Despite severe effects of ABZSO (> or = 1 microgram/ml) on bovine embryo development in vitro, it is beyond the scope of this study to speculate whether a therapeutic dosage of albendazole (10 mg/kg of body weight) would result in necessary concentrations of ABZSO in vivo to disrupt embryogenesis.

Effect of Embryo Density on In Vitro Development and Gene Expression in Bovine In Vitro-fertilized Embryos Cultured in a Microwell System

PubMed Central

SUGIMURA, Satoshi; AKAI, Tomonori; HASHIYADA, Yutaka; AIKAWA, Yoshio; OHTAKE, Masaki; MATSUDA, Hideo; KOBAYASHI, Shuji; KOBAYASHI, Eiji; KONISHI, Kazuyuki; IMAI, Kei

2012-01-01

Abstract To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 µl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density. PMID:23154384

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

PubMed

van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

2017-11-25

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

Fresh embryo donation for human embryonic stem cell (hESC) research: the experiences and values of IVF couples asked to be embryo donors

PubMed Central

Haimes, E.; Taylor, K.

2009-01-01

BACKGROUND This article reports on an investigation of the views of IVF couples asked to donate fresh embryos for research and contributes to the debates on: the acceptability of human embryonic stem cell (hESC) research, the moral status of the human embryo and embryo donation for research. METHODS A hypothesis-generating design was followed. All IVF couples in one UK clinic who were asked to donate embryos in 1 year were contacted 6 weeks after their pregnancy result. Forty four in-depth interviews were conducted. RESULTS Interviewees were preoccupied with IVF treatment and the request to donate was a secondary consideration. They used a complex and dynamic system of embryo classification. Initially, all embryos were important but then their focus shifted to those that had most potential to produce a baby. At that point, ‘other’ embryos were less important though they later realise that they did not know what happened to them. Guessing that these embryos went to research, interviewees preferred not to contemplate what that might entail. The embryos that caused interviewees most concern were good quality embryos that might have produced a baby but went to research instead. ‘The’ embryo, the morally laden, but abstract, entity, did not play a central role in their decision-making. CONCLUSIONS This study, despite missing those who refuse to donate embryos, suggests that debates on embryo donation for hESC research should include the views of embryo donors and should consider the social, as well as the moral, status of the human embryo. PMID:19502616

[Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

PubMed

Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

2007-09-01

To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae).

PubMed

Correia, Sandra M; Canhoto, Jorge M

2010-06-01

The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.

Different effectiveness of closed embryo culture system with time-lapse imaging (EmbryoScope(TM)) in comparison to standard manual embryology in good and poor prognosis patients: a prospectively randomized pilot study.

PubMed

Wu, Yan-Guang; Lazzaroni-Tealdi, Emanuela; Wang, Qi; Zhang, Lin; Barad, David H; Kushnir, Vitaly A; Darmon, Sarah K; Albertini, David F; Gleicher, Norbert

2016-08-24

Previously manual human embryology in many in vitro fertilization (IVF) centers is rapidly being replaced by closed embryo incubation systems with time-lapse imaging. Whether such systems perform comparably to manual embryology in different IVF patient populations has, however, never before been investigated. We, therefore, prospectively compared embryo quality following closed system culture with time-lapse photography (EmbryoScope™) and standard embryology. We performed a two-part prospectively randomized study in IVF (clinical trial # NCT92256309). Part A involved 31 infertile poor prognosis patients prospectively randomized to EmbryoScope™ and standard embryology. Part B involved embryos from 17 egg donor-recipient cycles resulting in large egg/embryo numbers, thus permitting prospectively alternative embryo assignments to EmbryoScope™ and standard embryology. We then compared pregnancy rates and embryo quality on day-3 after fertilization and embryologist time utilized per processed embryo. Part A revealed in poor prognosis patients no differences in day-3 embryo scores, implantation and clinical pregnancy rates between EmbryoScope™ and standard embryology. The EmbryoScope™, however, more than doubled embryology staff time (P < 0.0001). In Part B, embryos grown in the EmbyoScope™ demonstrated significantly poorer day-3 quality (depending on embryo parameter between P = 0.005 and P = 0.01). Suspicion that conical culture dishes of the EmbryoScope™ (EmbryoSlide™) may be the cause was disproven when standard culture dishes demonstrated no outcome difference in standard incubation. Though due to small patient numbers preliminary, this study raises concerns about the mostly uncontrolled introduction of closed incubation systems with time lapse imaging into routine clinical embryology. Appropriately designed and powered prospectively randomized studies appear urgently needed in well-defined patient populations before the uncontrolled utilization of these instruments further expands. NCT02246309 Registered September 18, 2014.

Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro.

PubMed

Rho, G J; Johnson, W H; Betteridge, K J

1998-10-15

The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively.

Factors affecting embryo viability and uterine receptivity: insights from an analysis of the UK registry data.

PubMed

Roberts, Stephen A; Hann, Mark; Brison, Daniel R

2016-02-01

Many studies have identified prognostic factors for IVF treatment outcome; however, little information is available on the mechanism of their action. Embryo-uterus models have the potential to distinguish between factors acting on the embryo directly and those acting through the uterine environment. Here we apply embryo-uterus models to comprehensive UK registry data from two periods, 2000-2005 and 2007-2011, containing 139,444 and 226,542 embryo transfer cycles, respectively. Given this large dataset, the embryo-uterus model is capable of distinguishing between uterine and embryo effects. Maternal age is the predominant predictor of live birth and acts on both the embryo and uterine components, but with larger effects on the embryo. Prolonged embryo culture is associated with greater embryo viability, reflecting the greater degree of selection, but is also associated with greater uterine receptivity. Cryopreserved embryos are less viable and were associated with poorer uterine receptivity. This work suggests that, in addition to the direct effects of in-vitro culture on the embryonic environment during the first few days of the embryo's life, the delay in transfer after extended culture or cryopreservation can lead to an altered uterine environment for the embryo after transfer. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Clinical evaluation of frozen/thawed embryo transfer following transport of oocytes and embryos

PubMed Central

2004-01-01

Background and Aims:  We evaluated the efficacy of the transport oocyte/embryo frozen/thawed embryo transfer method, in which oocytes or embryos were transported from satellite clinics to the main assisted reproductive technology (ART) center, and surplus embryos were placed in cryopreservation. Methods:  We evaluated 41 cycles in 34 patients in the transport oocyte group (TO group). In the TO group the oocytes were collected at the satellite clinics, transported to the main ART center and underwent in vitro fertilization or intracytoplasmic sperm injection. Surplus embryos were used for frozen/thawed embryo transfer. We also evaluated 17 cycles in 10 patients in the transport embryo group (TE group), where surplus embryos were transported to the main ART center and used for frozen/thawed embryo transfer; and 189 cycles in 134 patients in the center group (C group), where surplus embryos collected at the same time at the main ART center were used for frozen/thawed embryo transfer. Oocytes were transported from satellite clinics in HEPES buffered human tubal fluid (HTF) culture medium, and embryos in 30% synthetic serum substitute + HEPES buffered HTF, using a portable incubator we devised. Results:  The proportions of undamaged embryos after freeze/thawing were 47% for the C group, 46% for the TO group, and 46% for the TE group. The numbers of embryos transferred were 2.0 ± 0.7 for the C group, 2.0 ± 0.6 for the TO group, and 2.2 ± 0.4 for the TE group. The rate of embryo transfer was 63% for the C group, 68% for the TO group, and 76% for the TE group. Pregnancy rates per patient were 16% for the C group, 24% for the TO group, and 40% for the TE group. The embryo survival rates (number of embryos with ≥50% viable blastomeres/total number of embryos) were 55% for the C group, 60% for the TO group, and 54% for the TE group. No significant differences were seen between the C group and either the TO or TE groups in any of these parameters. Conclusions:  Favorable results were achieved with the transport oocyte/embryo frozen/thawed embryo transfer method, and it is suitable for widespread clinical application. (Reprod Med Biol 2004; 3: 1–8) PMID:29662379

Direct embryo tagging and identification system by attachment of biofunctionalized polysilicon barcodes to the zona pellucida of mouse embryos.

PubMed

Novo, Sergi; Penon, Oriol; Barrios, Leonardo; Nogués, Carme; Santaló, Josep; Durán, Sara; Gómez-Matínez, Rodrigo; Samitier, Josep; Plaza, José Antonio; Pérez-García, Luisa; Ibáñez, Elena

2013-06-01

Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of cultured embryos? The results achieved provide a proof of concept for a direct embryo tagging system using biofunctionalized polysilicon barcodes, which could help to minimize the risk of mismatching errors (mix-ups) in human assisted reproduction technologies. Even though the occurrence of mix-ups is rare, several cases have been reported in fertility clinics around the world. Measures to prevent the risk of mix-ups in human assisted reproduction technologies are therefore required. Mouse embryos were tagged with 10 barcodes and the effectiveness of the tagging system was tested during fresh in vitro culture (n=140) and after embryo cryopreservation (n = 84). Finally, the full-term development of tagged embryos was evaluated (n =105). Mouse pronuclear embryos were individually rolled over wheat germ agglutinin-biofunctionalized polysilicon barcodes to distribute them uniformly around the ZONA PELLUCIDA surface. Embryo viability and retention of barcodes were determined during 96 h of culture. The identification of tagged embryos was performed every 24 h in an inverted microscope and without embryo manipulation to simulate an automatic reading procedure. Full-term development of the tagged embryos was assessed after their transfer to pseudo-pregnant females. To test the validity of the embryo tagging system after a cryopreservation process, tagged embryos were frozen at the 2-cell stage using a slow freezing protocol, and followed in culture for 72 h after thawing. Neither the in vitro or in vivo development of tagged embryos was adversely affected. The tagging system also proved effective during an embryo cryopreservation process. Global identification rates higher than 96 and 92% in fresh and frozen-thawed tagged embryos, respectively, were obtained when simulating an automatic barcode reading system, although these rates could be increased to 100% by simply rotating the embryos during the reading process. The direct embryo tagging developed here has exclusively been tested in mouse embryos. Its effectiveness in other species, such as the human, is currently being tested. The direct embryo tagging system developed here, once tested in human embryos, could provide fertility clinics with a novel tool to reduce the risk of mix-ups in human assisted reproduction technologies.

Effect of embryo density on in vitro developmental characteristics of bovine preimplantative embryos with respect to micro and macroenvironments.

PubMed

Hoelker, M; Rings, F; Lund, Q; Phatsara, C; Schellander, K; Tesfaye, D

2010-10-01

To overcome developmental problems as a consequence of single embryo culture, the Well of the Well (WOW) culture system has been developed. In this study, we aimed to examine the effect of embryo densities with respect to both microenvironment and macroenvironment on developmental rates and embryo quality to get a deeper insight into developmentally important mechanisms. WOW diameter and depth significantly affected developmental rates (p < 0.05). WOWs with diameter of 500 μm reached significantly higher blastocyst rates (32.5 vs 21.1% vs 20.3%) compared to embryos cultured in WOWs of 300 μm diameter or plain cultured controls. Embryos cultured in WOWs with 700 μm depth reached significant higher developmental rates compared with embryos cultured in WOWs of 300 μm depth and control embryos (30.6 vs 22.6% vs 20.3%). Correlation of the embryo per WOW volume with developmental rates was higher (r(2) = 0.92, p = 0.0004) than correlation of WOW diameter or WOW depth with developmental rates. However, the embryo per WOW volume did not affect differential cell counts. An embryo per culture dish volume of 1 : 30 μl was identified to be optimal when the embryo per WOW volume was 1 : 0.27 μl increasing developmental rates up to the level of mass embryo production. Giving the opportunity to track each embryo over the complete culture period while keeping high developmental rates with normal mitotic dynamics, the results of this work will provide benefit for the single culture of embryos in human assisted reproduction, mammalian embryos with high economic interest as well as for scientific purpose. © 2009 Blackwell Verlag GmbH.

Climatic factors affecting quantity and quality grade of in vivo derived embryos of cattle.

PubMed

Chinchilla-Vargas, Josué; Jahnke, Marianna M; Dohlman, Tyler M; Rothschild, Max F; Gunn, Patrick J

2018-05-01

The present study investigated the effects of climatic variables on the quality grade and quantity of in vivo derived cattle embryos in the Midwestern United States. Climatic information included greatest and least daily temperature, average daily wind speed and average temperature-humidity index for each of the 765 records. The response variables included the number of ovarian structures, viable embryos, quality grade 1 embryos, quality grade 2 embryos, quality grade 3 embryos, freezable embryos (sum of quality grade 1 and quality grade 2 embryos), transferable embryos (sum of quality grade 1-3 embryos), degenerate embryos and unfertilized ova. Measures for variables among the breeds of donors and sires grouped by geographical origin were compared. A negative effect of greater temperatures during the early embryonic development stage tended (P < 0.10) to be associated with a decrease in the quality of embryos recovered. Interestingly, the greater the Temperature-Humidity Index (THI) during the early ovarian antral follicular development stage 40-45 days prior to ovulation was associated with a tendency for greater numbers of total number of freezable and transferable embryos recovered per uterine flushing (P < 0.10). Increased wind speed at the early antral follicular phase 40-45 days prior to ovulation was associated with an increase in the percentage of quality grade 1 embryos recovered (P < 0.05). Wind speed during the estrous synchronization period was also associated with a lesser number of embryos recovered (P < 0.05). This retrospective study confirms that climatic variables have significant effects on the in vivo production of cattle embryos and that wind speed should be considered in future analyses of factors affecting embryo quality. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparison of different cryopreservation methods for horse and donkey embryos.

PubMed

Pérez-Marín, C C; Vizuete, G; Vazquez-Martinez, R; Galisteo, J J

2018-05-01

Few studies have been published about cryopreservation and embryo assessment in horses and donkeys. To evaluate the viability of embryos collected from mares and jennies that were cryopreserved by slow freezing or by vitrification. Randomised controlled experiment. Horse (n=19) and donkey (n=16) embryos (≤300 μm) were recovered on days 6.5-7.5 post-ovulation and assigned to control or cryopreservation protocols of slow freezing or vitrification. For slow freezing, 1.5 mol/L ethylene glycol (EG) was used. For vitrification, horse embryos were exposed to 1.4 mol/L glycerol, 1.4 mol/L glycerol + 3.6 mol/L EG and 3.4 mol/L glycerol + 4.6 mol/L EG, using Fibreplug or a 0.25 mL straw; donkey embryos were vitrified using Fibreplug with similar EG-glycerol solutions to above or 7.0 mol/L EG. Dead cells, apoptotic and fragmented nuclei, and cytoskeleton quality were assessed on thawed/warmed embryos. A significant decrease in embryo quality was observed after cryopreservation (P<0.05). Although the percentage of dead cells was lower (P<0.05) in control than in cryopreserved embryos, no differences were observed between freezing protocols used for horse or donkey embryos. While no differences were detected in the number of apoptotic cells in warmed horse embryos, in donkey embryos a higher incidence of apoptosis was measured after vitrification with EG-glycerol in Fibreplug (P<0.05). Vitrified horse embryos had a significantly (P<0.05) higher percentage of nonviable cells than donkey embryo. Actin cytoskeleton quality did not differ between treatments. Difficulties in obtaining a large number of embryos meant that the number of embryos per group was low. Vitrified horse and donkey embryos did not show higher susceptibility to cell damage than those preserved by slow freezing, whether using straws or Fibreplug. However, Fibreplug with EG 7 mol/L resulted in fewer nonviable and apoptotic cells in donkey embryos. Donkey embryos showed lower susceptibility to vitrification than horse embryos. THE SUMMARY IS AVAILABLE IN SPANISH - SEE SUPPORTING INFORMATION. © 2017 EVJ Ltd.

Secretion of interferon-tau by bovine embryos in long-term culture: comparison of in vivo derived, in vitro produced, nuclear transfer and demi-embryos.

PubMed

Stojkovic, M; Büttner, M; Zakhartchenko, V; Riedl, J; Reichenbach, H D; Wenigerkind, H; Brem, G; Wolf, E

1999-04-30

Interferon-tau (IFNtau) is the pregnancy recognition signal of bovine embryos, inhibiting luteolysis. We studied trophoblastic growth and IFNtau secretion of embryos with different developmental potential, i.e., in vivo derived and in vitro produced embryos, cloned embryos and demi-embryos, to evaluate if the ability of secreting IFNtau might be responsible for differences in pregnancy rates after transfer of these categories of embryos to recipients. Day 8 embryos of excellent quality were individually placed in microdrops of buffalo rat liver cell-conditioned medium and maintained for up to 23 days. Embryos were observed on Days 11, 15, 19 and 23, the mean diameter (2r) of attached and spherical embryos was measured, and their trophoblastic area was calculated as r2pi or 4r2pi, respectively. Simultaneously, medium was changed and the IFNtau levels of conditioned media were determined using a bioassay of antiviral activity. Trophoblastic area was smaller (P < 0.05) in demi-embryos than in all other groups, which exhibited similar trophoblastic growth until Day 19. However, on Day 23 trophoblastic area of in vivo derived embryos was more than twice (P < 0.05) as large as those of in vitro produced and nuclear transfer (NT) embryos. IFNtau levels increased only slowly with time in culture of demi-embryos. By contrast, the level of IFNtau doubled from Day 11 to Day 15 in conditioned media from all other groups of embryos. The linear increase in IFNtau production of vivo and in vitro derived embryos continued until the end of the culture period, whereas conditioned media from NT embryos contained significantly (P < 0.05) less IFNtau activity on Days 19 and 23 than those of the former two groups. Our results demonstrate different capabilities of secreting IFNtau for in vivo derived and in vitro produced embryos vs. NT and demi-embryos, which may--at least part--be responsible for the differences in pregnancy rates after transfer to recipients.

9 CFR 98.20 - Embryos refused entry.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...

9 CFR 98.16 - The embryo collection unit.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...

9 CFR 98.16 - The embryo collection unit.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...

9 CFR 98.20 - Embryos refused entry.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...

9 CFR 98.20 - Embryos refused entry.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...

9 CFR 98.16 - The embryo collection unit.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...

9 CFR 98.20 - Embryos refused entry.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...

9 CFR 98.16 - The embryo collection unit.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...

9 CFR 98.20 - Embryos refused entry.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Embryos refused entry. 98.20 Section... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.20 Embryos refused entry. If any embryos are determined to be ineligible...

9 CFR 98.16 - The embryo collection unit.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false The embryo collection unit. 98.16... CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.16 The embryo collection unit. Ruminant and swine embryos may be...

In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.

PubMed

Isobe, Tomohiro; Ikebata, Yoshihisa; Do, Lanh Thi Kim; Tanihara, Fuminori; Taniguchi, Masayasu; Otoi, Takeshige

2015-07-01

The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients. © 2014 Japanese Society of Animal Science.

Early morphological nuclear events and developmental capacity of embryos reconstructed with fetal fibroblasts at the M or G1 phase after intracytoplasmic nuclear injection in cattle.

PubMed

Ideta, Atsushi; Urakawa, Manami; Aoyagi, Yoshito; Saeki, Kazuhiro

2005-04-01

We examined morphological nuclear events during the first cell cycle of bovine embryos reconstructed with somatic cells at the M and G1 phases (M-embryos and G1-embryos, respectively) by intracytoplasmic nuclear injection, and the subsequent development of these embryos in vitro and in vivo. Bovine fetal fibroblasts (BFFs) at the M or G1 phase were directly injected into enucleated oocytes, and activated immediately. Only half (48%) of the M-embryos extruded polar body-like cells (PBCs) at 6 h post injection (hpi). At 15 to 19 hpi, 54% of the M-embryos formed a single pronucleus-like nucleus. Nuclear envelope-breakdown, premature chromosome condensation and single nuclear clusters were observed in most of the G1-embryos (88%) within 30 min following the nuclear injection. At 15 to 19 hpi, single pronucleus-like nuclei were formed in most G1-embryos (83%). The potential of G1-embryos to develop to blastocysts was significantly higher than that of M-embryos (31% vs 16%). Three of five recipients following transfer of blastocysts derived from the G1-embryos became pregnant on Day 30, and one recipient delivered a calf. Our results indicate that almost a half of the M-embryos failed to extrude PBCs and that the G1-embryos developed to blastocysts at a higher rate than the M-embryos.

Embryo deaths in reproduction and embryo research: a reply to Murphy's double effect argument.

PubMed

Devolder, Katrien

2013-08-01

The majority of embryos created in natural reproduction die spontaneously within a few weeks of conception. Some have argued that, therefore, if one believes the embryo is a person (in the normative sense) one should find 'natural' reproduction morally problematic. An extension of this argument holds that, if one accepts embryo deaths in natural reproduction, consistency requires that one also accepts embryo deaths that occur in (i) assisted reproduction via in vitro fertilisation (IVF) and (ii) embryo research. In a recent paper in this journal, Timothy Murphy criticises both the initial argument and its extension. Murphy argues that double-effect reasoning can justify embryo deaths both in natural reproduction and IVF, but not in embryo research. Thus, according to Murphy, one can, without being inconsistent, (1) believe the embryo is a person and accept natural reproduction and IVF, and (2) accept natural reproduction and IVF, while rejecting embryo research on the ground that it involves embryo deaths. I show that Murphy's argument is problematic because double effect cannot justify embryo deaths in standard IVF practices. The problem is that the proportionality criterion of double effect is not met by such practices. Thus, Murphy's argument fails to support (1) and (2). An implication of his argument failing to support (2) is that it does not defeat the position I have defended in the past-that if one accepts standard IVF practices one should also accept embryo research, including research with embryos created solely for that purpose.

An economic assessment of embryo diagnostics (Dx) - the costs of introducing non-invasive embryo diagnostics into IVF standard treatment practices.

PubMed

Fugel, Hans-Joerg; Connolly, Mark; Nuijten, Mark

2014-10-09

New techniques in assessing oocytes and embryo quality are currently explored to improve pregnancy and delivery rates per embryo transfer. While a better understanding of embryo quality could help optimize the existing "in vitro fertilization" (IVF) therapy schemes, it is essential to address the economic viability of such technologies in the healthcare setting. An Embryo-Dx economic model was constructed to assess the cost-effectiveness of 3 different IVF strategies from a payer's perspective; it compares Embryo-Dx with single embryo transfer (SET) to elective single embryo transfer (eSET) and to double embryo transfer (DET) treatment practices. The introduction of a new non-invasive embryo technology (Embryo-Dx) associated with a cost up to €460 is cost-effective compared to eSET and DET based on the cost per live birth. The model assumed that Embryo-Dx will improve ongoing pregnancy rate/realize an absolute improvement in live births of 9% in this case. This study shows that improved embryo diagnosis combined with SET may have the potential to reduce the cost per live birth per couple treated in IVF treatment practices. The results of this study are likely more sensitive to changes in the ongoing pregnancy rate and consequently the live birth rate than the diagnosis costs. The introduction of a validated Embryo-Dx technology will further support a move towards increased eSET procedures in IVF clinical practice and vice versa.

Applying embryo cryopreservation technologies to the production of domestic and black-footed cats.

PubMed

Pope, C E; Gómez, M C; Galiguis, J; Dresser, B l

2012-12-01

Our objectives were (i) compare in vitro development of early cleavage stage domestic cat embryos after cryopreservation by minimal volume vitrification vs a standard slow, controlled-rate method, (ii) determine viability of vitrified domestic cat embryos by oviductal transfer into synchronous recipients and (iii) evaluate in vivo survival of black-footed cat (BFC, Felis nigripes) embryos after intra- and inter-species transfer. In vitro-derived (IVM/IVF) cat embryos were used to evaluate in vitro development after controlled-rate cryopreservation vs vitrification vs controls. Blastocyst development was similar in both groups of cryopreserved embryos (22-26%), but it was lower (p < 0.05) than that of fresh embryos (50%). After embryo transfer, four of eight recipients of vitrified embryos established pregnancies--three of six (50%) and one of two (50%) that received embryos from in vivo- and in vitro-matured oocytes, respectively. Three male and two female kittens weighing from 51 to 124 g (mean = 88 g) were delivered on days 61-65 of gestation. In BFC, four intra-species embryo transfer procedures were carried out--two recipients received fresh day 2 embryos (n = 5, 8) and two recipients received embryos that had been cryopreserved on day 1 (n = 6) or 2 (n = 8). A 2-year-old recipient of cryopreserved embryos established pregnancy and delivered two live male kittens. Subsequently, five cryopreserved BFC embryos were transferred to a domestic cat recipient. On day 29, the recipient was determined to be pregnant and delivered naturally a live, healthy female BFC kitten on day 66. In summary, in vivo survival of vitrified domestic cat embryos was shown by the births of kittens after transfer into recipients. Also, we demonstrated that sperm and embryo cryopreservation could be combined with intra- and inter-species embryo transfer and integrated into the array of assisted reproductive techniques used successfully for propagation of a rare and vulnerable felid species, the black-footed cat. © 2012 Blackwell Verlag GmbH.

Embryo loss in cattle between Days 7 and 16 of pregnancy.

PubMed

Berg, D K; van Leeuwen, J; Beaumont, S; Berg, M; Pfeffer, P L

2010-01-15

Embryo loss between embryonic Days 7 and 16 (Day 0=day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P=0.03) as more embryos were transferred, particularly at later stages (Day 14, P<0.01). The average length of embryos decreased by approximately 5% for every additional embryo transferred (P<0.0001). These effects may be linked to embryonic migration. Embryo mortality inherent to the embryo during the second week of pregnancy was 24%. Additionally, 9% of Day 14 embryos were of inferior quality, as they did not contain an epiblast. Combining embryo and recipient causes but excluding infection effects, embryonic loss of IVP embryos during the second week of pregnancy amounted to 26% (heifers) or 34% (parous dairy cows). The length of embryos doubled every day between Days 9 and 16, with a 4.4-fold range in sizes representing two thirds of the variation in length. Embryos retrieved from heifers were twice the size of those incubated in parous cows (P<0.0001), indicating faster embryonic development/trophoblast proliferation in heifers. Whereas season did not affect embryo recoveries, length was lower (50%) in winter (winter-autumn, P<0.05; winter-spring, P<0.001). Lastly, transuterine migration in cattle, when transferring multiple embryos, commenced at Day 14 (4%) and had occurred in all recipients by Day 16 (38% of embryos found contralaterally).

Nontransmission of porcine circovirus 2 (PCV2) by embryo transfer.

PubMed

Bielanski, A; Algire, J; Lalonde, A; Garceac, A; Pollard, J W; Plante, C

2013-07-15

Two experiments were conducted to determine the association of porcine circovirus type 2 (PCV2) with embryos and the risk of viral transmission by embryo transfer. In the first experiment, 240 embryos from uninfected donors were exposed to PCV2a 10(4)TCID50/mL in vitro before transfer to seronegative recipients; in the second experiment, 384 embryos recovered from infected donors, 10 days after donor inoculation with PCV2, were transferred to seronegative recipients. In total, 1120 embryos and/or ova were collected from 37 viral-free donors (experiment 1) and 1019 from 59 PCV2-infected donors (experiment 2) (P < 0.01). The washing and/or disinfection procedure recommended by the International Embryo Transfer Society was applied to embryos in both experiments. Transfer of embryos experimentally exposed in vitro to high titers of virus caused seroconversion of recipients (58%; N = 7/12) and their piglets (81%; N = 13/16). Postmortem, PCV2 DNA was detected in various organs of embryo transfer recipients and their embryo transfer-derived piglets. In contrast, the transfer of embryos recovered from infectious PCV2 donors did not result in the seroconversion of embryo recipients (N = 24) or their embryo transfer-derived piglets (N = 76). Neither PCV2 DNA nor infectious virus was detected in the tissues of either recipients or embryo transfer-derived piglets collected postmortem in the second experiment. The results obtained in this study indicate that the transmission of PCV2 from infected donors by embryo transfer is unlikely if the sanitary recommendations of the International Embryo Transfer Society are followed. In practical terms, this means that embryo transfer can be successfully used for the intentional elimination of PCV2 and to create virus-free offspring for the safe exchange of swine genetic materials. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Cryopreservation of day 2-3 embryos by vitrification yields better outcome than slow freezing.

PubMed

Levron, Jacob; Leibovitz, Oshrit; Brengauz, Masha; Gitman, Hila; Yerushalmi, Gil M; Katorza, Eldad; Gat, Itai; Elizur, Shai E

2014-03-01

To compare the outcome of vitrification versus slow freezing cryopreservation for cleavage stage day 2-3 embryos. A retrospective observational study. All thawed embryos assisted reproduction cycles between January 2010 and December 2012 at a single IVF laboratory of a Tertiary Medical Center. Five hundred and thirty-nine cycles of day 2-3 thawed embryos. In 327 of the thawed cycles, the embryos were vitrified and in 212 of the cycles the embryos were derived from slow freezing embryos. Embryo survival rate, blastomere surviving rate and pregnancy rate. Embryo survival rate was significantly higher after vitrification compared with slow freezing (81.6%, 647/793 versus 70.0%, 393/562 embryos, p < 0.0001). The clinical pregnancy rate per ET was significantly higher following vitrification compared to slow freezing, 20.0%, 63/314 versus 11.9%, 23/193, respectively (p = 0.02). Vitrification of day 2-3 cleavage stage embryos yields better cycle outcome in all the parameters compared to slow freezing.

Timing embryo biopsy for PGD - before or after cryopreservation?

PubMed

Shinar, S; Kornecki, N; Schwartz, T; Mey-Raz, N; Amir, H; Almog, B; Shavit, T; Hasson, J

2016-09-01

Pre-implantation genetic diagnosis (PGD) is required in order to screen and diagnose embryos of patients at risk of having a genetically affected offspring. A biopsy to diagnose the genetic profile of the embryo may be performed either before or after cryopreservation. The aim of this study was to determine which biopsy timing yields higher embryo survival rates. Retrospective cohort study of all PGD patients in a public IVF unit between 2010 and 2013. Inclusion criteria were patients with good-quality embryos available for cryopreservation by the slow freezing method. Embryos were divided into two groups: biopsy before and biopsy after cryopreservation. The primary outcome was embryo survival rates post thawing. Sixty-five patients met inclusion criteria. 145 embryos were biopsied before cryopreservation and 228 embryos were cryopreserved and biopsied after thawing. Embryo survival was significantly greater in the latter group (77% vs. 68%, p < 0.0001). Cryopreservation preceding biopsy results in better embryo survival compared to biopsy before cryopreservation.

Goose embryonic development from oviposition through 16 hours of incubation.

PubMed

Lukaszewicz, E; Lason, M; Rosenberger, J; Kowalczyk, A; Bakst, M

2017-06-01

Normal tables provide an objective step-wise description of the morphological development of an embryo. Such tables have been described for the chicken, turkey, quail, and duck embryos, but there is no such staging table for goose embryos. As the goose has one of the longest incubation periods of all the poultry species and embryo mortality during incubation is relatively high, a normal table of goose embryo development would be useful in assessing the morpho-genetic status of the goose embryo before and during incubation. In this study, embryos were isolated from commercial White Koluda goose eggs stored no longer than four days in a cool room (18°C) prior to incubation and after 4, 8, 12, and 16 h of incubation. Embryo staging was based on the normal tables described for the chicken by Eyal-Giladi and Kochav (EGK) and Hamburger and Hamilton (HH). Goose embryos from unincubated eggs were at Stage X and XI EGK and after 16 h of incubation the majority of embryos were between Stages 2 and 4 HH. Our results suggest that while the stage of development of the embryo in the unincubated goose egg is similar to that reported for the chicken, although the diameter of goose embryo is slighter larger. Following incubation, a goose embryo advances more slowly than a chicken embryo up to 16 h of incubation. © 2016 Poultry Science Association Inc.

In Amnio MRI of Mouse Embryos

PubMed Central

Roberts, Thomas A.; Norris, Francesca C.; Carnaghan, Helen; Savery, Dawn; Wells, Jack A.; Siow, Bernard; Scambler, Peter J.; Pierro, Agostino; De Coppi, Paolo; Eaton, Simon; Lythgoe, Mark F.

2014-01-01

Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community. PMID:25330230

Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human Day 3 embryos.

PubMed

Van Landuyt, L; Van de Velde, H; De Vos, A; Haentjens, P; Blockeel, C; Tournaye, H; Verheyen, G

2013-11-01

Is the effect of cell loss on further cleavage and implantation different for vitrified than for slowly frozen Day 3 embryos? Vitrified embryos develop better overnight than slowly frozen embryos, regardless of the number of cells lost, but have similar implantation potential if further cleavage occurs overnight. After slow-freezing, similar implantation rates have been obtained for intact 4-cell embryos or 4-cell embryos with 1 cell damaged. For slowly frozen Day 3 embryos, lower implantation rates have been observed when at least 25% of cells were lost. Other studies reported similar implantation potential for 7- to 8-cell embryos with 0, 1 or 2 cells damaged. No data are available on further development of vitrified embryos in relation to cell damage. Survival and overnight cleavage were retrospectively assessed for 7664 slowly frozen Day 3 embryos (study period: January 2004-December 2008) and 1827 vitrified embryos (study period: April 2010-September 2011). Overnight cleavage was assessed according to cell stage at cryopreservation and post-thaw cell loss for both protocols. The relationship between cell loss and implantation rate was analysed in a subgroup of single-embryo transfers (SETs) with 780 slowly frozen and 294 vitrified embryos. Embryos with ≥6 blastomeres and ≤20% fragmentation were cryopreserved using slow controlled freezing [with dimethyl sulphoxide (DMSO) as cryoprotectant] or closed vitrification [with DMSO-ethylene glycol (EG)-sucrose (S) as cryoprotectants]. Only embryos with ≥50% of cells intact after thawing were cultured overnight and were only transferred if further cleaved. For each outcome, logistic regression analysis was performed. Survival was 94 and 64% after vitrification and slow-freezing respectively. Logistic regression analysis showed that overnight cleavage of surviving embryos was higher after vitrification than after slow-freezing (P < 0.001) and decreased according to the degree of cell damage (P < 0.001). If the embryo continued to cleave after thawing, there was no effect of the number of cells lost or the cryopreservation method on its implantation potential. The implantation rates of embryos with 0, 1 or 2 cells damaged were, respectively, 21% (n = 114), 21% (n = 28) and 20% (n = 12) after slow-freezing and 20% (n = 50), 21% (n = 5) and 27% (n = 4) after vitrification. This analysis is retrospective and study periods for vitrification and slow-freezing are different. The number of SETs with vitrified embryos is limited. However, a large number of vitrified embryos were available to analyse the further cleavage of surviving embryos. Although it is not proved that vitrified embryos are more viable than slowly frozen embryos in terms of pregnancy outcome, vitrification yields higher survival rates, better overnight development and higher transfer rates per embryo warmed. This increases the number of frozen transfer cycles originating from a single treatment and might result in a better cumulative clinical outcome. Based on the present data, the policy to warm an extra embryo before overnight culture depends on the cell stage at cryopreservation and the cell damage after warming. For 8-cell embryos, up to two cells may be damaged compared with only one cell for 6- to 7-cell embryos, before an additional embryo is warmed. none.

Zygotic and somatic embryo morphogenesis in Pinus pinaster: comparative histological and histochemical study.

PubMed

Tereso, Susana; Zoglauer, Kurt; Milhinhos, Ana; Miguel, Célia; Oliveira, M Margarida

2007-05-01

We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.

In vitro and in vivo survival of bisected sheep embryos derived from frozen-thawed unsorted, and frozen-thawed sex-sorted and refrozen-thawed ram spermatozoa.

PubMed

Morton, Katherine M; Rowe, Anthony M; Chis Maxwell, W M; Evans, Gareth

2006-04-15

Ovine IVP embryos were derived from frozen-thawed unsorted and frozen-thawed sex-sorted spermatozoa that had been refrozen and thawed. The embryos were bisected and cultured in vitro, or transferred to recipient ewes to determine their survival in vitro and in vivo. Oocyte progression to the blastocyst stage was similar for unsorted (97/232, 41.8%) and sex-sorted spermatozoa (113/286, 39.5%; P > 0.05). Embryo survival in vitro post-bisection was similar for demi-embryos derived from unsorted and sex-sorted sperm, and embryos bisected at the blastocyst and expanded blastocyst stage (P > 0.05). A higher proportion of recipient ewes were pregnant at Day 63 after transfer of two intact embryos derived from unsorted (17/21, 80.9%) than two demi-embryos derived from unsorted (5/15, 33.3%) or sex-sorted spermatozoa (7/17, 41.2%). The number of fetuses per original embryo at Day 63 did not differ among groups (unsorted intact: 23/42, 54.8%; unsorted demi: 7/15, 46.7%; sex-sorted demi: 10/17, 58.8%) and twin pregnancies were observed in all groups. Embryo survival to term was high, and was not significantly different among intact (unsorted: 22/42, 52.4%) and demi-embryos (unsorted: 4/15, 26.7%; sex-sorted spermatozoa: 7/17, 41.2%; P > 0.05). Dizygotic twins (n = 6 sets) were born after the transfer of two intact embryos derived from unsorted spermatozoa, but only singleton lambs resulted from the transfer of demi-embryos. In conclusion, bisected IVP embryos successfully developed into morphologically normal lambs. However, embryo survival to term was neither increased nor decreased by embryo bisection.

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system.

PubMed

Tagawa, M; Matoba, S; Narita, M; Saito, N; Nagai, T; Imai, K

2008-03-15

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.

Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation.

PubMed

Velasquez, Alejandra E; Castro, Fidel O; Veraguas, Daniel; Cox, Jose F; Lara, Evelyn; Briones, Mario; Rodriguez-Alvarez, Lleretny

2016-02-01

Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7-9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

Effects of laser polar-body biopsy on embryo quality.

PubMed

Levin, Ishai; Almog, Benny; Shwartz, Tamar; Gold, Veronica; Ben-Yosef, Dalit; Shaubi, Michal; Amit, Ami; Malcov, Mira

2012-05-01

To evaluate the effect of laser polar-body biopsy (PBB) for preimplantation genetic diagnosis on embryo quality. Retrospective case-control analysis. The quality of 145 embryos after PBB was compared to 276 embryos of the same group of women without biopsy. University-based tertiary-care medical center. Women with inherited genetics disease. Laser PBB of IVF embryos for genetic diagnosis. The study and control embryos were compared for fertilization rate, pronuclear grading, and cleavage-stage parameters on days 1, 2, and 3 after oocyte retrieval. The study embryos demonstrated higher rates of cleavage arrest (3.6% vs. 0.7%), higher rate of significant fragmentation on day 2 (9.5% vs. 3.0%), and lower rate of good cleavage embryos on day 2 (69.1% vs. 78.4%) compared with control embryos. On day 3, the study embryos had lower cleavage rates (six or more blastomeres; 56.5% vs. 74.5%), higher fragmentation (11.7% vs. 3.9%), higher rate of embryos presenting inferior cleavage pattern (57.2% vs. 38.5%), and lower mean blastomere number (5.8 ± 2.1 vs. 6.6 ± 1.9) compared with control embryos. Polar-body biopsy may have a negative effect on embryo quality. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Transcriptomic and proteomic analysis reveals mechanisms of embryo abortion during chrysanthemum cross breeding.

PubMed

Zhang, Fengjiao; Wang, Zhiquan; Dong, Wen; Sun, Chunqing; Wang, Haibin; Song, Aiping; He, Lizhong; Fang, Weimin; Chen, Fadi; Teng, Nianjun

2014-10-07

Embryo abortion is the main cause of failure in chrysanthemum cross breeding, and the genes and proteins associated with embryo abortion are poorly understood. Here, we applied RNA sequencing and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic profiles of normal and abortive embryos. More than 68,000 annotated unigenes and 700 proteins were obtained from normal and abortive embryos. Functional analysis showed that 140 differentially expressed genes (DEGs) and 41 differentially expressed proteins (DEPs) were involved in embryo abortion. Most DEGs and DEPs associated with cell death, protein degradation, reactive oxygen species scavenging, and stress-response transcriptional factors were significantly up-regulated in abortive embryos relative to normal embryos. In contrast, most genes and proteins related to cell division and expansion, the cytoskeleton, protein synthesis and energy metabolism were significantly down-regulated in abortive embryos. Furthermore, abortive embryos had the highest activity of three executioner caspase-like enzymes. These results indicate that embryo abortion may be related to programmed cell death and the senescence- or death-associated genes or proteins contribute to embryo abortion. This adds to our understanding of embryo abortion and will aid in the cross breeding of chrysanthemum and other crops in the future.

Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

PubMed

Balbach, Sebastian T; Boiani, Michele

2015-01-01

Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system

PubMed Central

Men, Hongsheng; Zhao, Chongbei; Wei, Si; Murphy, Clifton N.; Spate, Lee; Liu, Yang; Walters, Eric M.; Samuel, Melissa S.; Prather, Randall S.; Critser, John K.

2011-01-01

As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN2). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a “closed” system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease. PMID:21458047

[Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

PubMed

Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

2014-06-01

To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

Fiber optic light-scattering measurement system for evaluation of embryo viability: light-scattering characteristics from live mouse embryo

NASA Astrophysics Data System (ADS)

Itoh, Harumi; Arai, Tsunenori; Kikuchi, Makoto

1997-06-01

We measured angular distribution of the light scattering from live mouse embryo with 632.8nm in wavelength to evaluate the embryo viability. We aim to measure the mitochondrial density in human embryo which have relation to the embryo viability. We have constructed the light scattering measurement system to detect the mitochondrial density non-invasively. We have employed two optical fibers for the illumination and sensing to change the angle between these fibers. There were two dips on the scattering angular distribution from the embryo. These dips existed on 30 and 85 deg. We calculated the scattering angular pattern by Mie theory to fit the measured scattering estimated scattering size and density. The best fitting was obtained when the particle size and density were 0.9 micrometers and 1010 particles per ml, respectively. These values coincided with the approximated values of mitochondrial in the embryo. The measured light scattering may mainly originated from mitochondria in spite of the existence of the various scattering particles in the embryo. Since our simple scattering measurement may offer the mitochondrial density in the embryo, it might become the practical method of human embryo on in vitro fertilization-embryo transfer.

Survey of 243 ART patients having made a final disposition decision about their surplus cryopreserved embryos: the crucial role of symbolic embryo representation.

PubMed

Bruno, C; Dudkiewicz-Sibony, C; Berthaut, I; Weil, E; Brunet, L; Fortier, C; Pfeffer, J; Ravel, C; Fauque, P; Mathieu, E; Antoine, J M; Kotti, S; Mandelbaum, J

2016-07-01

In couples who have chosen and confirmed the fate of surplus frozen embryos, which factors influence their decision, with a special emphasis on their symbolic representation of the embryo(s)? Embryo representation and gamete donation use significantly influence the fate of surplus cryopreserved embryos. Previous studies report difficulties for couples to decide whether or not to continue storing their frozen embryo(s) and different factors have been already highlighted which influence their decision, including embryo conceptualization, information and support provided by the medical institution, quality of embryo(s) and life events. Little is known, however, about couples who definitely decided to stop their parental project and finalized the process of decision-making about the fate of their cryopreserved embryo(s). This prospective study was conducted over a period of 3 years (2007-2010) and included IVF/ICSI patients with surplus frozen embryos, who made a final embryo disposition decision. Among the 280 eligible IVF/ICSI patients, 247 agreed to participate in the study. According to the available options, 91 persons chose to 'stop cryopreservation', 77 chose donation to 'research' and 48 'embryo donation' to infertile couples. Furthermore, 31 participants who chose embryo donation for a parental project were refused by the center as not compatible with their mandatory medical conditions. Among them, 27 participants then selected donation to research as a new option and were included in a fourth group: 'donation to research after Refusal of Embryo Donation for parental project' or 'research-RED' (n = 27). Four participants chose 'stop cryopreservation', however, given the small number of subjects this latter group was not included in the analysis. In all, 243 participants who made a final choice concerning the fate of their cryopreserved embryos were included in this study. Participants were sent a letter of invitation to a semi-structured interview of 30 min with a psychologist. Interviews were conducted separately for each partner, including a questionnaire with a common part and a specific part, according to the chosen option, and allowing a quantitative evaluation. A multivariate logistic regression model was used to assess the link between their embryo representation and their decision about their embryos' fate. After adjustment for age, gender, gamete donation, number of children and the different embryo representations, a choice to 'stop cryopreservation' is more frequent if the embryo is represented as a child [odds ratio (OR) adjusted = 3.29, 95% confidence interval (CI) = 1.62-6.66], P = 0.0009. Representing the embryo as a project prompts patients to choose 'donation to research' [OR adjusted = 3.76, 95% CI = 1.56-9.06], P = 0.0032. Respondents are more likely to choose 'embryo donation' if they represent the embryo as a potential person [OR adjusted = 3.77, 95% CI = 1.45-9.80], P = 0.0064. Furthermore, patients who benefited from gamete donation are ∼10 times more likely to donate their embryos to another couple [OR adjusted = 10.62, 95% CI = 3.99-28.30], P < 0.0001. For more than half the participants (57%) the decision-making was easy, however, deciding to stop cryopreservation was significantly more difficult than choosing research or embryo donation (P < 0.0001). Socio-economic status, moral and religious affiliations are known to influence the choice of couples but analyzing these factors was not an aim of the present study. When couples definitely decide to stop their parental project, the embryo symbolic representation remains the main factor that influences the fate of their frozen embryo(s). Moreover, this representation can evolve when influenced by external events and information provided. In order to support patients who are making this difficult decision, it could be helpful to explore this symbolic representation early in the IVF/ICSI procedure, before surplus embryo freezing, as a new tool enhancing the accuracy of counseling. this study was supported by a grant from the 'Agence de la biomedicine (ABM)', the national regulatory ART agency, under the authority of the French Ministry of Health. The authors have no conflict of interest to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Pollen source effects on growth of kernel structures and embryo chemical compounds in maize.

PubMed

Tanaka, W; Mantese, A I; Maddonni, G A

2009-08-01

Previous studies have reported effects of pollen source on the oil concentration of maize (Zea mays) kernels through modifications to both the embryo/kernel ratio and embryo oil concentration. The present study expands upon previous analyses by addressing pollen source effects on the growth of kernel structures (i.e. pericarp, endosperm and embryo), allocation of embryo chemical constituents (i.e. oil, protein, starch and soluble sugars), and the anatomy and histology of the embryos. Maize kernels with different oil concentration were obtained from pollinations with two parental genotypes of contrasting oil concentration. The dynamics of the growth of kernel structures and allocation of embryo chemical constituents were analysed during the post-flowering period. Mature kernels were dissected to study the anatomy (embryonic axis and scutellum) and histology [cell number and cell size of the scutellums, presence of sub-cellular structures in scutellum tissue (starch granules, oil and protein bodies)] of the embryos. Plants of all crosses exhibited a similar kernel number and kernel weight. Pollen source modified neither the growth period of kernel structures, nor pericarp growth rate. By contrast, pollen source determined a trade-off between embryo and endosperm growth rates, which impacted on the embryo/kernel ratio of mature kernels. Modifications to the embryo size were mediated by scutellum cell number. Pollen source also affected (P < 0.01) allocation of embryo chemical compounds. Negative correlations among embryo oil concentration and those of starch (r = 0.98, P < 0.01) and soluble sugars (r = 0.95, P < 0.05) were found. Coincidently, embryos with low oil concentration had an increased (P < 0.05-0.10) scutellum cell area occupied by starch granules and fewer oil bodies. The effects of pollen source on both embryo/kernel ratio and allocation of embryo chemicals seems to be related to the early established sink strength (i.e. sink size and sink activity) of the embryos.

Transcriptional regulators TRIM28, SETDB1, and TP53 are aberrantly expressed in porcine embryos produced by in vitro fertilization in comparison to in vivo- and somatic-cell nuclear transfer-derived embryos

PubMed Central

Hamm, Jennifer; Tessanne, Kim; Murphy, Clifton N; Prather, Randall S

2014-01-01

In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos. Mol. Reprod. Dev. 81: 552–556, 2014. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:24659575

Development of whole and demi-embryos of mice in culture and in vivo after supercooled storage.

PubMed

Fuku, E; Fiser, P S; Marcus, G J; Sasada, H; Downey, B R

1993-12-01

Demi-embryos (produced by destroying 1 or 2 blastomeres of 2- or 4-cell embryos, respectively) and intact mouse embryos were cultured to the blastocyst stage, stored at -5 degrees C for 48 h, then cultured for 24 h and transferred into pseudopregnant recipients. Supercooled storage did not impair the developmental potential of whole or demi-embryos in vitro, nor was there a difference between whole and demi-embryos with respect to growth in vitro. Similarly, there was no effect of supercooling on development of intact or demi embryos after transfer into pseudopregnant recipient mice, but fewer recipients of demi-embryos remained pregnant (P < 0.05). This was considered to be partly due to the lesser ability of demi-embryos to maintain luteal function and establish pregnancy.

Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

PubMed

Noli, Laila; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

2017-03-01

Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. A very limited number of studies have been conducted in humans and non-human primates. The published material, especially the studies with human embryos, is controversial. Some reports suggest that twinning technology will find clinical use in reproductive medicine in the future, whereas others conclude the opposite that human twin embryos created in vitro are unsuitable not only for clinical, but also for research, purposes. The blastomere biopsy technique of embryo splitting seems to be unsuitable for either clinical or research purposes; however, embryo bisection, a preferable method of cloning in veterinary medicine, has not yet been tested on human embryos. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Live birth in a woman with recurrent implantation failure and adenomyosis following transfer of refrozen-warmed embryos.

PubMed

Safari, Somayyeh; Faramarzi, Azita; Agha-Rahimi, Azam; Khalili, Mohammad Ali

2016-09-01

The aim was to report a healthy live birth using re-vitrified-warmed cleavage-stage embryos derived from supernumerary warmed embryos after frozen embryo transfer (ET) in a patient with recurrent implantation failure (RIF). The case was a 39-year-old female with a history of polycystic ovarian syndrome and adenomyosis, along with RIF. After ovarian hyperstimulation, 33 cumulus-oocyte complexes were retrieved and fertilized with conventional in vitro fertilization and intracytoplasmic sperm injection. Because of the risk of ovarian hyperstimulation syndrome, 16 grade B and C embryos were vitrified. After 3 and 6 months, 3 and 4 B-C warmed embryos were transferred to the uterus, respectively. However, implantation did not take place. Ten months later, four embryos were warmed, two grade B 8-cell embryos were transferred, and two embryos were re-vitrified. One year later, the two re-vitrified cleavage-stage embryos were warmed, which resulted in a successful live birth. This finding showed that following first warming, it is feasible to refreeze supernumerary warmed embryos for subsequent ET in patients with a history of RIF.

Factors affecting survivability of transferred whole and demi-embryos in a commercial dairy herd.

PubMed

Arave, C W; Bunch, T D; Mickelsen, C H; Warnick, K

1987-09-01

Sixty Holstein donor cows were superovulated and embryos were collected during a 6-d (27 cows) and a 4-d (33 cows) period approximately 60 d apart. Forty-three donor cows yielded embryos. Ninety-one embryos graded 1 or 2 were split and transferred to 181 recipient Holsteins. Demi-embryos were graded 2, 2-, 3 and 3- prior to transfer. Pregnancy and calving percentages were similar for all demi-embryo grades, averaging 59 and 53% from the two donor groups, respectively. Twin demi-embryo pregnancies averaged 36 and 19% for embryos split at the compacted morula and blastocyst stages, respectively. Twin demi-embryo calvings averaged 30 and 15% for these same groups. Progesterone levels of recipients (of either whole or demi-embryos) of second period donors were measured. Pregnancy rate increased generally with level of progesterone; however, calving percentage was slightly greater for recipients with intermediate levels of progesterone (2-6 ng/ml). Multiparous cow (20) recipients of demi-embryos had 45% pregnancy and 40% calving, while nulliparous heifer (161) recipients averaged 59 and 53% pregnancy and calving, respectively.

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models

PubMed Central

2011-01-01

Background PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. Methods Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. Results PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control). Conclusions PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner. PMID:21569635

Novel embryo selection techniques to increase embryo implantation in IVF attempts.

PubMed

Sigalos, George Α; Triantafyllidou, Olga; Vlahos, Nikos F

2016-11-01

The final success of an IVF attempt depends on several steps and decisions taken during the ovarian stimulation, the oocyte retrieval, the embryo culture and the embryo transfer. The final selection of the embryos most likely to implant is the final step in this process and the responsibility of the lab. Apart from strict morphologic criteria that historically have been used in embryo selection, additional information on genetic, metabolomic and morphokinetic characteristics of the embryo is recently combined to morphology to select the embryo most likely to produce a pregnancy. In this manuscript, we review the most recent information on the current methods used for embryo selection presenting the predictive capability of each one. A literature search was performed on Pubmed, Medline and Cochrane Database of Systematic Reviews for published studies using appropriate key words and phrases with no limits placed on time. It seems that the combination of morphologic criteria in conjunction to embryo kinetics as documented by time-lapse technology provides the most reliable information on embryo quality. Blastocyst biopsy with subsequent comprehensive chromosome analysis allows the selection of the euploid embryos with the higher implantation potential. Embryo time-lapse imaging and blastocyst biopsy combined to comprehensive chromosome analysis are the most promising technologies to increase pregnancy rates and reduce the possibility of multiple pregnancies. However, further studies will demonstrate the capability of routinely using these technologies to significantly improve IVF outcomes.

Unaltered timing of embryo development in women with polycystic ovarian syndrome (PCOS): a time-lapse study.

PubMed

Sundvall, Linda; Kirkegaard, Kirstine; Ingerslev, Hans Jakob; Knudsen, Ulla Breth

2015-07-01

Polycystic ovarian syndrome (PCOS) is a common cause of female infertility. Factors other than anovulation, such as low embryo quality have been suggested to contribute to the infertility in these women. This 2-year retrospective study used timelapse technology to investigate the PCOS-influence on timing of development in the pre-implantation embryo (primary endpoint). The secondary outcome measure was live birth rates after elective single-embryo transfer. In total, 313 embryos from 43 PCOS women, and 1075 embryos from 174 non-PCOS women undergoing assisted reproduction were included. All embryos were monitored until day 6. Differences in embryo kinetics were tested in a covariance regression model to account for potential confounding variables: female age, BMI, fertilization method and male infertility. Time to initiate compaction and reach the morula stage as well as the duration of the 4th cleavage division was significantly shorter in PCOS embryos compared with non-PCOS embryos. No other kinetic differences were found at any time-points annotated. The proportion of multi-nucleated cells at the 2-cell stage was significantly higher in PCOS embryos compared with non-PCOS embryos. The live birth rates were comparable between the two groups. The findings suggest that the causative factor for subfertility in PCOS is not related to timing of development in the pre-implantation embryo.

Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks

ERIC Educational Resources Information Center

Wellner, Karen L.

2014-01-01

In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…

Non-invasive metabolomic profiling of embryo culture media and morphology grading to predict implantation outcome in frozen-thawed embryo transfer cycles.

PubMed

Li, Xiong; Xu, Yan; Fu, Jing; Zhang, Wen-Bi; Liu, Su-Ying; Sun, Xiao-Xi

2015-11-01

Assessment of embryo viability is a crucial component of in vitro fertilization and currently relies largely on embryo morphology and cleavage rate. Because morphological assessment remains highly subjective, it can be unreliable in predicting embryo viability. This study investigated the metabolomic profiling of embryo culture media using near-infrared (NIR) spectroscopy for predicting the implantation potential of human embryos in frozen-thawed embryo transfer (FET) cycles. Spent embryo culture media was collected on day 4 after thawed embryo transfer (n = 621) and analysed using NIR spectroscopy. Viability scores were calculated using a predictive multivariate algorithm of fresh embryos with known pregnancy outcomes. The mean viability indices of embryos resulting in clinical pregnancy following FET were significantly higher than those of non-implanted embryos and differed between the 0, 50, and 100 % implantation groups. Notably, the 0 % group index was significantly lower than the 100 % implantation group index (-0.787 ± 0.382 vs. 1.064 ± 0.331, P < 0.01). To predict implantation outcomes, we examined the area under the ROC curve (AUCROC), which was significantly higher for the viability than for the morphology score (0.94 vs. 0.55; P < 0.01); however, the AUCROCs for the composite and viability scores did not differ significantly (0.92 vs. 0.94; P > 0.05). NIR metabolomic profiling of thawed embryo culture media is independent of morphology and correlates with embryo implantation potential in FET cycles. The viability score alone or in conjunction with morphologic grading is a more objective marker for implantation outcome in FET cycles than morphology alone.

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: influence of the maternally inherited components.

PubMed

Mognetti, B; Leppens, G; Sakkas, D

1996-04-01

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In-view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose.

Photoautotrophic Culture of Coffea arabusta Somatic Embryos: Photosynthetic Ability and Growth of Different Stage Embryos

PubMed Central

AFREEN, F.; ZOBAYED, S. M. A.; KOZAI, T.

2002-01-01

Coffea arabusta somatic embryos were cultured and development of stomata, rate of CO2 fixation or production, chlorophyll content and chlorophyll fluorescence were studied in embryos at different stages of development. Cotyledonary and germinated embryos have photosynthetic capacity, although pretreatment at a high photosynthetic photon flux (PPF) (100 µmol m–2 s–1) for 14 d increased photosynthetic ability. Except in a very small number of cases, stomata did not develop fully in precotyledonary stage embryos and were absent in torpedo stage embryos. Low chlorophyll content (90–130 µg g–1 fresh mass) was noted in torpedo and precotyledonary stage embryos compared with cotyledonary and germinated embryos (300–500 µg g–1 fresh mass). Due to the absence of stomata and low chlorophyll content in the torpedo and precotyledonary stage embryos, the photosynthetic rate was low and, in some cases, CO2 production was observed. These data suggest that the cotyledonary stage is the earliest stage that can be cultured photoautotrophically to ensure plantlet development. When grown photoautotrophically (in a sugar‐free medium with CO2 enrichment in the culture headspace and high photosynthetic photon flux), torpedo and precotyledonary stage embryos lost 20–25 % of their initial dry mass after 60 d of culture. However, in cotyledonary and germinated embryos, the dry mass of each embryo increased by 10 and 50 %, respectively. By using a porous supporting material, growth (especially root growth) was increased in cotyledonary stage embryos. In addition, photoautotrophic conditions, high PPF (100–150 µmol m–2 s–1) and increased CO2 concentration (1100 µmol mol–1) were found to be necessary for the development of plantlets from cotyledonary stage embryos. PMID:12125763

Cost-effectiveness of single versus double embryo transfer in IVF in relation to female age.

PubMed

van Loendersloot, Laura L; Moolenaar, Lobke M; van Wely, Madelon; Repping, Sjoerd; Bossuyt, Patrick M; Hompes, Peter G A; van der Veen, Fulco; Mol, Ben Willem J

2017-07-01

To evaluate the cost-effectiveness of single embryo transfer followed by an additional frozen-thawed single embryo transfer, if more embryos are available, as compared to double embryo transfer in relation to female age. We used a decision tree model to evaluate the costs from a healthcare provider perspective and the pregnancy rates of two embryo transfer policies: one fresh single embryo transfer followed by an additional frozen-thawed single embryo transfer, if more embryos are available (strategy I), and double embryo transfer (strategy II). The analysis was performed on an intention-to-treat basis. Sensitivity analyses were carried out to evaluate the robustness of our model and to identify which model parameters had the strongest impact on the results. SET followed by an additional frozen-thawed single embryo transfer if available was dominant, less costly and more effective, over DET in women under 32 years. In women aged 32 or older DET was more effective than SET followed by an additional frozen-thawed single embryo transfer if available but also more costly. SET followed by an additional frozen-thawed single embryo transfer should be the preferred strategy in women under 32 undergoing IVF. The choice for SET followed by an additional frozen-thawed single embryo transfer or DET in women aged 32 or older depends on individual patient preferences and on how much society is willing to pay for an extra child. There is a strong need for a randomized clinical trial comparing the cost and effects of SET followed by an additional frozen-thawed single embryo transfer and DET in the latter category of women. Copyright © 2017 Elsevier B.V. All rights reserved.

Laser confers less embryo exposure than acid tyrode for embryo biopsy in preimplantation genetic diagnosis cycles: a randomized study.

PubMed

Geber, Selmo; Bossi, Renata; Lisboa, Cintia B; Valle, Marcelo; Sampaio, Marcos

2011-04-28

We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome.

'New embryos' - new challenges for the ethics of stem cell research.

PubMed

Holm, Søren

2008-01-01

Among the many ethical issues raised by human embryonic stem cell research (in the following all references to 'stem cells' should be read as references to human embryonic stem cells), two have gained specific prominence: (1) whether stem cell research is ethically problematic because it entails the destruction of human embryos and (2) what kind of control embryo donors should have over the stem cell lines derived from their embryos. In the present paper, I will analyse how these two issues are engaged by various attempts to derive stem cells from anomalous embryos (e.g. embryos in cleavage arrest, embryos not implanted following pre-implantation genetic diagnosis or embryos created by altered nuclear transfer) or in ways that are claimed to be non-destructive for the embryo (e.g. blastocyst or blastomere biopsy). Copyright 2008 S. Karger AG, Basel.

Inbreeding effects on in vitro embryo production traits in Guzerá cattle.

PubMed

Perez, B C; Balieiro, J C C; Ventura, R V; Bruneli, F A T; Peixoto, M G C D

2017-11-01

Inbreeding has been associated with the impairment of reproductive performance in many cattle breeds. Although the usage of reproductive biotechnologies has been increasing in bovine populations, not much attention has been given to the impact of inbreeding over cow's performance on artificial reproduction. The objective of this study was to estimate the impact of inbreeding on in vitro embryo production in a Guzerá breed population. The inbreeding coefficient (F), calculated as half of the co-ancestry of the individual's parents, was used as an estimate of inbreeding. The inbreeding coefficients of the donor, sire (used on in vitro fertilization) and of the embryos were included, separately, in the proposed models either as classificatory or continuous variables (linear and quadratic effects). The percentage of non-inbred individuals (or embryos) and mean F of donors, embryos and sires were 29.38%; 35.76%; 42.86% and 1.98±2.68; 1.32±3.13; 2.08±2.79, respectively. Two different models were considered, one for oocyte production traits and other for embryo production traits. The increase of F of the donor significantly (P<0.05) impaired the number of viable oocytes (N OV), number of grade I oocytes (N GI) and number of cleaved embryos (N CLV). Moreover, the donor's F influenced the percentage of grade I oocytes (P GI), percentage of viable embryos (P EMB) and percentage of cleaved embryos that turned into embryos (P CXE). No significant (P>0.05) effects were observed for the sire (father of the embryos) inbreeding coefficient over the traits analysed. Embryo's F influenced (P<0.05) the number of viable embryos (N EMB), percentage of viable embryos (P EMB) and percentage of cleaved embryos that turn into embryos (P CXE). Results suggested that an increase in the inbreeding coefficient might impair the embryos ability to survive through challenges imposed by the in vitro environment. Submitting highly inbred Guzerá female donors to in vitro embryo production may, in the long-term, have negative implications on the number of embryos obtained per cow and increase the relative costs of the improvement programmes based on this technology. High levels of inbreeding should be avoided when selecting Guzerá female donors and planning in vitro fertilization mating.

A mathematical model of in vivo bovine blastocyst developmental to gestational Day 15.

PubMed

Shorten, P R; Donnison, M; McDonald, R M; Meier, S; Ledgard, A M; Berg, D

2018-06-20

Bovine embryo growth involves a complex interaction between the developing embryo and the growth-promoting potential of the uterine environment. We have previously established links between embryonic factors (embryo stage, embryo gene expression), maternal factors (progesterone, body condition score), and embryonic growth to 8 d after bulk transfer of Day 7 in vitro-produced blastocysts. In this study we recovered blastocysts on Days 7 and 15 after artificial insemination to test the hypothesis that in vivo and in vitro embryos follow a similar growth program. We conducted our study using 4 commercial farms and repeated our study over 2 yr (2014, 2015), with data available from 2 of the 4 farms in the second year. Morphological and gene expression measurements (196 candidate genes) of the Day 7 embryos were measured and the progesterone concentration of the cows were measured throughout the reproductive cycle as a reflection of the state of the uterine environment. These data were also used to assess the interaction between the uterine environment and the developing embryo and to examine how well Day 7 embryo stage can be predicted from the Day 7 gene expression profile. Progesterone was not a strong predictor of in vivo embryo growth to Day 15. This contrasts with a range of Day 7 embryo transfer studies which demonstrated that progesterone is a very good predictor of embryo growth to Day 15. Our analysis demonstrates that in vivo embryos are 3 times less sensitive to progesterone than in vitro-transferred embryos (up to Day 15). This highlights that caution must be applied when extrapolating the results of in vitro embryo transfer studies to the in vivo situation. The similar variance in measured and predicted (based on Day 15 length) Day 7 embryo stage indicate low stochastic perturbations for in vivo embryo growth (large stochastic growth effects would generate a significantly larger standard deviation in measured embryo length on Day 15). We also identified that Day 7 embryo stage could be predicted based on the Day 7 gene expression profile (58% overall success rate for classification of 5 embryo stages). Our analysis also associated genes with each developmental stage and demonstrates the high level of temporal regulation of genes that occurs during early embryonic development. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cellular damage suffered by equine embryos after exposure to cryoprotectants or cryopreservation by slow-freezing or vitrification.

PubMed

Hendriks, W K; Roelen, B A J; Colenbrander, B; Stout, T A E

2015-11-01

Equine embryos are cryopreserved by slow-freezing or vitrification. While small embryos (<300 μm) survive cryopreservation reasonably well, larger embryos do not. It is not clear if slow-freezing or vitrification is less damaging to horse embryos. To compare the type and extent of cellular damage suffered by small and large embryos during cryopreservation by slow-freezing vs. vitrification. Sixty-three Day 6.5-7 embryos were subdivided by size and assigned to one of 5 treatments: control, exposure to slow-freezing or vitrification cryoprotectants (CPs), and cryopreservation by either technique. After thawing/CP removal, embryos were stained with fluorescent stains for various parameters of cellular integrity, and assessed by multiphoton microscopy. Exposing large embryos to vitrification CPs resulted in more dead cells (6.8 ± 1.3%: 95% confidence interval [CI], 3.1-10.4%) than exposure to slow-freezing media (0.3 ± 0.1%; 95% CI 0.0-0.6%: P = 0.001). Cryopreservation by either technique induced cell death and cytoskeleton disruption. Vitrification of small embryos resulted in a higher proportion of cells with fragmented or condensed (apoptotic) nuclei (P = 0.002) than slow-freezing (6.7 ± 1.5%, 95% CI 3.0-10.4% vs. 5.0 ± 2.1%, 95% CI 4.0-14.0%). Slow-freezing resulted in a higher incidence of disintegrated embryos (P = 0.01) than vitrification. Mitochondrial activity was low in control embryos, and was not differentially affected by cryopreservation technique, whereas vitrification changed mitochondrial distribution from a homogenous crystalline pattern in control embryos to a heterogeneous granulated distribution in vitrified embryos (P = 0.05). Cryopreservation caused more cellular damage to large embryos than smaller ones. While vitrification is more practical, it is not advisable for large embryos due to a higher incidence of dead cells. The choice is less obvious for small embryos, as vitrification led to occasionally very high percentages of dead or damaged cells, but a lower incidence of embryo disintegration. Modifications that reduce the level of cellular damage induced by vitrification are required before it can be considered the method of choice for cryopreserving equine embryos. © 2014 EVJ Ltd.

9 CFR 98.11 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... Health Inspection Service of the United States Department of Agriculture. Collection of embryos. Embryos removed from a single donor dam in one operation. Embryo. The initial stages of development of an animal...

9 CFR 98.11 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... Health Inspection Service of the United States Department of Agriculture. Collection of embryos. Embryos removed from a single donor dam in one operation. Embryo. The initial stages of development of an animal...

9 CFR 98.11 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... Health Inspection Service of the United States Department of Agriculture. Collection of embryos. Embryos removed from a single donor dam in one operation. Embryo. The initial stages of development of an animal...

Transcriptome analysis of PCOS arrested 2-cell embryos.

PubMed

Lu, Cuiling; Chi, Hongbin; Wang, Yapeng; Feng, Xue; Wang, Lina; Huang, Shuo; Yan, Liying; Lin, Shengli; Liu, Ping; Qiao, Jie

2018-06-18

In an attempt to explore the early developmental arrest in embryos from polycystic ovarian syndrome (PCOS) patients, we sequenced the transcriptome profiles of PCOS arrested 2-cell embryos, non-PCOS arrested 2-cell embryos and non-arrested 2-cell embryos using single-cell RNA-Seq technique. Differential expression analysis was performed using the DEGSeq R package. Gene Ontology (GO) enrichment was analyzed using the GOseq R package. Data revealed 62 differentially expressed genes between non-PCOS arrested and PCOS arrested embryos and 2217 differentially expressed genes between PCOS arrested and non-arrested 2-cell embryos. A total of 49 differently expressed genes (DEGs) were annotated with GO terms in the up-regulated genes between PCOS arrested and non-PCOS arrested embryos after GO enrichment. A total of 29 DEGs were annotated with GO terms in the down-regulated genes between PCOS arrested and non-arrested 2-cell embryos after GO enrichment. These data can provide a reference for screening specific genes involved in the arrest of PCOS embryos.

Number of embryos for transfer following in-vitro fertilisation or intra-cytoplasmic sperm injection.

PubMed

Pandian, Z; Bhattacharya, S; Ozturk, O; Serour, G I; Templeton, A

2004-10-18

The traditional reliance on the transfer of multiple embryos during in vitro fertilisation (IVF) in order to maximise the chance of pregnancy, has resulted in increasing rates of multiple pregnancies. Women undergoing IVF had a 20 - fold increased risk of twins and 400 - fold increased risk of higher order pregnancies (Martin 1998). The maternal and perinatal morbidity and mortality as well as national health service costs associated with multiple pregnancies is significantly high in comparison with singleton births (Luke 1992; Callahan 1994; Goldfarb 1996). Single embryo transfer is now being considered as an effective means of reducing this iatrogenic complication. This systematic review evaluates the effectiveness of elective two embryo transfer in comparison with single and more than two embryo transfer following IVF and ICSI (intra cytoplasmic sperm injection) treatment. The aim of this review is to determine, whether in couples who undergo IVF/ICSI: (1) the elective transfer of two embryos improves the probability of livebirth compared with: (a) Single embryo transfer, (b) Three embryo transfer or (c) Four embryo transfer.(2) the elective transfer of three embryos improves the probability of livebirth compared with: (a) Single embryo transfer, or (b) Four embryo transfer, We searched the Cochrane Menstrual Disorders and Subfertility Group's trials register (searched June 2003), the Cochrane Central Register of Controlled Trials (The Cochrane Library, Issue 4, 2003), MEDLINE (1970 to 2003), EMBASE (1985 to 2003) and reference lists of articles. We also handsearched relevant conference proceedings and contacted researchers in the field. Only randomised controlled trials were included. Two reviewers independently assessed eligibility and quality of trials. We found no studies that compared a policy of transferring multiple embryos on one cycle versus a policy of cryo- preservation and transfer of a single embryo over multiple cycles. We also found no trials comparing transfer of two versus three embryos. Three small, poorly reported trials compared transfer of two versus one embryo in a single cycle, and one small, poorly reported trial compared transfer of two versus four embryos in a single cycle. The clinical pregnancy rate per woman/couple associated with two embryo transfer was significantly higher compared to single embryo transfer (OR 2.08, 95% CI 1.24 to 3.50; test for overall effect p = 0.006). The live birth rate per woman/couple associated with two embryo transfer was also significantly higher than that associated with single embryo transfer (OR 1.90, 95% CI 1.12 to 3.22, test for overall effect p=0.02). The multiple pregnancy rate was significantly lower in women who had single embryo transfer (OR 9.97, 95% CI 2.61 to 38.19; p = 0.0008). The effectiveness of double embryo transfer versus four embryo transfer was tested in a single trial. There was no statistically significant differences in the clinical pregnancy rate (OR 0.75, 95% CI 0.26 to 2.16; p=0.6), and multiple pregnancy rates (OR 0.44. 95% CI 0.10 to 1.97; p = 0.28) between the two groups. The livebirth rate in the four embryo transfer group was higher compared to the two embryo transfer group, but the results were not statistically significant (OR 0.35, 95% CI 0.11 to 1.05; p = 0.06). The results of this systematic review suggest that live birth and pregnancy rates following single embryo transfer are lower than those following double embryo transfer as are the chances of multiple pregnancy including twins. As such, it is unlikely that the conclusions are robust enough to catalyse a change in clinical practice. The studies included are limited by their small sample size, so that even large differences might be hidden. Cumulative livebirth rates are seldom reported. The data were inadequate to draw conclusions about single embryo transfer and first frozen single embryo transfer (1FZET) or subsequent single frozen embryo transfers. Until more evidence is available single embryo transfer may not be the preferred choice for all patients undergoing IVF/ICSI. Clinicians may need to individualise protocols for couples based on their risks of multiple pregnancy. A definitive pragmatic, large multi centre randomised controlled trial comparing single embryo versus double embryo transfer in terms of clinical and cost effectiveness as well as acceptability is required. The primary outcome measured should be cumulative livebirth per woman/couple.

Impact of PCOS on early embryo cleavage kinetics.

PubMed

Wissing, M L; Bjerge, M R; Olesen, A I G; Hoest, T; Mikkelsen, A L

2014-04-01

This study investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage and cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t₂, t₃, t₄, t₅, t₆, t₇, t₈, t(M), t(B)) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. Compared with controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown, t₂, t₃, t₄ and t₇ but not at t₅, t₆, t₈, t(M) or t(B). Embryos from hyperandrogenic PCOS women had developed slower from fertilization to the 8-cell stage compared with embryos from controls. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Array comparative genomic hybridization screening in IVF significantly reduces number of embryos available for cryopreservation

PubMed Central

Liu, Jiaen; Yang, Zhihong; Salem, Shala A; Rahil, Tayyab; Collins, Gary S; Liu, Xiaohong; Salem, Rifaat D

2012-01-01

Objective During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation. Methods First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization day 5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation. Results Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis). Conclusion While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation. PMID:22816070

Progestin implants can rescue demi-embryo pregnancies in goats: a case study.

PubMed

Beckett, D M; Oppenheim, S M; Moyer, A L; BonDurant, R H; Rowe, J D; Anderson, G B

1999-06-01

Survival after transfer of demi-embryos (i.e., half-embryos produced by embryo splitting) to recipients usually is lower than survival after transfer of intact embryos. Reduced survival after demi-embryo transfer could be due to loss of viability after splitting, failure of a viable demi-embryo to prevent corpus luteum (CL) regression in the recipient female, or a combination of factors. From a retrospective analysis of pregnancy and embryo survival rates after demi-embryo transfer in sheep and goats, we report the rescue of caprine demi-embryo pregnancies in which CL regression occurred at the end of diestrus despite the presence of a viable conceptus in the uterus with progestin implants. Day 5 or 6 morulae and blastocysts were flushed from superovulated ewes and does and split into demi-embryos of approximately equal halves. Demi-embryos were either transferred fresh to synchronized recipients of the homologous species or frozen in liquid nitrogen. Approximately half of the recipient does and ewes were treated with norgestomet implants on Day 10 of the embryo transfer cycle and again 2 wk later. Serum collected on Day 25 from recipients with implants was assayed for progesterone to determine if a CL of pregnancy had been maintained. Pregnancy was diagnosed by ultrasonography on Day 35 of gestation. Corpus luteum regression occurred despite the presence of a viable conceptus in the uterus in 6 of 55 progestin-treated caprine demi-embryo recipients and in 0 of 66 ovine demi-embryo recipients. Five of the caprine pregnancies were maintained to term with norgestomet implants and produced 5 live kids. The sixth fetus, which was carried by a progestin implant-treated 8-mo-old doeling, died at approximately 50 d of gestation. These results suggest that, at least in goats, some demi-embryos may provide inadequate signaling for maternal recognition of pregnancy, and such pregnancies can be rescued with progestin treatment to the doe.

Interaction of carboxylated CdSe/ZnS quantum dots with fish embryos: Towards understanding of nanoparticles toxicity.

PubMed

Rotomskis, Ričardas; Jurgelėnė, Živilė; Stankevičius, Mantas; Stankevičiūtė, Milda; Kazlauskienė, Nijolė; Jokšas, Kęstutis; Montvydienė, Danguolė; Kulvietis, Vytautas; Karabanovas, Vitalijus

2018-09-01

Due to colloidal instability even with protective coatings, nanoparticles tend to aggregate in complex environments and possibly interact with biota. In this study, visualization of quantum dots (QDs) interaction with rainbow trout (Oncorhynchus mykiss) embryos was performed. Studies on zebrafish (Danio rerio) and pearl gourami (Trichogaster leerii) embryos have shown that QDs interact with embryos in a general manner and their affects are independent on the type of the embryo. It was demonstrated that carboxylated CdSe/ZnS QDs (4 nM) were aggregating in accumulation media and formed agglomerates on the surface of fish embryos under 1-12 days incubation in deep-well water. Detailed analysis of QDs distribution on fish embryos surface and investigation of the penetration of QDs through embryo's membrane showed that the chorion protects embryos from the penetration through the chorion and the accumulation of nanoparticles inside the embryos. Confocal microscopy and spectroscopy studies on rainbow trout embryos demonstrated that QDs cause chorion damage, due to QDs aggregation on the surface of chorion, even the formation of the agglomerates at the outer part of the embryos and/or with the mucus were detected. Aggregation of QDs and formation of agglomerates on the outer part of the embryo's membrane caused the intervention of the aggregates to the chorion and even partially destroyed the embryo's chorion. The incorporation of QDs in chorion was confirmed by two methods: in living embryos from a 3D reconstruction view, and in slices of embryos from a histology view. The damage of chorion integrity might have adverse effects on embryonic development. Moreover, for the first time the toxic effect of QDs was separated from the heavy metal toxicity, which is most commonly discussed in the literature to the toxicity of the QDs. Copyright © 2018 Elsevier B.V. All rights reserved.

9 CFR 98.8 - Inspection.

Code of Federal Regulations, 2013 CFR

2013-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...

9 CFR 98.17 - Procedures.

Code of Federal Regulations, 2013 CFR

2013-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... donor dam is bred to produce embryos for importation to the United States, the donor dam must be housed at an embryo collection unit. (2) The donor dam must remain at the embryo collection unit until the...

9 CFR 98.8 - Inspection.

Code of Federal Regulations, 2012 CFR

2012-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...

9 CFR 98.8 - Inspection.

Code of Federal Regulations, 2011 CFR

2011-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...

9 CFR 98.17 - Procedures.

Code of Federal Regulations, 2012 CFR

2012-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... donor dam is bred to produce embryos for importation to the United States, the donor dam must be housed at an embryo collection unit. (2) The donor dam must remain at the embryo collection unit until the...

9 CFR 98.8 - Inspection.

Code of Federal Regulations, 2010 CFR

2010-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...

9 CFR 98.8 - Inspection.

Code of Federal Regulations, 2014 CFR

2014-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.8 Inspection. Any embryo offered for entry into the United States in accordance with this subpart and documents accompanying the embryo shall be subject to...

9 CFR 98.17 - Procedures.

Code of Federal Regulations, 2014 CFR

2014-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth... donor dam is bred to produce embryos for importation to the United States, the donor dam must be housed at an embryo collection unit. (2) The donor dam must remain at the embryo collection unit until the...

Feminists on the inalienability of human embryos.

PubMed

McLeod, Carolyn; Baylis, Francoise

2006-01-01

The feminist literature against the commodification of embryos in human embryo research includes an argument to the effect that embryos are "intimately connected" to persons, or morally inalienable from them. We explore why embryos might be inalienable to persons and why feminists might find this view appealing. But, ultimately, as feminists, we reject this view because it is inconsistent with full respect for women's reproductive autonomy and with a feminist conception of persons as relational, embodied beings. Overall, feminists should avoid claims about embryos' being inalienable to persons in arguments for or against the commodification of human embryos.

Laser confers less embryo exposure than acid tyrode for embryo biopsy in preimplantation genetic diagnosis cycles: a randomized study

PubMed Central

2011-01-01

We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome. PMID:21527004

Cryopreservation of embryos and oocytes in human assisted reproduction.

PubMed

Konc, János; Kanyó, Katalin; Kriston, Rita; Somoskői, Bence; Cseh, Sándor

2014-01-01

Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized.

Teratogenic and toxic effects of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.) P. Karst. (higher Basidiomycetes), on zebrafish embryo as model.

PubMed

Dulay, Rich Milton R; Kalaw, Sofronio P; Reyes, Renato G; Alfonso, Noel F; Eguchi, Fumio

2012-01-01

This paper highlights the teratogenic and toxic effects of Ganoderma lucidum (Lingzhi or Reishi mushroom) extract on zebrafish embryos. Hatchability, malformations, and lethality rate of zebrafish embryos were assessed to provide valuable information regarding the potential teratogenic activity of G. lucidum. Hatching was completed 48 h post treatment application (hpta) at 1% or lower concentrations of extract and embryo water. The hatching rate of embryos treated with 5% or higher concentrations was significantly lower (p> 0.05) than the control. Tail malformation was the most marked morphological abnormality in embryos at 72 hpta, which was obviously caused by 1% extract (55.56% tail malformation) and was observed in all embryos exposed to 5% of extract. Growth retardation was evident in embryos exposed to 5%, 10%, and 20%. However, lethal effect of extract of G. lucidum was dependent on dose and time of exposure. Mortality rates of embryos treated with 5% (44.44%) or higher concentrations of the extract was significantly higher (p > 0.05) than that of the control embryos at 72 hpta. These results suggest that G. lucidum extract has lethal and sub-lethal effects on zebrafish embryos.

Effects of growth hormone on the ultrastructure of bovine preimplantation embryos.

PubMed

Kölle, Sabine; Stojkovic, Miodrag; Reese, Sven; Reichenbach, Horst-Dieter; Wolf, Eckhard; Sinowatz, Fred

2004-07-01

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.

Cooling strategies for brazilian flounder Paralichthys orbignyanus embryos.

PubMed

Varela, A S; Cardoso, T F; Fernandes E Silva, E; Goularte, K L; Okamoto, M H; Sampaio, L A; Jardim, R D; Corcini, C D

Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). Control embryos were maintained at 23 degree C and treated embryos were cooled to 15 degree C, 10 degree C and 5 degree C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5 degree C for 30 min are recommended.

Is it time for a paradigm shift in understanding embryo selection?

PubMed

Gleicher, Norbert; Kushnir, Vitaly A; Barad, David H

2015-01-11

Embryo selection has been an integral feature of in vitro fertilization (IVF) almost since its inception. Since the advent of extended blastocyst stage embryo culture, and especially with increasing popularity of elective single embryo transfer (eSET), the concept of embryo selection has increasingly become a mainstay of routine IVF. We here, however, argue that embryo selection via blastocyst stage embryo transfer (BSET), as currently practiced, at best improves IVF outcomes only for a small minority of patients undergoing IVF cycles. For a large majority BSET is either ineffective or, indeed, may actually be harmful by decreasing IVF pregnancy chances. Overall, only a small minority of patients, thus, benefit from prolonged embryo culture, while BSET, as a tool to enhance IVF outcomes, is increasingly utilized as routine care in IVF for all patients. Since newer methods of embryo selection, like preimplantation genetic screening (PGS) and closed system embryo incubation with time-lapse photography are practically dependent on BSET, these concepts of embryo selection, currently increasingly adopted in mainstream IVF, require reconsideration. They, automatically, transfer the downsides of BSET, including decreases in IVF pregnancy chances in some patients, to these new procedures, and in addition raise serious questions about cost-effectiveness.

A Technique for Facile and Precise Transfer of Mouse Embryos

PubMed Central

Sarvari, Ali; Naderi, Mohammad Mehdi; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mehdi

2013-01-01

Background Successful Embryo Transfer (ET) technique is a fateful step of all efforts to achieve live births from in vitro produced embryos in assisted reproductive techniques or in knockout, transgenic or cloned animal projects. Small reproductive tract of mice and limitation of current techniques may not well satisfy the requirements for mass production of genetically modified mice. Genetic abnormalities of embryos, receptivity and uterine contractions, expulsion of embryos, blood, mucus or bacterial contamination on the transfer pipette tip, technical problems and even animal strain may affect embryo transfer outcome. Methods In this study, two techniques of embryo transfer in mice were compared. In conventional technique the oviduct wall was punctured with a 30-gauge needle and the loaded Pasteur pipette with embryos and medium was inserted into the hole. In new technique, embryos that were loaded in modified micropipette with minimal medium were transferred directly to the oviduct by manual piston micro-pump easily. Embryo viability was evaluated considering the percentage of live healthy newborns. Results Results of the two techniques were compared by t-test within the NPAR1WAY procedure of SAS software (ver. 9.2). The average live birth rates in the novel methods was significantly higher (42.4%) than the conventional method (21.7%, p<0.05). Conclusion In conclusion, using new embryo transfer technique improved birth rate by preventing embryos expulsion from the oviduct, saving time and easy transfer of embryos with minimum volume of medium. PMID:23626878

Short communication: expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos.

PubMed

Laporta, J; Driver, A; Khatib, H

2011-08-01

Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

PubMed

Kelley, Rebecca L; Gardner, David K

2017-05-01

Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P < 0.05). Reduction of media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P < 0.05), but not in 20% oxygen (55.2 ± 2.9 versus 57.1 ± 2.8). Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P < 0.05). Addition of embryo-conditioned media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P < 0.01). Single culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Cryotolerance of Day 2 or Day 6 in vitro produced ovine embryos after vitrification by Cryotop or Spatula methods.

PubMed

Dos Santos Neto, P C; Vilariño, M; Barrera, N; Cuadro, F; Crispo, M; Menchaca, A

2015-02-01

This study was conducted to evaluate the cryotolerance of in vitro produced ovine embryos submitted to vitrification at different developmental stages using two methods of minimum volume and rapid cooling rate. Embryos were vitrified at early stage (2 to 8-cells) on Day 2 or at advanced stage (morulae and blastocysts) on Day 6 after in vitro fertilization. Vitrification procedure consisted of the Cryotop (Day 2, n=165; Day 6, n=174) or the Spatula method (Day 2, n=165; Day 6, n=175). Non vitrified embryos were maintained in in vitro culture as a control group (n=408). Embryo survival was determined at 3h and 24h after warming, development and hatching rates were evaluated on Day 6 and Day 8 after fertilization, and total cell number was determined on expanded blastocysts. Embryo survival at 24h after warming increased as the developmental stage progressed (P<0.05) and was not affected by the vitrification method. The ability for hatching of survived embryos was not affected by the stage of the embryos at vitrification or by the vitrification method. Thus, the proportion of hatching from vitrified embryos was determined by the survival rate and was lower for Day 2 than Day 6 vitrified embryos. The percentage of blastocysts on Day 8 was lower for the embryos vitrified on Day 2 than Day 6 (P<0.05), and was lower for both days of vitrification than for non-vitrified embryos (P<0.05). No interaction of embryo stage by vitrification method was found (P=NS) and no significant difference was found in the blastocyst cell number among vitrified and non-vitrified embryos. In conclusion, both methods using minimum volume and ultra-rapid cooling rate allow acceptable survival and development rates in Day 2 and Day 6 in vitro produced embryos in sheep. Even though early stage embryos showed lower cryotolerance, those embryos that survive the vitrification-warming process show high development and hatching rates, similar to vitrification of morulae or blastocysts. Copyright © 2014 Elsevier Inc. All rights reserved.

Fertilization and early embryonic development in heifers and lactating cows in summer and lactating and dry cows in winter.

PubMed

Sartori, R; Sartor-Bergfelt, R; Mertens, S A; Guenther, J N; Parrish, J J; Wiltbank, M C

2002-11-01

Two experiments in two seasons evaluated fertilization rate and embryonic development in dairy cattle. Experiment 1 (summer) compared lactating Holstein cows (n = 27; 97.3 +/- 4.1 d postpartum [dppl; 40.0 +/- 1.5 kg milk/d) to nulliparous heifers (n = 28; 11 to 17 mo old). Experiment 2 (winter) compared lactating cows (n = 27; 46.4 +/- 1.6 dpp; 45.9 +/- 1.4 kg milk/d) to dry cows (n = 26). Inseminations based on estrus included combined semen from four high-fertility bulls. Embryos and oocytes recovered 5 d after ovulation were evaluated for fertilization, embryo quality (1 = excellent to 5 = degenerate), nuclei/embryo, and accessory sperm. In experiment 1, 21 embryos and 17 unfertilized oocytes (UFO) were recovered from lactating cows versus 32 embryos and no UFO from heifers (55% vs. 100% fertilization). Embryos from lactating cows had inferior quality scores (3.8 +/- 0.4 vs. 2.2 +/- 0.3), fewer nuclei/embryo (19.3 +/- 3.7 vs. 36.8 +/- 3.0) but more accessory sperm (37.3 +/- 5.8 vs. 22.4 +/- 5.5/embryo) than embryos from heifers. Sperm were attached to 80% of UFO (17.8 +/- 12.1 sperm/UFO). In experiment 2, lactating cows yielded 36 embryos and 5 UFO versus 34 embryos and 4 UFO from dry cows (87.8 vs. 89.5% fertilization). Embryo quality from lactating cows was inferior to dry cows (3.1 +/- 0.3 vs. 2.2 +/- 0.3), but embryos had similar numbers of nuclei (27.2 +/- 2.7 vs. 30.6 +/- 2.1) and accessory sperm (42.0 +/- 9.4 vs. 36.5 +/- 6.3). From 53% of the flushings from lactating cows and 28% from dry cows, only nonviable embryos were collected. Thus, embryos of lactating dairy cows were detectably inferior to embryos from nonlactating females as early as 5 d after ovulation, with a surprisingly high percentage of nonviable embryos. In addition, fertilization rate was reduced only in summer, apparently due to an effect of heat stress on the oocyte.

A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

PubMed

Zhao, Shuan; Liu, Zhen-Xing; Gao, Hui; Wu, Yi; Fang, Yuan; Wu, Shuai-Shuai; Li, Ming-Jie; Bai, Jia-Hua; Liu, Yan; Evans, Alexander; Zeng, Shen-Ming

2015-07-15

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation. Copyright © 2015 Elsevier Inc. All rights reserved.

PGS-FISH in reproductive medicine and perspective directions for improvement: a systematic review.

PubMed

Zamora, Sandra; Clavero, Ana; Gonzalvo, M Carmen; de Dios Luna Del Castillo, Juan; Roldán-Nofuentes, Jose Antonio; Mozas, Juan; Castilla, Jose Antonio

2011-08-01

Embryo selection can be carried out via morphological criteria or by using genetic studies based on Preimplantation Genetic Screening. In the present study, we evaluate the clinical validity of Preimplantation Genetic Screening with fluorescence in situ hybridization (PGS-FISH) compared with morphological embryo criteria. A systematic review was made of the bibliography, with the following goals: firstly, to determine the prevalence of embryo chromosome alteration in clinical situations in which the PGS-FISH technique has been used; secondly, to calculate the statistics of diagnostic efficiency (negative Likelihood Ratio), using 2 × 2 tables, derived from PGS-FISH. The results obtained were compared with those obtained from embryo morphology. We calculated the probability of transferring at least one chromosome-normal embryo when it was selected using either morphological criteria or PGS-FISH, and considered what diagnostic performance should be expected of an embryo selection test with respect to achieving greater clinical validity than that obtained from embryo morphology. After an embryo morphology selection that produced a negative result (normal morphology), the likelihood of embryo aneuploidies was found to range from a pre-test value of 65% (prevalence of embryo chromosome alteration registered in all the study groups) to a post-test value of 55% (Confidence interval: 50-61), while after PGS-FISH with a negative result (euploid), the post-test probability was 42% (Confidence interval: 35-49) (p < 0.05). The probability of transferring at least one euploid embryo was the same whether 3 embryos were selected according to morphological criteria or whether 2, selected by PGS-FISH, were transferred. Any embryo selection test, if it is to provide greater clinical validity than embryo morphology, must present a LR-value of 0.40 (Confidence interval: 0.32-0.51) in single embryo transfer, and 0.06 (CI: 0.05-0.07) in double embryo transfer. With currently available technology, and taking into account the number of embryos to be transferred, the clinical validity of PGS-FISH, although superior to that of morphological criteria, does not appear to be clinically relevant.

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo

2014-02-21

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesusmore » monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.« less

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation.

PubMed

Yang, Feikun; Hao, Ru; Kessler, Barbara; Brem, Gottfried; Wolf, Eckhard; Zakhartchenko, Valeri

2007-01-01

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

Digital Microfluidic Dynamic Culture of Mammalian Embryos on an Electrowetting on Dielectric (EWOD) Chip

PubMed Central

Huang, Hong-Yuan; Shen, Hsien-Hua; Tien, Chang-Hung; Li, Chin-Jung; Fan, Shih-Kang; Liu, Cheng-Hsien; Hsu, Wen-Syang; Yao, Da-Jeng

2015-01-01

Current human fertilization in vitro (IVF) bypasses the female oviduct and manually inseminates, fertilizes and cultivates embryos in a static microdrop containing appropriate chemical compounds. A microfluidic microchannel system for IVF is considered to provide an improved in-vivo-mimicking environment to enhance the development in a culture system for an embryo before implantation. We demonstrate a novel digitalized microfluidic device powered with electrowetting on a dielectric (EWOD) to culture an embryo in vitro in a single droplet in a microfluidic environment to mimic the environment in vivo for development of the embryo and to culture the embryos with good development and live births. Our results show that the dynamic culture powered with EWOD can manipulate a single droplet containing one mouse embryo and culture to the blastocyst stage. The rate of embryo cleavage to a hatching blastocyst with a dynamic culture is significantly greater than that with a traditional static culture (p<0.05). The EWOD chip enhances the culture of mouse embryos in a dynamic environment. To test the reproductive outcome of the embryos collected from an EWOD chip as a culture system, we transferred embryos to pseudo-pregnant female mice and produced live births. These results demonstrate that an EWOD-based microfluidic device is capable of culturing mammalian embryos in a microfluidic biological manner, presaging future clinical application. PMID:25933003

Digital Microfluidic Dynamic Culture of Mammalian Embryos on an Electrowetting on Dielectric (EWOD) Chip.

PubMed

Huang, Hong-Yuan; Shen, Hsien-Hua; Tien, Chang-Hung; Li, Chin-Jung; Fan, Shih-Kang; Liu, Cheng-Hsien; Hsu, Wen-Syang; Yao, Da-Jeng

2015-01-01

Current human fertilization in vitro (IVF) bypasses the female oviduct and manually inseminates, fertilizes and cultivates embryos in a static microdrop containing appropriate chemical compounds. A microfluidic microchannel system for IVF is considered to provide an improved in-vivo-mimicking environment to enhance the development in a culture system for an embryo before implantation. We demonstrate a novel digitalized microfluidic device powered with electrowetting on a dielectric (EWOD) to culture an embryo in vitro in a single droplet in a microfluidic environment to mimic the environment in vivo for development of the embryo and to culture the embryos with good development and live births. Our results show that the dynamic culture powered with EWOD can manipulate a single droplet containing one mouse embryo and culture to the blastocyst stage. The rate of embryo cleavage to a hatching blastocyst with a dynamic culture is significantly greater than that with a traditional static culture (p<0.05). The EWOD chip enhances the culture of mouse embryos in a dynamic environment. To test the reproductive outcome of the embryos collected from an EWOD chip as a culture system, we transferred embryos to pseudo-pregnant female mice and produced live births. These results demonstrate that an EWOD-based microfluidic device is capable of culturing mammalian embryos in a microfluidic biological manner, presaging future clinical application.

Spatial distribution of the capacity to initiate a secondary embryo in the 32-cell embryo of Xenopus laevis.

PubMed

Kageura, H

1990-12-01

To examine the spatial distribution of dorsal determinants in the early embryos of Xenopus laevis, individual cells from the 32-cell embryo were transplanted into the same tier of the ventral side of a synchronous recipient. Their abilities to initiate a secondary embryo were measured by the incidence of secondary embryos and by the length of the secondary axis relative to the primary embryo. The ability was found to be localized in all cells (A1, B1, C1, and D1) of the dorsal most column and in the vegetal cells (C2 and D2) of the dorsolateral column. Transplanted C1 (subequatorial) cells caused the highest incidence of a secondary embryo and the average relative length of the secondary embryo was also greatest. Effectiveness decreased in the order: D1, B1, D2, C2, and A1. When these results were compared with Dale and Slack's fate map of the 32-cell embryo, it was concluded that the distribution of dorsal determinants is unique and does not coincide with the prospective regions for any tissues, though it is somewhat similar to the prospective region of dorsal endoderm or notochord. From these results it seems that dorsal determinants do not determine a particular tissue in an embryo but rather the "dorsal" region of an embryo.

Selection of Norway spruce somatic embryos by computer vision

NASA Astrophysics Data System (ADS)

Hamalainen, Jari J.; Jokinen, Kari J.

1993-05-01

A computer vision system was developed for the classification of plant somatic embryos. The embryos are in a Petri dish that is transferred with constant speed and they are recognized as they pass a line scan camera. A classification algorithm needs to be installed for every plant species. This paper describes an algorithm for the recognition of Norway spruce (Picea abies) embryos. A short review of conifer micropropagation by somatic embryogenesis is also given. The recognition algorithm is based on features calculated from the boundary of the object. Only part of the boundary corresponding to the developing cotyledons (2 - 15) and the straight sides of the embryo are used for recognition. An index of the length of the cotyledons describes the developmental stage of the embryo. The testing set for classifier performance consisted of 118 embryos and 478 nonembryos. With the classification tolerances chosen 69% of the objects classified as embryos by a human classifier were selected and 31$% rejected. Less than 1% of the nonembryos were classified as embryos. The basic features developed can probably be easily adapted for the recognition of other conifer somatic embryos.

Elucidating the origin of chromosomal aberrations in IVF embryos by preimplantation genetic analysis.

PubMed

Frumkin, Tsvia; Malcov, Mira; Yaron, Yuval; Ben-Yosef, Dalit

2008-01-30

Preimplantation genetic screening (PGS) has been proposed as a method for improving success rates in patients with repeated IVF failures. This approach is based on the hypothesis that such failures are the result of aneuploid embryos. It has been suggested that FISH analysis of blastomeres removed from preimplantation embryos represent the chromosomal constitution of the entire embryo. However, it is not yet clear whether it also represents the chromosomal constitution of the implanted embryo. PGS reanalysis on day 5 of embryos designated as "aneuploid" on day 3 may demonstrate a high rate of mosaicism for chromosomal aberration. Some of these mosaic embryos are capable of developing into normal embryos by "self-correction". Others, however, may accumulate additional chromosomal anomalies. It is therefore concluded that the chromosomal constitution of a preimplantation embryo may evolve during early cleavages. Meiotic and post zygotic mitotic errors may account for these chromosomal aberrations. This review will focus on elucidating the origin of chromosomal changes during preimplantation embryo development by studying their chromosomal constitution at different stages.

Trichostatin A (TSA) improves the development of rabbit-rabbit intraspecies cloned embryos, but not rabbit-human interspecies cloned embryos.

PubMed

Shi, Li-Hong; Miao, Yi-Liang; Ouyang, Ying-Chun; Huang, Jun-Cheng; Lei, Zi-Li; Yang, Ji-Wen; Han, Zhi-Ming; Song, Xiang-Fen; Sun, Qing-Yuan; Chen, Da-Yuan

2008-03-01

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development. (c) 2008 Wiley-Liss, Inc.

Fluorescence-based visualization of autophagic activity predicts mouse embryo viability

NASA Astrophysics Data System (ADS)

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Kito, Seiji; Minami, Naojiro; Kubota, Toshiro; Sato, Ken; Kokubo, Toshiaki

2014-03-01

Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality.

Assessing embryo development using swept source optical coherence tomography

NASA Astrophysics Data System (ADS)

Caujolle, S.; Cernat, R.; Silvestri, G.; Marques, M. J.; Bradu, A.; Feuchter, T.; Robinson, G.; Griffin, D.; Podoleanu, A.

2018-03-01

A detailed assessment of embryo development would assist biologists with selecting the most suitable embryos for transfer leading to higher pregnancy rates. Currently, only low resolution microscopy is employed to perform this assessment. Although this method delivers some information on the embryo surface morphology, no specific details are shown related to its inner structure. Using a Master-Slave Swept-Source Optical Coherence Tomography (SS-OCT), images of bovine embryos from day 7 after fertilization were collected from different depths. The dynamic changes inside the embryos were examined, in detail and in real-time from several depths. To prove our ability to characterize the morphology, a single embryo was imaged over 26 hours. The embryo was deprived of its life support environment, leading to its death. Over this period, clear morphological changes were observed.

Clinical significance of intercellular contact at the four-cell stage of human embryos, and the use of abnormal cleavage patterns to identify embryos with low implantation potential: a time-lapse study.

PubMed

Liu, Yanhe; Chapple, Vincent; Feenan, Katie; Roberts, Peter; Matson, Phillip

2015-06-01

To investigate the clinical significance of intercellular contact point (ICCP) in four-cell stage human embryos and the effectiveness of morphology and abnormal cleavage patterns in identifying embryos with low implantation potential. Retrospective cohort study. Private IVF center. A total of 223 consecutive IVF and intracytoplasmic sperm injection treatment cycles, with all resulting embryos cultured in the Embryoscope, and a subset of 207 cycles analyzed for ICCP number where good-quality four-cell embryos were available on day 2 (n = 373 IVF and n = 392 intracytoplasmic sperm injection embryos). None. Morphologic score on day 3, embryo morphokinetic parameters, incidence of abnormal biological events, and known implantation results. Of 765 good-quality four-cell embryos, 89 (11.6%) failed to achieve six ICCPs; 166 of 765 (21.7%) initially had fewer than six ICCPs but were able to establish six ICCPs before subsequent division. Embryos with fewer than six ICCPs at the end of four-cell stage had a lower implantation rate (5.0% vs. 38.5%), with lower embryology performance in both conventional and morphokinetic assessments, compared with embryos achieving six ICCPs by the end of four-cell stage. Deselecting embryos with poor morphology, direct cleavage, reverse cleavage, and fewer than six ICCPs at the four-cell stage led to a significantly improved implantation rate (33.6% vs. 22.4%). Embryos with fewer than six ICCPs at the end of the four-cell stage show compromised subsequent development and reduced implantation potential. Deselection of embryos with poor morphology and abnormal cleavage revealed via time-lapse imaging could provide the basis of a qualitative algorithm for embryo selection. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yingying; State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080; Graduate University of Chinese Academy of Sciences, Beijing 100049

2010-07-02

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilizedmore » embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo.« less

Embryo apoptosis identification: Oocyte grade or cleavage stage?

PubMed Central

Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

2015-01-01

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm.

PubMed

Tian, Yongsheng; Chen, Zhangfan; Tang, Jiang; Duan, Huimin; Zhai, Jieming; Li, Bo; Ma, Wenhui; Liu, Jiangchun; Hou, Yunxia; Sun, Zhengxiang

2017-04-01

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16-22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16-22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16-22 somite stage embryos survived when cooled at -25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at -25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at -140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (-196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques. Copyright © 2017 Elsevier Inc. All rights reserved.

The legal status of embryos and implications for reproductive technologies and biotechnology research.

PubMed

Bowens, Krietta Kai

2006-01-01

The legal status of embryos in American law is changing. At present, most states do not afford embryos the same protections as a born person, but some states are attempting to change this standard. Granting embryos the same legal status as born human beings poses a significant problem for industries that work with embryos, especially fertility treatment facilities and scientists researching stem cell and gene therapy technologies. This paper describes the methods of defining embryos in American law, and discusses the implications of granting embryos the same rights as born persons for the reproductive technology and scientific research industries.

Successful pregnancies from vitrified embryos in the dromedary camel: Avoidance of a possible toxic effect of sucrose on embryos.

PubMed

Herrid, M; Billah, M; Skidmore, J A

2017-12-01

Successful embryo cryopreservation facilitates the wider application of assisted reproduction technologies and also provides a useful method for gene banking of valuable genetics. Unfortunately attempts to establish an effective cryopreservation protocol for camelid embryos have been unsuccessful. In the current study, a modified vitrification protocol with three steps was investigated, whereby embryos were exposed to solutions containing increasing amounts of glycerol and ethylene glycol for fixed time periods. Embryos were then loaded into an Open Pull Straw (OPS) and plunged directly into liquid nitrogen for storage. Three experiments were designed to investigate the effect of 1) artificial shrinkage (AS) of embryos, 2) the addition of sucrose to the vitrification solutions, and 3) the replacement of sucrose by galactose in the warming solution, on the outcome of vitrification. The results showed that neither AS of hatched embryos prior to vitrification, nor the addition of sucrose into vitrification solutions improves the outcome of vitrification, while replacement of sucrose with galactose in warming solution increases the survival and developmental rates of vitrified embryos in culture. Transfer of vitrified embryos that were warmed in galactose resulted in a pregnancy rate of 42.8% per embryo or 46.1% per recipient. Collectively, these results suggest a possible species-specific toxic effect of sucrose on camel embryos, and that avoiding its use either in vitrification or warming solution is critical for establishing an effective protocol. This study may also be applicable to the vitrification of embryos of other camelid species including alpaca and llamas. Copyright © 2017 Elsevier B.V. All rights reserved.

Current status of in vitro embryo production in sheep and goats.

PubMed

Paramio, M-T; Izquierdo, D

2014-10-01

Sheep and goat production is an important economic activity in Spain with an increasing interest in milk production. Multiovulation and Embryo Transfer (MOET) and In vitro Embryo Production (IVEP) are assisted reproductive technologies aimed at increasing the genetic diffusion of females. In vitro embryo production is a multi-step methodology comprising the following procedures: (i) In vitro Maturation (IVM) of oocytes recovered directly from the follicles, (ii) In vitro Fertilization (IVF) or co-incubation of capacitated spermatozoa with in vitro matured oocytes and (iii) In vitro culture (IVC) of zygotes up to the blastocyst stage. In vitro embryo production from oocytes recovered from prepubertal females is called JIVET (Juvenile in vitro Embryo Transfer) and allows shortened generation intervals and increased genetic gain. Embryo production together with embryo cryoconservation would allow large-scale embryo marketing, a pathogen-free genetic movement and easier and cheaper germplasm commercial transactions. Commercial Embryo activity in small ruminants is low compared to cows in the European Union (data from the European Embryo Transfer Association) and in the world (data from the International Embryo Transfer Association). There is less IVEP research in small ruminants compared to other livestock species. The aim of this review was to provide an overview of the current status of IVEP of small ruminant with an emphasis on (i) description of the main methodologies currently used for IVM, IVF and IVC of embryos (ii) comparing procedures and outputs from JIVET and IVEP of adult females and (iii) the future research perspectives of this technology. © 2014 Blackwell Verlag GmbH.

[Effect of phytohemagglutinin (PHA) from Yunnan white kidney bean on development of mouse embryos].

PubMed

Zhang, Lifen; Wang, Changmei; Yang, Mingjie; Zhang, Tian; Wang, Minkang

2011-06-01

To study the effect of different concentration of phytohemagglutinin (PHA) on mouse embryo development. In experiment 1, crude and purified PHA extracted from Yunnan white kidney bean with different concentration were added into M16 culture medium, the final concentration of PHA were: 50, 100, 200, 500, 1 000, 2 000 and 5 000 mg x L(-1) respectively. 2-cell stage embryos were collected and cultured in PHA containing or control medium for 72-96 h and their development were recorded. In experiment 2, different stage of embryos from 1-cell to blastocyst were treated by different concentrations of PHA same as experiment 1 and 10 000 mg x L(-1) in culture medium for 24 h before washing and cultured in M16 + PVA without PHA to blastocyst or hatching blastocyst stage. Low concentrations PHA at 50-100 mg x L(-1) promoted embryo development and increased the number of blastocyst stage embryos. In contrast, high concentrations of PHA (> 1 000 mg x L(-1)) blocked the embryos development from 1-cell to blastocyst stage and showed apoptosis morphology or death. Depending on the concentrations, PHA from white kidney bean shown promotion or inhibition on mouse embryo development. 1-cell stage embryo shown more sensitive to PHA treatment than that of later stage embryos. Pretreatment 24 h in PHA containing medium can influence the further development of embryos. Low concentrations of PHA is benefit to embryo development, but high concentrations of PHA (> 1 000 mg x L(-1)) will block of the development of embryos.

Pre-persons, commodities or cyborgs: the legal construction and representation of the embryo.

PubMed

Fox, M

2000-01-01

This paper explores how embryos have been represented in law. It argues that two main models have underpinned legal discourse concerning the embryo. One discourse, which has become increasingly prevalent, views embryos as legal subjects or persons. Such representations are facilitated by technological developments such as ultrasound imaging. In addition to influencing Parliamentary debate prior to the passage of the Human Fertilisation and Embryology Act 1990, images of embryos as persons feature prominently in popular culture, including advertising and films, and this discourse came to the fore in the 'orphaned embryo' debate in 1996. The main opposing discourse dismisses embryos as commodifiable objects, which fits with a trend towards legal recognition that reproductive materials such as sperm may be classified as property which may be donated or sold. In the case of cryopreserved embryos these competing perspectives have resulted in litigation over the status of frozen embryos. In this paper I argue that it might be productive to shift the debate from this polarised dispute over whether embryos matter or not, whether they are pre-persons or commodities. Instead, I suggest that we should attempt to locate them in a biotechnological milieu, where cyborg metaphors may be utilised, and questions of how we should treat embryos would be contextualized alongside our response to other cyborgs.

Attitudes of couples towards the destination of surplus embryos: results among couples with cryopreserved embryos in Switzerland.

PubMed

Mohler-Kuo, Meichun; Zellweger, Ueli; Duran, Aysun; Hohl, Michael K; Gutzwiller, Felix; Mutsch, Margot

2009-08-01

The purpose of this study was to investigate attitudes towards the donation of surplus embryos among couples with cryopreserved embryos/zygotes, and to identify correlates associated with attitudes toward the destinations of surplus embryos/zygotes. Eleven of 19 Swiss in vitro fertilization (IVF) centers in existence in 2004 participated in the survey. Questionnaires were sent to 888 eligible couples; 458 men (52%) and 468 women (53%) returned them. Fifty-two percent of the participants supported the donation of surplus embryos to other couples, but divided opinions on the disclosure of biological parents' identities were identified. About 70% of participants indicated that donations of surplus embryos for medical research or therapy should be allowed, following strict regulations. Multiple logistic regression analyses revealed couples' position on the moral status of an embryo as the strongest predictor of attitudes toward all destinations of surplus embryos. Having children due to IVF/Intra-Cytoplasmic Sperm Injection (ICSI) treatment was negatively associated with attitudes towards donations to other couples. Perceived importance of religion, age >40, being a resident of the French-speaking region and unsuccessful IVF/ICSI treatment experiences were predictive of supporting donations for medical research. Swiss couples with cryopreserved embryos/zygotes are open to different options related to donating, rather than discarding, surplus embryos.

Transient Overexpression of adh8a Increases Allyl Alcohol Toxicity in Zebrafish Embryos

PubMed Central

Klüver, Nils; Ortmann, Julia; Paschke, Heidrun; Renner, Patrick; Ritter, Axel P.; Scholz, Stefan

2014-01-01

Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in metabolization. PMID:24594943

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.

PubMed

Flores, Luis E; Hildebrandt, Thomas B; Kühl, Anja A; Drews, Barbara

2014-05-10

Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9-11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert's membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart beat. Corresponding histology showed apoptotic cells in the embryo while the placenta was still intact. In the subsequent resorption process first the embryo and then its membranes disappeared. Our results provide a temporal time course of embryo resorption. With this method, animals exhibiting embryo resorption can be targeted, enabling the investigation of underlying mechanisms before the onset of total embryo disintegration.

Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation.

PubMed

Wessels, Cara; Penrose, Lindsay; Ahmad, Khaliq; Prien, Samuel

2017-11-01

Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15-20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P < 0.05). This suggests NEAT is successful in determining blastocysts viability in cryopreserved mice blastocysts. At a commercial ovine facility, NEAT was performed on fourteen frozen and thawed ovine blastocysts. Blastocysts of similar descent times were paired and transferred into recipient ewes as twins. Pregnancy was later confirmed by blood test and multiple gestation outcomes were determined at lambing. Six of seven recipient ewes were pregnant and all pregnant ewes delivered lambs without complication. Four ewes delivered twin lambs and two ewes delivered singletons, which totals ten of the fourteen (71%) blastocysts surviving to term. This pregnancy rate is comparable to expected to pregnancy rates in a commercial setting. The blastocysts which did not establish pregnancy demonstrated less buoyancy versus those blastocysts which established pregnancies which survived to term (P < 0.05). These results suggest NEAT can identify which blastocysts survive cryopreservation, thus significantly reduce the transfer of non-viable embryos. Further studies on a larger scale commercial setting will evaluate the efficacy of NEAT. Copyright © 2017 Elsevier Inc. All rights reserved.

Relationships between oxygen consumption rate, viability, and subsequent development of in vivo-derived porcine embryos.

PubMed

Sakagami, N; Nishida, K; Akiyama, K; Abe, H; Hoshi, H; Suzuki, C; Yoshioka, K

2015-01-01

Oxygen consumption rate of in vivo-derived porcine embryos was measured, and its value as an objective method for the assessment of embryo quality was evaluated. Embryos were surgically collected 5 or 6 days after artificial insemination (AI), and oxygen consumption rate of embryos was measured using an embryo respirometer. The average oxygen consumption rate (F × 10(14)/mol s(-1)) of the embryos that developed to the compacted morula stage on Day 5 (Day 0 = the day of artificial insemination) was 0.58 ± 0.03 (mean ± standard error of the mean). The Day-6 embryos had consumption rates of 0.56 ± 0.13, 0.87 ± 0.06, and 1.13 ± 0.07 at the early blastocyst, blastocyst, and expanded blastocyst stages, respectively, showing a gradual increase as the embryos developed. Just after collection, the average oxygen consumption rates of embryos that hatched and of those that did not hatch after culture were 0.60 ± 0.04 and 0.50 ± 0.04 for Day 5 (P = 0.08) and 1.05 ± 0.09 and 0.77 ± 0.05 for Day 6 (P < 0.05), respectively. The value and probability of discrimination by measuring the oxygen consumption rates of embryos to predict their hatching ability after culture were 0.56 and 63.6% for Day-5 embryos and 0.91 and 68.4% for Day-6 blastocysts, respectively. When Day-5 embryos were classified based on the oxygen consumption rate and then transferred non-surgically to recipient sows, three of the seven sows, to which embryos having a high oxygen consumption rate (≥ 0.59) were transferred, became pregnant and farrowed a total of 20 piglets. However, none of the four sows, to which embryos having low oxygen consumption rate (< 0.59) were transferred, became pregnant. These results suggest that the viability of in vivo-derived porcine embryos and subsequent development can be estimated by measuring the oxygen consumption rate. Copyright © 2015 Elsevier Inc. All rights reserved.

Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

PubMed

Kurosawa, Hiroki; Utsunomiya, Hiroki; Shiga, Naomi; Takahashi, Aiko; Ihara, Motomasa; Ishibashi, Masumi; Nishimoto, Mitsuo; Watanabe, Zen; Abe, Hiroyuki; Kumagai, Jin; Terada, Yukihiro; Igarashi, Hideki; Takahashi, Toshifumi; Fukui, Atsushi; Suganuma, Ryota; Tachibana, Masahito; Yaegashi, Nobuo

2016-10-01

Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality embryos by CERMs may enable the omission of long-term in vitro embryo culture to the blastocyst stage. CERMs is scalable technology that can be integrated into incubators and/or other embryo evaluation systems, such as the time-lapse systems, due to its chip-based architecture. Thus, CERMS would enable automatic measurement of oxygen consumption, under 5% CO2, in the near future, in order to reduce oxidative stress from exposure to atmospheric air. This study was supported by grants from the Health and Labor Sciences Research Grant (H24-Hisaichiiki-Shitei-016). The authors have no conflicts of interest. Not applicable. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

9 CFR 98.18 - Shipment of embryos to the United States.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Shipment of embryos to the United... IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.18 Shipment of embryos to the United States. (a) Release...

9 CFR 98.18 - Shipment of embryos to the United States.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Shipment of embryos to the United... IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.18 Shipment of embryos to the United States. (a) Release...

9 CFR 98.18 - Shipment of embryos to the United States.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Shipment of embryos to the United... IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.18 Shipment of embryos to the United States. (a) Release...

9 CFR 98.18 - Shipment of embryos to the United States.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Shipment of embryos to the United... IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.18 Shipment of embryos to the United States. (a) Release...

9 CFR 98.18 - Shipment of embryos to the United States.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Shipment of embryos to the United... IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.18 Shipment of embryos to the United States. (a) Release...

OpenSource lab-on-a-chip physiometer for accelerated zebrafish embryo biotests.

PubMed

Akagi, Jin; Hall, Chris J; Crosier, Kathryn E; Cooper, Jonathan M; Crosier, Philip S; Wlodkowic, Donald

2014-01-02

Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-time and/or time-lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post-analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real-time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype-based pharmaceutical screening. Copyright © 2014 John Wiley & Sons, Inc.

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium.

PubMed

Smith, D L; Krikorian, A D

1989-01-01

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

NASA Technical Reports Server (NTRS)

Smith, D. L.; Krikorian, A. D.

1989-01-01

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.

[Embryos and embryo-like entities: problem of definition in the draft of the Swiss embryonic research law].

PubMed

Bürgin, M T; Bürkli, P

2002-11-01

At the end of May 2002, the draft of the Swiss "Federal Act on Research on Surplus Embryos and Embryonic Stem Cells" (EFG, Embryonic Research Act) reached the pre-legislative consultation stage. Under certain conditions, it would allow research on "surplus" embryos from in-vitro fertilization, and the derivation of embryonic stem cells from surplus embryos for research purposes. The EFG draft defines an embryo as "the developing organism from the point of nuclear fusion until the completion of organ development". New technological developments show that embryo-like entities can also be created without nuclear fusion having taken place. It remains unclear how to treat embryonic entities that don't fall under the draft's narrow definition of an embryo. Expanding this definition would be a welcome improvement.

Tissue densities in developing avian embryos. [under acceleration stresses

NASA Technical Reports Server (NTRS)

Smith, A. H.; Abbott, U. K.; Morzenti, A.

1984-01-01

The density changes in the components of the incubated egg, the embryo, and the embryo's body parts were measured in the course of 21 days of incubation. In the first two-thirds of the incubation period there is a sequence of increasing density among egg contents: amniotic fluid, embryo, yolk, and albumin. As a result, the embryo is located at the bottom of the amniotic fluid, but at the top of the albumin. This position provides the embryo with mechanical protection and a proximity to the egg's air cell. The observed density changes and the asymmetry of these changes among various body parts of the embryo suggest a functional relationship. The density distributions among the body parts are particularly important in gravitational investigations of embryogenesis since they will produce forces tending to dislocate parts of the embryo.

Effect of sericin on preimplantation development of bovine embryos cultured individually.

PubMed

Isobe, T; Ikebata, Y; Onitsuka, T; Wittayarat, M; Sato, Y; Taniguchi, M; Otoi, T

2012-09-01

The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H(2)O(2)), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H(2)O(2). However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H(2)O(2). When embryos were exposed to 100 μm H(2)O(2) during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress. Copyright © 2012 Elsevier Inc. All rights reserved.

Influence of embryo handling and transfer method on pig cloning efficiency.

PubMed

Shi, Junsong; Zhou, Rong; Luo, Lvhua; Mai, Ranbiao; Zeng, Haiyu; He, Xiaoyan; Liu, Dewu; Zeng, Fang; Cai, Gengyuan; Ji, Hongmei; Tang, Fei; Wang, Qinglai; Wu, Zhenfang; Li, Zicong

2015-03-01

The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs. Copyright © 2015 Elsevier B.V. All rights reserved.

Exposure to high ambient temperatures alters embryology in rabbits

NASA Astrophysics Data System (ADS)

García, M. L.; Argente, M. J.

2017-09-01

High ambient temperatures are a determining factor in the deterioration of embryo quality and survival in mammals. The aim of this study was to evaluate the effect of heat stress on embryo development, embryonic size and size of the embryonic coats in rabbits. A total of 310 embryos from 33 females in thermal comfort zone and 264 embryos of 28 females in heat stress conditions were used in the experiment. The traits studied were ovulation rate, percentage of total embryos, percentage of normal embryos, embryo area, zona pellucida thickness and mucin coat thickness. Traits were measured at 24 and 48 h post-coitum (hpc); mucin coat thickness was only measured at 48 hpc. The embryos were classified as zygotes or two-cell embryos at 24 hpc, and 16-cells or early morulae at 48 hpc. The ovulation rate was one oocyte lower in heat stress conditions than in thermal comfort. Percentage of normal embryos was lower in heat stress conditions at 24 hpc (17.2%) and 48 hpc (13.2%). No differences in percentage of zygotes or two-cell embryos were found at 24 hpc. The embryo development and area was affected by heat stress at 48 hpc (10% higher percentage of 16-cells and 883 μm2 smaller, respectively). Zona pellucida was thicker under thermal stress at 24 hpc (1.2 μm) and 48 hpc (1.5 μm). No differences in mucin coat thickness were found. In conclusion, heat stress appears to alter embryology in rabbits.

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

PubMed

Skrzyszowska, M; Smorag, Z; Katska, L

1997-09-01

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (

Growth and metabolism of murine and bovine embryos in bovine uterine flushing-supplemented culture media.

PubMed Central

Rondeau, M; Guay, P; Goff, A K; Cooke, G M

1996-01-01

The aim of this study was to compare the development and metabolic activity of cultured murine and bovine embryos in 2 standard media (HAM F-10 and RPMI) in the presence or absence of bovine uterine flushings. Murine morulae (n = 653) and day 7 bovine embryos (n = 273) were cultured for 18 h or 36 h in either HAM F-10 or RPMI in the presence or absence of bovine uterine flushings. After culture, the development, quality, and metabolic activity (glucose utilization or methionine uptake and incorporation) of embryos was assessed. It was found that HAM F-10 (without uterine flushings) was a more suitable medium than RPMI for optimal development and metabolism of murine and bovine embryos. Poor quality and development, as well as decreased metabolism, were evident after culture of murine embryos in RPMI; in contrast, this medium had no adverse effects on bovine embryos in culture. Supplementation of HAM F-10 with bovine uterine flushings improved the growth of murine embryos and the protein synthesis (as measured by an increased methionine incorporation) for both murine and bovine embryos. However, supplementation with bovine uterine flushings could not overcome deficiencies of an inappropriate medium (RPMI) for murine embryos. Supplementation of a well-defined culture medium with uterine flushings increased metabolism of embryos in culture, and thus might help to increase pregnancy rates after transfer of such embryos to recipient cows. PMID:8825988

PGF₂α levels in Day 8 blood plasma are increased by the presence of one or more embryos in the uterus.

PubMed

Gomez, E; Martin, D; Carrocera, S; Muñoz, M

2015-08-01

In cattle, the detection of very early endometrial responses is considered to be hampered by the presence of only a single embryo. Therefore, we have previously developed a model of multiple embryo transfer to circumvent this hindrance. In this work, we analysed embryo-maternal interactions in the bovine uterus on day 8 of development while comparing the presence of multiple v. single embryos using embryo transfer and artificial insemination, respectively. Concentration of proteins (β-actin, NFkB, clusterin and immunoproteosome 20S β5i subunit-i20S), by western blot, and hexoses (glucose and fructose) were measured in paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus and were compared with UF obtained after artificial insemination. Prostaglandin (PG) F2 α and PGE2 concentrations were also analysed in blood plasma. The four proteins analysed and hexoses were unaffected by the presence of one or more embryos in the uterus. However, blood PGF2 α showed similar, significant increases with one or more embryos over cyclic animals; such changes were not observed in blood PGE2. Although multiple embryo transfer may appear to be non-physiological, we showed that the uterus, at the very early embryonic stages, does exhibit physiological reactions. Multiple embryo transfer can, therefore, be used for studies of very early embryo-maternal interactions in vivo in monotocous species.

Rethinking In Vitro Embryo Culture: New Developments in Culture Platforms and Potential to Improve Assisted Reproductive Technologies1

PubMed Central

Smith, Gary D.; Takayama, Shuichi; Swain, Jason E.

2011-01-01

ABSTRACT The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future. PMID:21998170

Can Chlamydia abortus be transmitted by embryo transfer in goats?

PubMed

Oseikria, M; Pellerin, J L; Rodolakis, A; Vorimore, F; Laroucau, K; Bruyas, J F; Roux, C; Michaud, S; Larrat, M; Fieni, F

2016-10-01

The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8-16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 × 10(7)Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 °C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at -20 °C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C. abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C. abortus would eliminate the bacteria from the ZP. Copyright © 2016 Elsevier Inc. All rights reserved.

Use of "excess" human embryos for stem cell research: protecting women's rights and health.

PubMed

Cohen, C B

2000-01-01

Proposed National Institutes of Health guidelines for stem cell research are too narrowly drawn and do not adequately protect the freedom of choice and health of women who donate embryos. They need to be expanded to cover not only the point of embryo donation, but also that of embryo creation. Guidelines are provided to ensure that donors undergoing hyperstimulation and egg retrieval gave voluntary informed consent to the production of embryos that might later prove in excess. A standard for determining when embryos have been overproduced is presented to address the possibility that additional embryos will be created for stem cell research in violation of the guidelines and at risk to women's health.

9 CFR 98.10a - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and...

9 CFR 85.10 - Interstate movement of swine semen and swine embryos for insemination of or implantation into swine.

Code of Federal Regulations, 2014 CFR

2014-01-01

... swine embryos for insemination of or implantation into swine. 85.10 Section 85.10 Animals and Animal... and swine embryos for insemination of or implantation into swine. Swine semen and swine embryos moved... collection of the semen or embryos or were members of a qualified pseudorabies negative herd, and had not...

9 CFR 98.21 - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.21 Embryos from sheep...

9 CFR 98.10a - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and...

10 CFR 835.206 - Limits for the embryo/fetus.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 4 2014-01-01 2014-01-01 false Limits for the embryo/fetus. 835.206 Section 835.206... Exposure § 835.206 Limits for the embryo/fetus. (a) The equivalent dose limit for the embryo/fetus from the... provided in § 835.206(a) shall be avoided. (c) If the equivalent dose to the embryo/fetus is determined to...

9 CFR 85.10 - Interstate movement of swine semen and swine embryos for insemination of or implantation into swine.

Code of Federal Regulations, 2013 CFR

2013-01-01

... swine embryos for insemination of or implantation into swine. 85.10 Section 85.10 Animals and Animal... and swine embryos for insemination of or implantation into swine. Swine semen and swine embryos moved... collection of the semen or embryos or were members of a qualified pseudorabies negative herd, and had not...

9 CFR 85.10 - Interstate movement of swine semen and swine embryos for insemination of or implantation into swine.

Code of Federal Regulations, 2012 CFR

2012-01-01

... swine embryos for insemination of or implantation into swine. 85.10 Section 85.10 Animals and Animal... and swine embryos for insemination of or implantation into swine. Swine semen and swine embryos moved... collection of the semen or embryos or were members of a qualified pseudorabies negative herd, and had not...

9 CFR 98.21 - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.21 Embryos from sheep...

9 CFR 98.21 - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.21 Embryos from sheep...

10 CFR 835.206 - Limits for the embryo/fetus.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 4 2011-01-01 2011-01-01 false Limits for the embryo/fetus. 835.206 Section 835.206... Exposure § 835.206 Limits for the embryo/fetus. (a) The equivalent dose limit for the embryo/fetus from the... provided in § 835.206(a) shall be avoided. (c) If the equivalent dose to the embryo/fetus is determined to...

10 CFR 835.206 - Limits for the embryo/fetus.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 4 2013-01-01 2013-01-01 false Limits for the embryo/fetus. 835.206 Section 835.206... Exposure § 835.206 Limits for the embryo/fetus. (a) The equivalent dose limit for the embryo/fetus from the... provided in § 835.206(a) shall be avoided. (c) If the equivalent dose to the embryo/fetus is determined to...

9 CFR 98.10a - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and...

9 CFR 98.21 - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.21 Embryos from sheep...

Competing Views of Embryos for the Twenty-First Century: Textbooks and Society

ERIC Educational Resources Information Center

Maienschein, Jane; Wellner, Karen

2013-01-01

It might seem that an embryo is an embryo, and that there would be a fact of the matter. That seems especially true with respect to the way embryos are presented in textbooks, including high school biology textbooks. This paper looks at three co-existing, competing, and often conflicting views of embryos. Then with a close study of twentieth…

9 CFR 98.21 - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos From Regions Where Rinderpest or Foot-and-Mouth Disease Exists § 98.21 Embryos from sheep...

10 CFR 835.206 - Limits for the embryo/fetus.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 4 2012-01-01 2012-01-01 false Limits for the embryo/fetus. 835.206 Section 835.206... Exposure § 835.206 Limits for the embryo/fetus. (a) The equivalent dose limit for the embryo/fetus from the... provided in § 835.206(a) shall be avoided. (c) If the equivalent dose to the embryo/fetus is determined to...

9 CFR 98.10a - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and...

9 CFR 98.10a - Embryos from sheep in regions other than Australia, Canada, and New Zealand.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Embryos from sheep in regions other... (INCLUDING POULTRY) AND ANIMAL PRODUCTS IMPORTATION OF CERTAIN ANIMAL EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and...

10 CFR 835.206 - Limits for the embryo/fetus.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false Limits for the embryo/fetus. 835.206 Section 835.206... Exposure § 835.206 Limits for the embryo/fetus. (a) The equivalent dose limit for the embryo/fetus from the... provided in § 835.206(a) shall be avoided. (c) If the equivalent dose to the embryo/fetus is determined to...

Death in the clinic: women's perceptions and experiences of discarding supernumerary IVF embryos.

PubMed

de Lacey, Sheryl

2017-03-01

Perspectives on the status of human embryos and whether they should be discarded differ globally. Some countries protect embryos in law while in other countries embryos 'die' or 'succumb' in assisted reproductive technology clinics on a daily basis. This study analyses interview data drawn from a larger qualitative study conducted in South Australia from 2004-2007. 21 women and 12 of 21 partners were interviewed about the decision they made to discard their embryos. The analysis reported here sought to examine the ways in which women constructed and experienced the decision to discard embryos. The article highlights the ways in which embryo discard is a contested discursive space. Embryo death is sequestered through their confinement in the laboratory and their invisibility to the naked eye. The clinic treated embryo discard as disposal of biological waste and failed to acknowledge the meaning of the event. By contrast women experienced emotional bereavement described as similar to early pregnancy loss, and described experiences of attachment and grief. For sensitive and compassionate care these differences in perceptions of embryo discard need to be addressed. © 2016 Foundation for the Sociology of Health & Illness.

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential.

PubMed

Pudakalakatti, Shivanand M; Uppangala, Shubhashree; D'Souza, Fiona; Kalthur, Guruprasad; Kumar, Pratap; Adiga, Satish Kumar; Atreya, Hanudatta S

2013-01-01

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5) ) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright © 2012 John Wiley & Sons, Ltd.

Critical reappraisal of embryo quality as a predictive parameter for pregnancy outcome: a pilot study.

PubMed

Campo, R; Binda, M M; Van Kerkhoven, G; Frederickx, V; Serneels, A; Roziers, P; Lopes, A S; Gordts, S; Puttemans, P; Gordts, S

2010-01-01

Pilot study to analyse the efficacy and embryo morphology using a new human embryo culture medium (GM501) versus the conventional used medium (ISM1). Over a four-month period, all patients at the Leuven Institute of Fertility and Embryology (LIFE) were -randomly allocated to have their embryos cultured in either the standard sequential culture medium ISM1 (control) or in a new universal medium (GM501) (study group). Primary outcome parameters were clinical pregnancy and live birth rate. The secondary outcome parameter was the correlation of embryo fragmentation rate with pregnancy outcome. We did not observe any differences between the ISM1 control group and GM501 study group with regard to fertilization, pregnancy, implantation rates, ongoing pregnancy, and babies born. The number of embryos with a minimal fragmentation rate (less than 30%) was significantly higher in the GM501 study group. Although a significant higher embryo fragmentation rate was seen in In vitro culture of embryos in GM501, pregnancy outcome results were comparable to those of embryos cultured in ISM1. According to our results the value of embryo morphological criteria as a parameter for pregnancy outcome should be examined and discussed again.

Preimplantation death of xenomitochondrial mouse embryo harbouring bovine mitochondria

PubMed Central

Kawahara, Manabu; Koyama, Shiori; Iimura, Satomi; Yamazaki, Wataru; Tanaka, Aiko; Kohri, Nanami; Sasaki, Keisuke; Takahashi, Masashi

2015-01-01

Mitochondria, cellular organelles playing essential roles in eukaryotic cell metabolism, are thought to have evolved from bacteria. The organization of mtDNA is remarkably uniform across species, reflecting its vital and conserved role in oxidative phosphorylation (OXPHOS). Our objectives were to evaluate the compatibility of xenogeneic mitochondria in the development of preimplantation embryos in mammals. Mouse embryos harbouring bovine mitochondria (mtB-M embryos) were prepared by the cell-fusion technique employing the haemagglutinating virus of Japan (HVJ). The mtB-M embryos showed developmental delay at embryonic days (E) 3.5 after insemination. Furthermore, none of the mtB-M embryos could implant into the maternal uterus after embryo transfer, whereas control mouse embryos into which mitochondria from another mouse had been transferred developed as well as did non-manipulated embryos. When we performed quantitative PCR (qPCR) of mouse and bovine ND5, we found that the mtB-M embryos contained 8.3% of bovine mitochondria at the blastocyst stage. Thus, contamination with mitochondria from another species induces embryonic lethality prior to implantation into the maternal uterus. The heteroplasmic state of these xenogeneic mitochondria could have detrimental effects on preimplantation development, leading to preservation of species-specific mitochondrial integrity in mammals. PMID:26416548

Administration of the nonsteroidal anti-inflammatory drug tolfenamic acid at embryo transfer improves maintenance of pregnancy and embryo survival in recipient mice.

PubMed

Schlapp, Geraldine; Goyeneche, Lucía; Fernández, Gabriel; Menchaca, Alejo; Crispo, Martina

2015-02-01

To evaluate the effect of the nonsteroidal anti-inflammatory drugs tolfenamic acid and flunixin meglumine in pregnancy rate and embryo survival of recipient mice subjected to embryo transfer. A total of 142 recipient females were transferred with 2,931 embryos and treated with a single injection of tolfenamic acid (1 mg/kg; n = 54 females with 1,129 embryos), flunixin meglumine (2.5 mg/kg; n = 46 females with 942 embryos), or bi-distilled water (10 mL/kg) as control group (n = 42 females with 860 embryos). Pregnancy was checked 2 weeks after embryo transfer, delivery was registered on the due date, and litter size was recorded on Day 7 after birth. Pregnancy rate of tolfenamic acid treated females was significantly higher than flunixin group (P < 0.05) and showed a tendency to be higher when compared to the control group (P = 0.06). The number of pups born from transferred embryos in pregnant females was significantly higher for both treatment groups compared to controls (P < 0.05). Number of pups from total transferred embryos was higher for both treatment groups (P < 0.05) when compared to controls. The use of tolfenamic acid at the time of embryo transfer improves both pregnancy rate and number of live pups in recipient mice, with optimal effects observed with flunixin meglumine. We suggest that the use of tolfenamic acid has beneficial effects on the maintenance of pregnancy and embryo survival in recipient mice, which should be taken into account for further studies in other mammalian females.

Quality of preimplantation embryos recovered in vivo from dairy cows in relation to their body condition.

PubMed

Makarevich, A V; Stádník, L; Kubovičová, E; Hegedüšová, Z; Holásek, R; Louda, F; Beran, J; Nejdlová, M

2016-06-01

This study examined the impact of cow body condition on the quality of bovine preimplantation embryos. The embryos (n = 107) were flushed from dairy cows and classified according to a five-point scale body condition score (BCS2 n = 17; BCS3 n = 31; BCS4 n = 11) on the 7th day after insemination and then analyzed for development, dead cell index (DCI), cell number and actin cytoskeleton quality. The highest embryo recovery rate (P < 0.05) was recorded in the BCS3 group and the lowest in the BCS4 group. More transferable (morula, blastocyst) embryos were obtained from the BCS4 cows (79%), compared with the BCS2 (64%) or BCS3 (63%) animals. However, cell numbers were higher in the BCS2 and BCS3 groups (P < 0.05) compared with the BCS4 embryos. Conversely, the DCI was lowest in the BCS2 (3.88%; P < 0.05) and highest in the BCS4 (6.56%) embryos. The proportion of embryos with the best actin quality (grade I) was higher in the BCS2 and BCS3 cows compared with the BCS4 group. Almost 25% of all embryos showed fragmented morphology and a higher DCI (5.65%) than normal morulas (1.76%). More fragmented embryos were revealed in the BCS2 (28.6%) and BCS4 (31.25%) groups, and less (19.15%) in the BCS3 group. The cell numbers in such embryos were lower in the BCS4 (22.57) than in the BCS2 (46.25) or BCS3 (42.4) groups. In conclusion, the body condition of dairy cows affects the quality of preimplantation embryos. A BCS over 3.0 resulted in a higher incidence of poor (fragmented) embryos.

Methanol as a cryoprotectant for equine embryos.

PubMed

Bass, L D; Denniston, D J; Maclellan, L J; McCue, P M; Seidel, G E; Squires, E L

2004-09-15

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.

Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

PubMed Central

Akagi, Jin; Khoshmanesh, Khashayar; Evans, Barbara; Hall, Chris J.; Crosier, Kathryn E.; Cooper, Jonathan M.; Crosier, Philip S.; Wlodkowic, Donald

2012-01-01

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale. PMID:22606275

Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

PubMed Central

Majidi Gharenaz, Nasrin; Movahedin, Mansoureh; Mazaheri, Zohreh; Pour beiranvand, Shahram

2016-01-01

Background: Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. Objective: The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. Materials and Methods: In this experimental study, 8 cell embryos (n=240) were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80), vitrified at 8 cell stage (n=80), vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80). Embryos were vitrified by using cryolock, (open system) described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Results: Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03). In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004), however expression of Bax and Bcl-2 (apoptotic) genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003), but it was similar between re-vitrified and vitrified embryos. Conclusion: Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage. PMID:27679826

The relevance and use of mouse embryo bioassays for quality control in an assisted reproductive technology program.

PubMed

Scott, L F; Sundaram, S G; Smith, S

1993-09-01

To define both the limits of a mouse embryo bioassay for quality control in an assisted reproductive technology (ART) program and the areas where it can be effectively used. Embryos at the pronuclear and two-cell stage from three different strains of mice were used to assess the effectiveness of this assay for media quality control using five different media routinely used in ART. Pronuclear and two-cell embryos from CD-1 mice were used to test the ability of a mouse embryo bioassay to control for water quality, contaminants in the culture system, and fluctuations in the environmental conditions using a medium, culture system, and scoring technique that were optimized for this strain. The mouse embryo bioassay is not effective in differentiating media appropriate for supporting human embryo development since the development of mouse embryos in vitro is strain, stage, and media related. However, CD-1 embryos were shown to be sensitive to variations in water quality, pH, temperature, incubator conditions, and contaminants in the system when grown in a protein-free medium optimized for their development. Both total blastocyst number and the cell count in the blastocysts were affected. Pronuclear embryos were more sensitive to perturbations in the culture system than two-cell embryos. A mouse embryo bioassay can be effectively used as a means of quality control of water, chemicals, and contact materials and for technique standardization and training in an assisted reproduction program. All the conditions of the test should be defined, pronuclear embryos should be used, and the end point should be fully expanded blastocysts and/or cell numbers in these blastocysts where appropriate.

Use of the Coelomic Grafting Technique for Prolonged ex utero Cultivation of Late Preprimitive Streak-Stage Rabbit Embryos.

PubMed

Püschel, Bernd; Männer, Jörg

2016-01-01

Due to its morphological similarity with the early human embryo, the pregastrulation-stage rabbit may represent an appropriate mammalian model for studying processes involved in early human development. The usability of mammalian embryos for experimental studies depends on the availability of whole embryo culture methods facilitating prolonged ex utero development. While currently used culture methods yield high success rates for embryos from primitive streak stages onward, the success rate of extended cultivation of preprimitive streak-stage mammalian embryos is low for all previously established methods and for all studied species. This limits the usability of preprimitive streak-stage rabbit embryos in experimental embryology. We have tested whether the extraembryonic coelom of 4-day-old chick embryos may be used for prolonged ex utero culture of preprimitive streak-stage rabbit embryos (stage 2, 6.2 days post coitum). We found that, within this environment, stage 2 rabbit blastocysts can be cultured at decreasing success rates (55% after 1 day, 35% after 2 days, 15% after 3 days) up to a maximum of 72 h. Grafted blastocysts can continue development from the onset of gastrulation to early organogenesis and thereby form all structures characterizing age-matched controls (e.g. neural tube, somites, beating heart). Compared to normal controls, successfully cultured embryos developed at a slower rate and finally showed some structural and gross morphological anomalies. The method presented here was originally developed for whole embryo culture of mouse embryos by Gluecksohn-Schoenheimer in 1941. It is a simple and inexpensive method that may represent a useful extension to presently available ex utero culture systems for rabbit embryos. © 2016 S. Karger AG, Basel.

The relationship between oxygen consumption rate and viability of in vivo-derived pig embryos vitrified by the micro volume air cooling method.

PubMed

Sakagami, N; Nishida, K; Misumi, K; Hirayama, Y; Yamashita, S; Hoshi, H; Misawa, H; Akiyama, K; Suzuki, C; Yoshioka, K

2016-01-01

The aim of this study was to assess the viability of vitrified-warmed in vivo-derived pig embryos after measuring the oxygen consumption rate. Six days after artificial insemination, blastocysts were collected from gilts and vitrified by the micro volume air cooling method. The oxygen consumption rate was measured in 60 vitrified-warmed embryos, which were then cultured for 48h to assess the viability. The survival (re-expansion) rate of embryos after warming was 85.0%. The average oxygen consumption rate of embryos immediately after warming was greater in embryos which could re-expand during subsequent culture (F=0.75±0.04) than that in those which failed to re-expand (F=0.33±0.05). Moreover, the oxygen consumption rate of vitrified-warmed embryos was greater in the hatched (F=0.88±0.06) than that in the not-hatched group (F=0.53±0.04). When the oxygen consumption rate of the vitrified-warmed embryos and the numbers of viable and dead cells in embryos were determined, there was a positive correlation between the oxygen consumption rate and the number of live cells (P<0.01, r=0.538). A total of 29 vitrified embryos after warming and measuring the oxygen consumption rate were surgically transferred into uterine horns of two recipients. Both of the recipients become pregnant and farrowed 12 healthy piglets. These results demonstrate that the oxygen consumption rate of vitrified-warmed pig embryos can be related to the number of live cells and that the measurement of oxygen consumption of embryos after cryopreservation may be useful for estimating embryo survivability. Copyright © 2015 Elsevier B.V. All rights reserved.

Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423.

PubMed

Liu, Beibei; Su, Shengzhong; Wu, Ying; Li, Ying; Shan, Xiaohui; Li, Shipeng; Liu, Hongkui; Dong, Haixiao; Ding, Meiqi; Han, Junyou; Yuan, Yaping

2015-07-01

Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Is cryopreservation of embryos a legitimate surrogate marker of embryo quality in studies of assisted reproductive technology conducted using national databases?

PubMed

Stern, Judy E; Lieberman, Ellice S; Macaluso, Maurizio; Racowsky, Catherine

2012-04-01

To investigate whether cryopreservation of supernumerary embryos is a good surrogate for embryo quality. Retrospective study of 6,859 assisted reproductive technology (ART) cycles from women aged <35 years with two fresh day 3 embryos transferred. National Society for Assisted Reproductive Technology Clinic Outcome Reporting System data from 2006-2008. Women undergoing ART. None. Embryo quality (good, fair, or poor), cell number, and live births were compared for cycles with and without cryopreservation, using χ(2) to evaluate statistical significance. The association of freezing with embryo quality was examined using multiple logistic regression after adjusting for confounders (patient age, oocyte yield, intracytoplasmic sperm injection [ICSI], assisted hatching, male factor infertility). Cycles with cryopreservation were more likely to have two embryos of good quality transferred (81.3% vs. 48.5%) and had more 8-cell embryos transferred (76.0% vs. 50.1%). Relative to cycles with two good embryos (good-good), the adjusted odds ratios (OR) for cryopreservation were: good-fair (OR = 0.301, 95% confidence interval [CI] = 0.257-0.354), fair-fair (OR = 0.308, 95% CI = 0.258-0.367), and any poor (OR = 0.058, 95% CI = 0.040-0.083). The live birth rate was 52.4% for cycles with freezing and 40.6% for cycles without. Embryo quality and cell number were both associated with embryo cryopreservation. However, although cryopreservation was a strong marker for good quality, not having cryopreservation did not reliably indicate poor quality, as almost half of those cycles had two good quality embryos. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

[Cryopreservation of mouse embryos in ethylene glycol-based solutions: a search for the optimal and simple protocols].

PubMed

Luo, Ming-Jiu; Liu, Na; Miao, De-Qiang; Lan, Guo-Cheng; Suo-Feng; Chang, Zhong-Le; Tan, Jing-He

2005-09-01

Although ethylene glycol (EG) has been widely used for embryo cryopreservation in domestic animals, few attempts were made to use this molecule to freeze mouse and human embryos. In the few studies that used EG for slow-freezing of mouse and human embryos, complicated protocols for human embryos were used, and the protocols need to be simplified. Besides, freezing mouse morula with EG as a cryoprotectant has not been reported. In this paper, we studied the effects of embryo stages, EG concentration, duration and procedure of equilibration, sucrose supplementation and EG removal after thawing on the development of thawed mouse embryos, using the simple freezing and thawing procedures for bovine embryos. The blastulation and hatching rates (81.92% +/- 2.24% and 68.56% +/- 2.43%, respectively) of the thawed late compact morulae were significantly (P < 0.05) higher than those of embryos frozen-thawed at other stages. When mouse late compact morulae were frozen with different concentrations of EG, the highest rates of blastocyst formation and hatching were obtained with 1.8mol/L EG. The blastulation rate was significantly higher when late morulae were equilibrated in 1.8 mol/L EG for 10 min prior to freezing than when they were equilibrated for 30 min, and the hatching rate of embryos exposed to EG for 10 min was significantly higher than that of embryos exposed for 20 and 30 min. Both rates of blastocyst formation and hatching obtained with two-step equilibration were higher (P < 0.05) than with one-step equilibration in 1.8 mol/L EG. Addition of sucrose to the EG-based solution had no beneficial effects. On the contrary, an increased sucrose level (0.4 mol/L) in the solution impaired the development of the frozen-thawed embryos. In contrast, addition of 0.1 mol/L sucrose to the propylene glycol (PG)-based solution significantly improved the development of the frozen-thawed embryos. Elimination of the cryoprotectant after thawing did not improve the development of the thawed embryos. The cell numbers were less (P < 0.05) in blastocysts developed from the thawed morulae than in the in vivo derived ones. In summary, embryo stage, EG concentration, duration and procedure of equilibration and sucrose supplementation had marked effects on development of the thawed mouse embryos, and a protocol for cryopreservation of mouse embryos is recommended in which the late morulae are frozen in 1.8 mol/L EG using the simple freezing and thawing procedures of bovine embryos after a two-step equilibration and the embryos can be cultured or transferred without EG removal after thawing.

Planar embryos have poor prognosis in terms of blastocyst formation and implantation.

PubMed

Ebner, T; Maurer, M; Shebl, O; Moser, M; Mayer, R B; Duba, H C; Tews, G

2012-09-01

Normally, day-2 embryos show a crosswise arrangement of four cells with three blastomeres lying side by side. Cleavage anomalies include embryos that are characterized by a particular planar constellation of four blastomeres with presumed incomplete cleavage. Since little is known on the developmental fate of such conceptuses, within a 10-month period all consecutive patients were screened for day-2 planar embryos. A total of 64/2070 embryos with suboptimal blastomere configuration were detected (3.1%). In conventional IVF, planar embryos were significantly less frequent (0.7%) as compared with intracytoplasmic sperm injection (2.8%; P<0.05) and cases of testicular sperm extraction (5.4%; P<0.01). Interestingly, embryos with a cleavage anomaly showed better morphology both on day 2 (P<0.005) and day 3 (P<0.001). In contrast, blastocyst formation (P<0.001) and blastocyst quality (P=NS) was higher in tetrahedral embryos. There was a significant increase in implantation rate if tetrahedral embryos could be transferred compared with when planar embryos had to be transferred (P<0.01). It may be postulated that, in planar embryos, the mitotic spindle might have been affected, e.g. sperm centrosome composition or function, which in turn might have led to the observed cleavage anomaly. Normally, day-2 embryos show a crosswise arrangement of four cells with three blastomeres lying side by side. Cleavage anomalies include more planar embryos that are characterized by a particular flat constellation of four blastomeres with presumed premature cleavage (like a tetrafoliate clover). Since little is known on the developmental fate of such embryos within a 10-month study period, all consecutive patients were screened for the presence of day-2 planar embryos (study group). A total of 64 (out of 2070) embryos with abnormal blastomere configuration were detected (3.1%). Interestingly, in conventional IVF (0.7%), the presence of planar embryos was significantly less frequent as compared with intracytoplasmic sperm injection (2.8%; P<0.05) and cases of testicular biopsy (5.4%; P<0.01). Embryos from the study group showed better morphology both on day 2 (P<0.005) and day 3 (P<0.001). In contrast, blastocyst formation (survival to day 5 of preimplantation development) was higher in the normally cleaved control group (P<0.001) and so was blastocyst quality; however, the latter parameter did not reach level of significance. This was also reflected in a significantly higher implantation rate in the control group (P<0.01). Based on present data, it may be postulated that, in planar embryos, the mitotic spindle (which involves the sperm centrosome) might have been affected, which in turn might have led to an incomplete cleavage. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Morphological evaluation of Day 8 embryos developed during induced aluteal cycles in the mare.

PubMed

Leisinger, C A; Medina, V; Markle, M L; Paccamonti, D L; Pinto, C R F

2018-01-01

A novel in vivo model utilizing serial administrations of PGF 2α to induce aluteal cycles in the mare was used to evaluate the effects of progesterone-deprivation on the morphology of in vivo preimplantation embryos. We hypothesized that equine embryos produced during induced aluteal cycles (AL) would be developmentally affected, characterized by earlier embryo stage at collection, smaller embryo diameter, and lower quality grade, compared with those collected on the same day post-ovulation from control cycles during diestrus (high progesterone; > 4 ng/mL). Seven cyclic mares with a median age of 6.5 years (range 3-16) were utilized in a crossover design. Mares in estrus were artificially inseminated to a fertile stallion and randomly assigned to control or AL groups. Mares received either saline solution (control mares) or PGF 2α (AL mares), twice daily on days 0, 1, and 2 and once daily on days 3 and 4. Serial blood samples were collected daily during estrus and until the day of embryo collection 8 days after ovulation. Mares were monitored until they returned to estrus, and artificially inseminated. Mares were switched to the opposite treatment group only after a successful embryo collection occurred during the previous cycle. Only cycles that produced embryos were used for analyses. No significant rise in progesterone was observed in the AL group with mean concentrations of plasma progesterone remaining <1.0 ng/mL from ovulation until embryo collection on Day 8. This is in sharp contrast to the control (luteal) cycle where a post-ovulatory rise in plasma progesterone was observed. The mean daily concentrations of plasma progesterone were significantly higher in control vs. AL group beginning at Day 3 and remained so until Day 8. The mean (±SEM) embryo diameter of AL embryos was 171 ± 5 μm compared to 756 ± 99 μm for control embryos. The majority of the Day 8 AL embryos were classified as morulas (3/9) or early blastocysts (5/9) with only 2 embryos of quality grade 1 compared to the Day 8 control embryos that were mostly expanded blastocysts (6/7) with 5 of 6 being of quality grade 1. This study shows that serial administrations of PGF 2α were able to prevent significant rises in plasma progesterone, thus inducing aluteal cycles characterized by a progesterone-deprived environment for developing embryos. Embryos collected from induced aluteal cycles were adversely affected as demonstrated by a lower quality grade, smaller diameter and earlier embryo stage at collection when compared to control embryos. Copyright © 2017 Elsevier Inc. All rights reserved.

Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program.

PubMed

Herrera, C; Morikawa, M I; Bello, M B; von Meyeren, M; Centeno, J Eusebio; Dufourq, P; Martinez, M M; Llorente, J

2014-03-15

Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300-1000, and >1000 μm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7-10 hours and 63% for 1-2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the results obtained using ultrasonography in all pregnancies assessed (11/11, 100%); untreated biopsy samples were correctly diagnosed in 36 of 41 assessed pregnancies (87.8%), although the difference between treated and untreated biopsy samples was not significant. Our results demonstrated that biopsied embryos can remain in holding medium before being transferred, until gender diagnosis by PGD is complete (7-10 hours), without affecting pregnancy rates. This simplifies the management of an embryo transfer program willing to incorporate PGD for gender selection, by transferring only embryos of the desired sex. Embryo biopsy can be performed in a clinical setting on embryos of different sizes, without affecting their viability. Additionally, we showed that pretreating biopsy samples with a short incubation at 95 °C improved the overall efficiency of embryo sex determination. Copyright © 2014 Elsevier Inc. All rights reserved.

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

PubMed

Galli, C; Colleoni, S; Duchi, R; Lagutina, I; Lazzari, G

2007-03-01

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Jun-Xue; Kang, Jin-Dan; Li, Suo

2015-01-02

Highlights: • First explored that the effects of PXD101 on the development of SCNT embryos in vitro. • 0.5 μM PXD101 treated for 24 h improved the development of porcine SCNT embryos. • Level of AcH3K9 was significantly higher than control group at early stages. - Abstract: In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with variousmore » concentrations of PXD101 for 24 h. Treatment with 0.5 μM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P < 0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 μM PXD101 for various amounts of times following activation. Treatment for 24 h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P < 0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.« less

Cryosurvival and pregnancy rates after exposure of IVF-derived Bos indicus embryos to forskolin before vitrification.

PubMed

Sanches, B V; Marinho, L S R; Filho, B D O; Pontes, J H F; Basso, A C; Meirinhos, M L G; Silva-Santos, K C; Ferreira, C R; Seneda, M M

2013-09-01

In vitro-produced (IVP) bovine embryos are more sensitive to cryopreservation than their in vivo counterparts due to their higher lipid concentrations, whereas Bos indicus IVP embryos are even more sensitive than Bos taurus IVP embryos. To examine the effects of a lipolytic agent, before vitrification of Bos indicus IVP embryos, on embryo survival, viability, and pregnancy rates, two experiments were conducted. In experiment 1, Bos indicus (Nelore) embryos were produced from abattoir-derived ovaries and allocated into two groups. In the treatment group, 10 μM of forskolin was added to the in vitro culture medium on Day 5 and incubated for 48 hours. On Day 7 of culture, IVP-expanded blastocysts from both the control (n = 101) and treatment (n = 112) groups were vitrified with ethylene glycol and DMSO via the Cryotop procedure. Although there was no significant difference between the rates of blastocoel reexpansion and hatching of the embryos exposed to forskolin (87.5% and 70.5%, respectively) compared with the control embryos (79.2% and 63.3%, respectively), the numerically superior rates of the embryos exposed to forskolin led to another experiment. In experiment 2, blastocysts produced from the ovum pick up were exposed or not exposed to the lipolytic agent and vitrified as in experiment 1. Embryos treated with forskolin had higher pregnancy rates than the control group (48.8% vs. 18.5%). In view of these results, 1908 Bos indicus embryos were produced from ovum pick up, exposed to the lipolytic agent, and blastocysts were transferred to recipients, and the pregnancy rates of the embryos of various breeds were compared. The mean pregnancy rate obtained was 43.2%. All data were analyzed by chi-square or by binary logistic regression (P ≤ 0.05). In conclusion, treatment with forskolin before vitrification improved cryotolerance of Bos indicus IVP embryos, resulting in good post-transfer pregnancy rates. Copyright © 2013 Elsevier Inc. All rights reserved.

The ovine uterus as a host for in vitro-produced bovine embryos.

PubMed

Rexroad, C E; Powell, A M

1999-07-15

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.

Effect of embryo source and recipient progesterone environment on embryo development in cattle.

PubMed

Lonergan, P; Woods, A; Fair, T; Carter, F; Rizos, D; Ward, F; Quinn, K; Evans, A

2007-01-01

The aim of the present study was to examine the effect of embryo source (in vivo v. in vitro) and the progesterone environment into which it was transferred on Day 7 on embryo survival and size on Day 13. Day 7 blastocysts were produced either in vivo using superovulation, artificial insemination and non-surgical embryo recovery or in vitro using in vitro maturation, fertilisation and culture. In order to produce animals with divergent progesterone concentrations, following synchronisation recipients were either superovulated (High progesterone; n = 10) or not (Control progesterone; n = 10). Ten blastocysts, produced either in vivo or in vitro, were transferred to each recipient on Day 7. Both groups were killed on Day 13. The mean progesterone concentration from Day 7 to Day 13 (the period when the embryos were in the uterus) in the High and Control progesterone recipients was 36.32 +/- 1.28 and 10.30 +/- 0.51 ng mL(-1), respectively. Of the in vivo embryos transferred, the overall recovery rate at Day 13 was 64%, which was higher (P < 0.001) than that of 20% for the in vitro embryos transferred. The mean area of embryos recovered from High progesterone recipients was 3.86 +/- 0.45 mm(2) (n = 28) compared with 1.66 +/- 0.38 mm(2) (n = 24) for embryos recovered from Control progesterone recipients (P < 0.001). Similarly, the origin of the embryo used for transfer affected embryo size on Day 13. In summary, the recovery rate of blastocysts was higher for in vivo- than in vitro-derived embryos. Blastocyst size was approximately 2.3-fold greater in recipients with high compared with normal progesterone. The present study lends strong support to the hypothesis that an earlier rise in progesterone after conception stimulates blastocyst growth and the development of competent embryos.

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors.

PubMed

Kimber, S J; Sneddon, S F; Bloor, D J; El-Bareg, A M; Hawkhead, J A; Metcalfe, A D; Houghton, F D; Leese, H J; Rutherford, A; Lieberman, B A; Brison, D R

2008-05-01

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

Analysis of compaction initiation in human embryos by using time-lapse cinematography.

PubMed

Iwata, Kyoko; Yumoto, Keitaro; Sugishima, Minako; Mizoguchi, Chizuru; Kai, Yoshiteru; Iba, Yumiko; Mio, Yasuyuki

2014-04-01

To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis).

A minimally invasive methodology based on morphometric parameters for day 2 embryo quality assessment.

PubMed

Molina, Inmaculada; Lázaro-Ibáñez, Elisa; Pertusa, Jose; Debón, Ana; Martínez-Sanchís, Juan Vicente; Pellicer, Antonio

2014-10-01

The risk of multiple pregnancy to maternal-fetal health can be minimized by reducing the number of embryos transferred. New tools for selecting embryos with the highest implantation potential should be developed. The aim of this study was to evaluate the ability of morphological and morphometric variables to predict implantation by analysing images of embryos. This was a retrospective study of 135 embryo photographs from 112 IVF-ICSI cycles carried out between January and March 2011. The embryos were photographed immediately before transfer using Cronus 3 software. Their images were analysed using the public program ImageJ. Significant effects (P < 0.05), and higher discriminant power to predict implantation were observed for the morphometric embryo variables compared with morphological ones. The features for successfully implanted embryos were as follows: four cells on day 2 of development; all blastomeres with circular shape (roundness factor greater than 0.9), an average zona pellucida thickness of 13 µm and an average of 17695.1 µm² for the embryo area. Embryo size, which is described by its area and the average roundness factor for each cell, provides two objective variables to consider when predicting implantation. This approach should be further investigated for its potential ability to improve embryo scoring. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Enhance beef cattle improvement by embryo biotechnologies.

PubMed

Wu, B; Zan, L

2012-10-01

Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer (in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions. © 2011 Blackwell Verlag GmbH.

Economic evaluations of single- versus double-embryo transfer in IVF.

PubMed

Fiddelers, A A A; Severens, J L; Dirksen, C D; Dumoulin, J C M; Land, J A; Evers, J L H

2007-01-01

Multiple pregnancies lead to complications and induce high costs. The most successful way to decrease multiple pregnancies in IVF is to transfer only one embryo, which might reduce the efficacy of treatment. The objective of this review is to determine which embryo-transfer policy is most cost-effective: elective single-embryo transfer (eSET) or double-embryo transfer (DET). Several databases were searched for (cost* or econ*) and (single embryo* or double embryo* or one embryo* or two embryo* or elect* embryo or multip* embryo*). On the basis of five exclusion criteria, titles and abstracts were screened by two individual reviewers. The remaining papers were read for further selection, and data were extracted from the selected studies. A total of 496 titles were identified through the searches and resulted in the selection of one observational study and three randomized studies. Study characteristics, total costs and probability of live births were extracted. Besides this, cost-effectiveness and incremental cost-effectiveness were derived. It can be concluded that DET is the most expensive strategy. DET is also most effective if performed in one fresh cycle. eSET is only preferred from a cost-effectiveness point of view when performed in good prognosis patients and when frozen/thawed cycles are included. If frozen/thawed cycles are excluded, the choice between eSET and DET depends on how much society is willing to pay for one extra successful pregnancy.

9 CFR 98.6 - Ports of entry.

Code of Federal Regulations, 2010 CFR

2010-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.6 Ports of entry. An embryo shall not be imported into the...

9 CFR 98.6 - Ports of entry.

Code of Federal Regulations, 2013 CFR

2013-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.6 Ports of entry. An embryo shall not be imported into the...

9 CFR 98.6 - Ports of entry.

Code of Federal Regulations, 2011 CFR

2011-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.6 Ports of entry. An embryo shall not be imported into the...

9 CFR 98.6 - Ports of entry.

Code of Federal Regulations, 2012 CFR

2012-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.6 Ports of entry. An embryo shall not be imported into the...

9 CFR 98.6 - Ports of entry.

Code of Federal Regulations, 2014 CFR

2014-01-01

... EMBRYOS AND ANIMAL SEMEN Ruminant and Swine Embryos from Regions Free of Rinderpest and Foot-and-Mouth Disease; and Embryos of Horses and Asses § 98.6 Ports of entry. An embryo shall not be imported into the...

Mortality, size of the gonads, and ultrastructure of primordial germ cell in chick embryos treated with gamma-irradiation or injected with donor cells.

PubMed

Maeda, T; Clark, M E; Etches, R J

1998-06-01

The effects of injection and/or gamma-irradiation prior to injection on mortality, size of the gonads, and ultrastructure of primordial germ cell (PGC) were examined after 5 d of incubation. The mortality of embryos injected with donor cells was significantly higher than that of control and irradiated embryos. All irradiated embryos were alive, although their development was delayed compared to those not exposed to irradiation. The size of the gonads of embryos injected with donor cells were similar to those of control embryos, however, the size of the gonads in irradiated embryos was significantly smaller than those of control embryos. The number of PGC in the gonads was significantly decreased by irradiation. There was no notable effect of irradiation or injection on the nuclei and cytoplasmic organelles in PGC.

[Embryo donation: why is there a delay in the implementation in France? A discussion on the practical, ethical and psychological dilemmas].

PubMed

Temstet, R; Devaux, A; Lourdel, E; Cabry, R; Brzakowski, M; Copin, H; Merviel, P

2011-01-01

Since 1999, French legislation has stipulated that embryo donation is one of the possibilities afforded to couples who have a surplus of cryopreserved embryos. Donation of embryos with no foreseeable future use by the genetic couple can therefore be given to infertile couples. In practice however, since the authorization of this novel Medically Assisted Reproduction technique, embryo donation is not widely performed in France even though it is not technically difficult. Why then is there reluctance towards the implementation of embryo donation in France? The aim of this article is to analyze the grounds for the delay in the realization of embryo donation in France. Our findings propose that a myriad of factors including organizational, ethical and psychological determinants have deterred the implementation of embryo donation in France. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Efficient harvesting methods for early-stage snake and turtle embryos.

PubMed

Matsubara, Yoshiyuki; Kuroiwa, Atsushi; Suzuki, Takayuki

2016-04-01

Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle. © 2016 Japanese Society of Developmental Biologists.

Embryo quality is the main factor affecting cumulative live birth rate after elective single embryo transfer in fresh stimulation cycles.

PubMed

Niinimäki, Maarit; Veleva, Zdravka; Martikainen, Hannu

2015-11-01

The study was aimed to evaluate which factors affect the cumulative live birth rate after elective single embryo transfer in women younger than 36 years. Additionally, number of children in women with more than one delivery per ovum pick-up after fresh elective single embryo transfer and subsequent frozen embryo transfers was assessed. Retrospective cohort study analysing data of a university hospital's infertility clinic in 2001-2010. A total of 739 IVF/ICSI cycles with elective single embryo transfer were included. Analyses were made per ovum pick-up including fresh and subsequent frozen embryo transfers. Factors affecting cumulative live birth rates were examined in uni- and multivariate analyses. A secondary endpoint was the number of children born after all treatments. In the fresh cycles, the live birth rate was 29.2% and the cumulative live birth rate was 51.3%, with a twin rate of 3.4%. In the multivariate analysis, having two (odds ratio (OR) 1.73; 95% confidence interval (CI) 1.12-2.67) or ≥3 top embryos (OR 2.66; 95% CI 1.79-3.95) was associated with higher odds for live birth after fresh and frozen embryo cycles. Age, body mass index, duration of infertility, diagnosis or total gonadotropin dose were not associated with the cumulative live birth rate. In cycles with one top embryo, the cumulative live birth rate was 40.2%, whereas it was 64.1% in those with at least three top embryos. Of women who had a live birth in the fresh cycle, 20.4% had more than one child after all frozen embryo transfers. Among women with three or more top embryos after ovum pick-up, 16.1% gave birth to more than one child. The cumulative live birth rate in this age group varies from 40% to 64% and is dependent on the quality of embryos. Women with three or more top embryos have good chance of having more than one child per ovum pick-up without elevated risk of multiple pregnancies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Hypoxia interferes with ABA metabolism and increases ABA sensitivity in embryos of dormant barley grains.

PubMed

Benech-Arnold, Roberto L; Gualano, Nicolas; Leymarie, Juliette; Côme, Daniel; Corbineau, Françoise

2006-01-01

Two mechanisms have been suggested as being responsible for dormancy in barley grain: (i) ABA in the embryo, and (ii) limitation of oxygen supply to the embryo by oxygen fixation as a result of the oxidation of phenolic compounds in the glumellae. The aim of the present work was to investigate whether hypoxia imposed by the glumellae interferes with ABA metabolism in the embryo, thus resulting in dormancy. In dormant and non-dormant grains incubated at 20 degrees C and in non-dormant grains incubated at 30 degrees C (i.e. when dormancy is not expressed), ABA content in the embryo decreased dramatically during the first 5 h of incubation before germination was detected. By contrast, germination of dormant grains was less than 2% within 48 h at 30 degrees C and embryo ABA content increased during the first hours of incubation and then remained 2-4 times higher than in embryos from grains in which dormancy was not expressed. Removal of the glumellae allowed germination of dormant grains at 30 degrees C and the embryos did not display the initial increase in ABA content. Incubation of de-hulled grains under 5% oxygen to mimic the effect of glumellae, restored the initial increase ABA in content and completely inhibited germination. Incubation of embryos isolated from dormant grains, in the presence of a wide range of ABA concentrations and under various oxygen tensions, revealed that hypoxia increased embryo sensitivity to ABA by 2-fold. This effect was more pronounced at 30 degrees C than at 20 degrees C. Furthermore, when embryos from dormant grains were incubated at 30 degrees C in the presence of 10 microM ABA, their endogenous ABA content remained constant after 48 h of incubation under air, while it increased dramatically in embryos incubated under hypoxia, indicating that the apparent increase in embryo ABA responsiveness induced by hypoxia was, in part, mediated by an inability of the embryo to inactivate ABA. Taken together these results suggest that hypoxia, either imposed artificially or by the glumellae, increases embryo sensitivity to ABA and interferes with ABA metabolism.

Fish embryo toxicity test: identification of compounds with weak toxicity and analysis of behavioral effects to improve prediction of acute toxicity for neurotoxic compounds.

PubMed

Klüver, Nils; König, Maria; Ortmann, Julia; Massei, Riccardo; Paschke, Albrecht; Kühne, Ralph; Scholz, Stefan

2015-06-02

The fish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, but concerns have been raised for its predictivity given that a few compounds have been shown to exhibit a weak acute toxicity in the fish embryo. In order to better define the applicability domain and improve the predictive capacity of the fish embryo test, we performed a systematic analysis of existing fish embryo and acute fish toxicity data. A correlation analysis of a total of 153 compounds identified 28 compounds with a weaker or no toxicity in the fish embryo test. Eleven of these compounds exhibited a neurotoxic mode of action. We selected a subset of eight compounds with weaker or no embryo toxicity (cyanazine, picloram, aldicarb, azinphos-methyl, dieldrin, diquat dibromide, endosulfan, and esfenvalerate) to study toxicokinetics and a neurotoxic mode of action as potential reasons for the deviating fish embryo toxicity. Published fish embryo LC50 values were confirmed by experimental analysis of zebrafish embryo LC50 according to OECD guideline 236. Except for diquat dibromide, internal concentration analysis did not indicate a potential relation of the low sensitivity of fish embryos to a limited uptake of the compounds. Analysis of locomotor activity of diquat dibromide and the neurotoxic compounds in 98 hpf embryos (exposed for 96 h) indicated a specific effect on behavior (embryonic movement) for the neurotoxic compounds. The EC50s of behavior for neurotoxic compounds were close to the acute fish toxicity LC50. Our data provided the first evidence that the applicability domain of the fish embryo test (LC50s determination) may exclude neurotoxic compounds. However, neurotoxic compounds could be identified by changes in embryonic locomotion. Although a quantitative prediction of acute fish toxicity LC50 using behavioral assays in fish embryos may not yet be possible, the identification of neurotoxicity could trigger the conduction of a conventional fish acute toxicity test or application of assessment factors while considering the very good fish embryo-acute fish toxicity correlation for other compounds.

Donation of surplus frozen pre-embryos to research in Israel: underlying motivations.

PubMed

Raz, Aviad; Amer-Alshiek, Jonia; Goren-Margalit, Mor; Jacobi, Gal; Hochberg, Alyssa; Amit, Ami; Azem, Foad; Amir, Hadar

2016-01-01

The high number of IVF procedures performed in Israel has had an unforeseen consequence: accumulation of large amounts of surplus frozen embryos. After five years that the frozen embryos are kept for free, patients need to make an embryo disposition decision. One option is donation for research. The donation rate in Israel is very low. Our aim was to understand the attitudes, values and perceptions of female IVF patients that decided to donate their surplus frozen embryos to research. The study setting was a tertiary IVF unit which during the 2000-2009 period treated 241 patients who had their frozen pre-embryos stored for more than five years. The study population consists of the 12 patients (from among the 241) who had decided to donate their excess frozen pre-embryos to research. In-depth interviews were carried out with 8 of those 12 patients. IVF patients who donated their surplus frozen pre-embryos to research viewed the frozen embryo as a valuable resource that does not have human identity yet. The majority expressed a gradualist approach to the human status of the embryo as requiring successful implantation and development in the uterus. All the respondents chose donation to research not because it was their first choice but because they did not want or were unable to use the pre-embryos in the future, in addition to not willing to thaw them. For many of the respondents, donation to research was accompanied by a sense of uncertainty. All would have preferred to donate their pre-embryos to infertile women or couples, an option which is currently prohibited in Israel. The moral reasoning behind decisions that patients make regarding excess pre-embryos is important for health care practitioners to consider when offering decision-making alternatives and counseling. For our respondents, the scarcity of donating excess frozen pre-embryos to research may reflect patients' preference for embryo donation to infertile couples. Recommended ways to increase donation to research may include public education and awareness, as well as targeted communication with IVF patients by multi-professional IVF unit teams comprised of a medical doctor and a professional trained in bioethics.

Developments in the storage of embryos in France and the limitations of the laws of bioethics. Analysis of procedures in 17 storage centres and the destiny of stored embryos.

PubMed

Moutel, Grégoire; Gregg, Edna; Meningaud, Jean Paul; Hervé, Christian

2002-01-01

1985 witnessed the first transfers of frozen embryos resulting in live births in France. Since this time the number of embryos obtained by in vitro fertilisation (IVF) has increased each year. In 1999 each IVF attempt obtains, on average, 4.5 embryos that can be successfully implanted. In this paper we consider only those couples who have successfully obtained embryos (either by ICSI or traditional IVF techniques). The aims of the study are: To show how developments in embryo production and conservation have influenced the number of embryos stored. To address the socio-medical and ethical issues raised and to provide practitioners with some thoughts for reflection when consulting with couples based on the study findings To discuss the results of our findings in the light of those ethical questions raised by the imminent revision of the Laws of Bioethics. In the first instance we did a retrospective analysis of quantitative data that 17 storage centres had collected over a period of 5 years. This period was marked by the implementation in 1994 of Laws described as Bioethics' Laws in France. During a second period we conducted a qualitative study regarding the fate of stored embryos. In order to do this, we began an analysis of the "status" of embryos and the decisions of those couples whose embryos were still in storage. For this a questionnaire was used. The number of embryos that remain in storage in the 17 storage centres has increased reaching a total of 17,592 embryos involving 3,888 couples. The results show a consistent and persistent increase in the number of embryos stored before and after 1994. The qualitative study shows that: 51% of couples with embryos in storage can no longer be found, 23.6% request a continuance of storage, 12% would accept donating their embryos to medical research, 9.1% would wish for other couples to take eventual ownership of the embryo in 7.2% of cases the storage centre has can provide no information concerning the continuing of storage of such embryos. The Bioethics Laws have therefore not succeeded in limiting the inflation in the number of embryos stored despite that the fact that this was one of the major concerns of those involved in formulating the laws and the medical professionals involved. Our study shows that the guidelines provided by these Laws remain ambiguous and that the objectives defined therein are thus difficult to achieve. Our study highlights the impact of such developments on the possible eventual "destiny" of such embryos, the behaviour of and the advice provided to couples, and the management practice of the storage centres involved. We present these results in the light of the laws established in 1994 in order to define not only the benefits but also the potential limitations of such legislation. No guidelines were laid down regarding the future of embryos belonging to a couple that no longer wishes to embark upon a "parental project". As a result it is not possible to terminate the conservation of such embryos which therefore remain in storage awaiting the revision of the current laws. The major objectives of our study assess the impact of the Laws and their impact on practice and to contribute to the debate which has yet to take place in the social and political arena.

Sex and PRNP genotype determination in preimplantation caprine embryos.

PubMed

Guignot, F; Perreau, C; Cavarroc, C; Touzé, J-L; Pougnard, J-L; Dupont, F; Beckers, J-F; Rémy, B; Babilliot, J-M; Bed'Hom, B; Lamorinière, J M; Mermillod, P; Baril, G

2011-08-01

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. © 2010 Blackwell Verlag GmbH.

Polar body based aneuploidy screening is poorly predictive of embryo ploidy and reproductive potential.

PubMed

Salvaggio, C N; Forman, E J; Garnsey, H M; Treff, N R; Scott, R T

2014-09-01

Polar body (polar body) biopsy represents one possible solution to performing comprehensive chromosome screening (CCS). This study adds to what is known about the predictive value of polar body based testing for the genetic status of the resulting embryo, but more importantly, provides the first evaluation of the predictive value for actual clinical outcomes after embryo transfer. SNP array was performed on first polar body, second polar body, and either a blastomere or trophectoderm biopsy, or the entire arrested embryo. Concordance of the polar body-based prediction with the observed diagnoses in the embryos was assessed. In addition, the predictive value of the polar body -based diagnosis for the specific clinical outcome of transferred embryos was evaluated through the use of DNA fingerprinting to track individual embryos. There were 459 embryos analyzed from 96 patients with a mean maternal age of 35.3. The polar body-based predictive value for the embryo based diagnosis was 70.3%. The blastocyst implantation predictive value of a euploid trophectoderm was higher than from euploid polar bodies (51% versus 40%). The cleavage stage embryo implantation predictive value of a euploid blastomere was also higher than from euploid polar bodies (31% versus 22%). Polar body based aneuploidy screening results were less predictive of actual clinical outcomes than direct embryo assessment and may not be adequate to improve sustained implantation rates. In nearly one-third of cases the polar body based analysis failed to predict the ploidy of the embryo. This imprecision may hinder efforts for polar body based CCS to improve IVF clinical outcomes.

Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.).

PubMed

Adu-Gyamfi, Raphael; Wetten, Andy; Marcelino Rodríguez López, Carlos

2016-01-01

While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation.

Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.)

PubMed Central

Adu-Gyamfi, Raphael; Wetten, Andy; Marcelino Rodríguez López, Carlos

2016-01-01

While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. Key message: Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation. PMID:27403857

In vitro development of donated frozen-thawed human embryos in a prototype static microfluidic device: a randomized controlled trial.

PubMed

Kieslinger, Dorit C; Hao, Zhenxia; Vergouw, Carlijn G; Kostelijk, Elisabeth H; Lambalk, Cornelis B; Le Gac, Séverine

2015-03-01

To compare the development of human embryos in microfluidic devices with culture in standard microdrop dishes, both under static conditions. Prospective randomized controlled trial. In vitro fertilization laboratory. One hundred eighteen donated frozen-thawed human day-4 embryos. Random allocation of embryos that fulfilled the inclusion criteria to single-embryo culture in a microfluidics device (n = 58) or standard microdrop dish (n = 60). Blastocyst formation rate and quality after 24, 28, 48, and 72 hours of culture. The percentage of frozen-thawed day-4 embryos that developed to the blastocyst stage did not differ significantly in the standard microdrop dishes and microfluidic devices after 28 hours of culture (53.3% vs. 58.6%) or at any of the other time points. The proportion of embryos that would have been suitable for embryo transfer was comparable after 28 hours of culture in the control dishes and microfluidic devices (90.0% vs. 93.1%). Furthermore, blastocyst quality was similar in the two study groups. This study shows that a microfluidic device can successfully support human blastocyst development in vitro under static culture conditions. Future studies need to clarify whether earlier stage embryos will benefit from the culture in microfluidic devices more than the tested day-4 embryos because many important steps in the development of human embryos already take place before day 4. Further improvements of the microfluidic device will include parallel culture of single embryos, application of medium refreshment, and built-in sensors. NTR3867. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Adjustments in cholinergic, adrenergic and purinergic control of cardiovascular function in snapping turtle embryos (Chelydra serpentina) incubated in chronic hypoxia.

PubMed

Eme, John; Rhen, Turk; Crossley, Dane A

2014-10-01

Adenosine is an endogenous nucleoside that acts via G-protein coupled receptors. In vertebrates, arterial or venous adenosine injection causes a rapid and large bradycardia through atrioventricular node block, a response mediated by adenosine receptors that inhibit adenylate cyclase and decrease cyclic AMP concentration. Chronic developmental hypoxia has been shown to alter cardioregulatory mechanisms in reptile embryos, but adenosine's role in mediating these responses is not known. We incubated snapping turtle embryos under chronic normoxic (N21; 21 % O2) or chronic hypoxic conditions (H10; 10 % O2) beginning at 20 % of embryonic incubation. H10 embryos at 90 % of incubation were hypotensive relative to N21 embryos in both normoxic and hypoxic conditions. Hypoxia caused a hypotensive bradycardia in both N21 and H10 embryos during the initial 30 min of exposure; however, f H and P m both trended towards increasing during the subsequent 30 min, and H10 embryos were tachycardic relative to N21 embryos in hypoxia. Following serial ≥1 h exposure to normoxic and hypoxic conditions, a single injection of adenosine (1 mg kg(-1)) was given. N21 and H10 embryos responded to adenosine injection with a rapid and large hypotensive bradycardia in both normoxia and hypoxia. Gene expression for adenosine receptors were quantified in cardiac tissue, and Adora1 mRNA was the predominant receptor subtype with transcript levels 30-82-fold higher than Adora2A or Adora2B. At 70 % of incubation, H10 embryos had lower Adora1 and Adora2B expression compared to N21 embryos. Expression of Adora1 and Adora2B decreased in N21 embryos during development and did not differ from H10 embryos at 90 % of incubation. Similar to previous results in normoxia, H10 embryos in hypoxia were chronically tachycardic compared to N21 embryos before and after complete cholinergic and adrenergic blockade. Chronic hypoxia altered the development of normal cholinergic and adrenergic tone, as well as adenosine receptor mRNA levels. This study demonstrates that adenosine may be a major regulator of heart rate in developing snapping turtle embryos, and that chronic hypoxic incubation alters the response to hypoxic exposure.

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy

PubMed Central

2014-01-01

Background Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. Methods In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. Results The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9–11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert’s membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart beat. Corresponding histology showed apoptotic cells in the embryo while the placenta was still intact. In the subsequent resorption process first the embryo and then its membranes disappeared. Conclusions Our results provide a temporal time course of embryo resorption. With this method, animals exhibiting embryo resorption can be targeted, enabling the investigation of underlying mechanisms before the onset of total embryo disintegration. PMID:24886361

Impact of follicular G-CSF quantification on subsequent embryo transfer decisions: a proof of concept study.

PubMed

Lédée, N; Gridelet, V; Ravet, S; Jouan, C; Gaspard, O; Wenders, F; Thonon, F; Hincourt, N; Dubois, M; Foidart, J M; Munaut, C; Perrier d'Hauterive, S

2013-02-01

Previous experiments have shown that granulocyte colony-stimulating factor (G-CSF), quantified in the follicular fluid (FF) of individual oocytes, correlates with the potential for an ongoing pregnancy of the corresponding fertilized oocytes among selected transferred embryos. Here we present a proof of concept study aimed at evaluating the impact of including FF G-CSF quantification in the embryo transfer decisions. FF G-CSF was quantified with the Luminex XMap technology in 523 individual FF samples corresponding to 116 fresh transferred embryos, 275 frozen embryos and 131 destroyed embryos from 78 patients undergoing ICSI. Follicular G-CSF was highly predictive of subsequent implantation. The receiving operator characteristics curve methodology showed its higher discriminatory power to predict ongoing pregnancy in multivariate logistic regression analysis for FF G-CSF compared with embryo morphology [0.77 (0.69-0.83), P < 0.001 versus 0.66 (0.58-0.73), P = 0.01)]. Embryos were classified by their FF G-CSF concentration: Class I over 30 pg/ml (a highest positive predictive value for implantation), Class II from 30 to 18.4 pg/ml and Class III <18.4 pg/ml (a highest negative predictive value). Embryos derived from Class I follicles had a significantly higher implantation rate (IR) than those from Class II and III follicles (36 versus 16.6 and 6%, P < 0.001). Embryos derived from Class I follicles with an optimal morphology reached an IR of 54%. Frozen-thawed embryos transfer derived from Class I follicles had an IR of 37% significantly higher than those from Class II and III follicles, respectively, of 8 and 5% (P < 0.001). Thirty-five per cent of the frozen embryos but also 10% of the destroyed embryos were derived from G-CSF Class I follicles. Non-optimal embryos appear to have been transferred in 28% (22/78) of the women, and their pregnancy rate was significantly lower than that of women who received at least one optimal embryo (18 versus 36%, P = 0.04). Monitoring FF G-CSF for the selection of embryos with a better potential for pregnancy might improve the effectiveness of IVF by reducing the time and cost required for obtaining a pregnancy.

Accurate and noninvasive embryos screening during in vitro fertilization (IVF) assisted by Raman analysis of embryos culture medium Accurate and noninvasive embryos screening during IVF

NASA Astrophysics Data System (ADS)

Shen, A. G.; Peng, J.; Zhao, Q. H.; Su, L.; Wang, X. H.; Hu, J. M.; Yang, J.

2012-04-01

In combination with morphological evaluation tests, we employ Raman spectroscopy to select higher potential reproductive embryos during in vitro fertilization (IVF) based on chemical composition of embryos culture medium. In this study, 57 Raman spectra are acquired from both higher and lower quality embryos culture medium (ECM) from 10 patients which have been preliminarily confirmed by clinical assay. Data are fit by using a linear combination model of least squares method in which 12 basis spectra represent the chemical features of ECM. The final fitting coefficients provide insight into the chemical compositions of culture medium samples and are subsequently used as criterion to evaluate the quality of embryos. The relative fitting coefficients ratios of sodium pyruvate/albumin and phenylalanine/albumin seem act as key roles in the embryo screening, attaining 85.7% accuracy in comparison with clinical pregnancy. The good results demonstrate that Raman spectroscopy therefore is an important candidate for an accurate and noninvasive screening of higher quality embryos, which potentially decrease the time-consuming clinical trials during IVF.

The evolution of porcine embryo in vitro production.

PubMed

Grupen, Christopher G

2014-01-01

The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology. Copyright © 2014 Elsevier Inc. All rights reserved.

Metabolomic Assessment of Embryo Viability

PubMed Central

Uyar, Asli; Seli, Emre

2014-01-01

Preimplantation embryo metabolism demonstrates distinctive characteristics associated with the developmental potential of embryos. On this basis, metabolite content of culture media was hypothesized to reflect the implantation potential of individual embryos. This hypothesis was tested in consecutive studies reporting a significant association between culture media metabolites and embryo development or clinical pregnancy. The need for a noninvasive, reliable, and rapid embryo assessment strategy promoted metabolomics studies in vitro fertilization (IVF) in an effort to increase success rates of single embryo transfers. With the advance of analytical techniques and bioinformatics, commercial instruments were developed to predict embryo viability using spectroscopic analysis of surplus culture media. However, despite the initial promising results from proof-of-principal studies, recent randomized controlled trials using commercial instruments failed to show a consistent benefit in improving pregnancy rates when metabolomics is used as an adjunct to morphology. At present, the application of metabolomics technology in clinical IVF laboratory requires the elimination of factors underlying inconsistent findings, when possible, and development of reliable predictive models accounting for all possible sources of bias throughout the embryo selection process. PMID:24515909

Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

PubMed

Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

2013-10-01

Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence. This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest. ClinicalTrials.gov Identifier: NCT01397136.

Effect of mastitis on luteal function and pregnancy rates in buffaloes.

PubMed

Mansour, Mohamed Mohsen; Hendawy, Amin O; Zeitoun, Moustafa M

2016-09-15

The aim of this study was to investigate the effects of mastitis on CL development and function and pregnancy rate in buffaloes. Sixty-six buffaloes (Bubalus bubalus) reared in a commercial farm at El-Beheira governorate, north of Egypt were used in this study. According to the visual observation of milk, physical examination of the udder and actual somatic cell count in milk, buffalo cows were divided into three groups: without mastitis (W), n = 23; subclinical mastitis (SC), n = 18; and clinical mastitis (C), n = 25. All buffalo cows were synchronized by double dose of PGF2α (11-day interval) and inseminated by frozen-thawed semen of fertile bull. Mean CL diameter was ultrasonically examined on Days 5, 9, 12, 16, 21, and 25 after artificial insemination (AI). Blood samples were taken on the days of ultrasonography for progesterone (P4) assay. Results indicated that pregnancy rates were lower (P < 0.05) in C (28.00%) and SC (55.56%) compared with W (69.57%) on Day 25 after first AI. Pregnancy rates reduced to 60.87%, 44.45%, and 16.00% in W, SC, and C, respectively, at Day 45 after insemination. Thus, the embryonic loss was 8.7%, 11.11%, and 12.00 % in W, SC, and C cows, respectively. Pregnancy rates decreased between 44.32% and 50.51% when mastitis occurred during Day -15 before to Day +30 after AI, compared with 59.22% in the uninfected cows. The diameter of CL was greater (P < 0.05) in W than SC and C cows starting at Day 9 postbreeding onward. Likewise, P4 concentrations on Days 9 through 25 after AI were greater (P < 0.05) in W cows as compared to SC and C cows. Positive correlations (P < 0.01) were found on Days 5, 9, 12, 16, 21, and 25 after AI between CL diameter and P4 concentrations. Similar trend was found among CL diameter, P4 concentrations, and pregnancy rate. Accordingly, incidence of mastitis revealed suppression to both CL diameter and function leading to significant reduction in pregnancy outcome of buffalo cows. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of quail and chicken embryos for the detection of botulinum toxin serotypes A, B, E and F activity

USDA-ARS?s Scientific Manuscript database

Comparison of quail (Coturnix japonica) and chicken (Gallus domesticus) embryos for the detection of BoNT/A activity was conducted using equal dosages of toxin/g of embryo (quail at 7 g and chickens at 48 g). Quail embryos were injected at 0, 0.5 to 50 ng adn chicken embryos at 0, 3.4 to 342 ng and...

Evaluation of quail and chicken embryos for the detection of botulism toxin serotypes A, B E and F activity.

USDA-ARS?s Scientific Manuscript database

Comparison of quail (Coturnix japonica) and chicken (Gallus domesticus) embryos for the detection of BoNT/A activity was conducted using equal dosages of toxin/g of embryo (quail at 7 g and chickens at 48 g). Quail embryos were injected at 0, 0.5 to 50 ng and chicken embryos at 0, 3.4 to 342 ng and...

[Traditional and modern approaches to culture of preimplantation mammalian embryos in vitro].

PubMed

Brusentsev, E Iu; Igonina, T N; Amstislavskiĭ, S Ia

2014-01-01

This review covers the basic principles and methods of in vitro culture of preimplantation mammalian embryos. The features of in vitro development of embryos of various species of animals with allowance for the composition of nutrient media are described, with special attention paid to those species that have traditionally been consideredas laboratory (i.e., mice, rats, and hamsters). The effects of suboptimal culturing conditions of preimplantation embryos on the formation of the phenotype of individuals developed from these embryos are discussed. New approaches to optimize the conditions of the development of preimplantation mammalian embryos in vitro are analyzed.

High-frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.).

PubMed

Nair, R Ramakrishnan; Dutta Gupta, S

2006-01-01

A high-frequency plantlet regeneration protocol was developed for black pepper (Piper nigrum L.) through cyclic secondary somatic embryogenesis. Secondary embryos formed from the radicular end of the primary somatic embryos which were originally derived from micropylar tissues of germinating seeds on growth regulator-free SH medium in the absence of light. The process of secondary embryogenesis continued in a cyclic manner from the root pole of newly formed embryos resulting in clumps of somatic embryos. Strength of the medium and sucrose concentration influenced the process of secondary embryogenesis and fresh weight of somatic embryo clumps. Full-strength SH medium supplemented with 1.5% sucrose produced significantly higher fresh weight and numbers of secondary somatic embryos while 3.0 and 4.5% sucrose in the medium favored further development of proliferated embryos into plantlets. Ontogeny of secondary embryos was established by histological analysis. Secondary embryogenic potential was influenced by the developmental stage of the explanted somatic embryo and stages up to "torpedo" were more suitable. A single-flask system was standardized for proliferation, maturation, germination and conversion of secondary somatic embryos in suspension cultures. The system of cyclic secondary somatic embryogenesis in black pepper described here represents a permanent source of embryogenic material that can be used for genetic manipulations of this crop species.

Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos.

PubMed

Terashita, Yukari; Yamagata, Kazuo; Tokoro, Mikiko; Itoi, Fumiaki; Wakayama, Sayaka; Li, Chong; Sato, Eimei; Tanemura, Kentaro; Wakayama, Teruhiko

2013-01-01

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

Chromosome analysis in embryos from young patients with previous parity.

PubMed

Kilani, Z; Magli, Mc; Qaddomi, E; Ferraretti, Ap; Shaban, M; Crippa, A; Haj Hassan, L; Shenfield, F; Gianaroli, L

2014-09-01

This study included 173 young couples of proven fertility who had previously undergone preimplantation genetic screening for chromosomes X and Y for family balancing. Several months later, when the outcome of the pregnancies was already known, the blastomeres from the corresponding embryos transferred were reanalysed by fluorescence in-situ hybridization (FISH) for chromosomes 13, 16, 18, 21, 22 with the aim of investigating correlation with embryo viability and the level of FISH sensitivity (embryos confirmed to be euploid). According to the results, informative in 152 couples, the proportion of euploid embryos was significantly lower in 53 nonpregnant women when compared with 99 women with term pregnancy (49% versus 75% respectively, P < 0.001). In addition, in 21 nonpregnant patients, all embryos transferred were found to be chromosomally abnormal. The level of FISH sensitivity was calculated in the group of term pregnancies where the number of euploid embryos was expected to exceed or match with the number of babies born. The resulting false-negative rate was 4.0% per patient and 1.9% per embryo. These findings confirmed the limited prediction power of embryo morphology on implantation but also the relevance of chromosomal abnormalities in causing embryo demise. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Embryo transfer practices in the United States: a survey of clinics registered with the Society for Assisted Reproductive Technology.

PubMed

Jungheim, Emily S; Ryan, Ginny L; Levens, Eric D; Cunningham, Alexandra F; Macones, George A; Carson, Kenneth R; Beltsos, Angeline N; Odem, Randall R

2010-09-01

To gain a better understanding of factors influencing clinicians' embryo transfer practices. Cross-sectional survey. Web-based survey conducted in December 2008 of individuals practicing IVF in centers registered with the Society for Assisted Reproductive Technology (SART). None. None. Prevalence of clinicians reporting following embryo transfer guidelines recommended by the American Society for Reproductive Medicine (ASRM), prevalence among these clinicians to deviate from ASRM guidelines in commonly encountered clinical scenarios, and practice patterns related to single embryo transfer. Six percent of respondents reported following their own, independent guidelines for the number of embryos to transfer after IVF. Of the 94% of respondents who reported routinely following ASRM embryo transfer guidelines, 52% would deviate from these guidelines for patient request, 51% for cycles involving the transfer of frozen embryos, and 70% for patients with previously failed IVF cycles. All respondents reported routinely discussing the risks of multiple gestations associated with standard embryo transfer practices, whereas only 34% reported routinely discussing single embryo transfer with all patients. Although the majority of clinicians responding to our survey reported following ASRM embryo transfer guidelines, at least half would deviate from these guidelines in a number of different situations. Copyright (c) 2010 American Society for Reproductive Medicine. All rights reserved.

Critical reappraisal of embryo quality as a predictive parameter for pregnancy outcome: a pilot study

PubMed Central

Campo, R.; Binda, M.M.; Van Kerkhoven, G.; Frederickx, V.; Serneels, A.; Roziers, P.; Lopes, A.S.; Gordts, S.; Puttemans, P.; Gordts, S.

2010-01-01

Aim of the study: Pilot study to analyse the efficacy and embryo morphology using a new human embryo culture medium (GM501) versus the conventional used medium (ISM1). Methods: Over a four-month period, all patients at the Leuven Institute of Fertility and Embryology (LIFE) were randomly allocated to have their embryos cultured in either the standard sequential culture medium ISM1 (control) or in a new universal medium (GM501) (study group). Primary outcome parameters were clinical pregnancy and live birth rate. The secondary outcome parameter was the correlation of embryo fragmentation rate with pregnancy outcome. Results: We did not observe any differences between the ISM1 control group and GM501 study group with regard to fertilization, pregnancy, implantation rates, ongoing pregnancy, and babies born. The number of embryos with a minimal fragmentation rate (less than 30%) was significantly higher in the GM501 study group. Conclusion: Although a significant higher embryo fragmentation rate was seen in In vitro culture of embryos in GM501, pregnancy outcome results were comparable to those of embryos cultured in ISM1. According to our results the value of embryo morphological criteria as a parameter for pregnancy outcome should be examined and discussed again. PMID:25009716

A Higher Ovarian Response after Stimulation for IVF Is Related to a Higher Number of Euploid Embryos.

PubMed

Labarta, Elena; Bosch, Ernesto; Mercader, Amparo; Alamá, Pilar; Mateu, Emilia; Pellicer, Antonio

2017-01-01

This study has analysed the relationship between ovarian response and the number of euploid embryos. This is a post hoc analysis of a subset of data generated during a prospective cohort study previously published. Forty-six oocyte donors were subjected to ovarian stimulation with 150 IU of rFSH and 75 IU of hp-hMG in a GnRH agonist long protocol. Preimplantation genetic screening was performed in all viable embryos. We observed a positive relationship between ovarian response and the number of euploid embryos. When ovarian response was above the median (≥17 oocytes), the mean number of euploid embryos per donor was 5.0 ± 2.4, while when <17 oocytes were obtained the mean number of euploid embryos was 2.7 ± 1.4 ( p = 0.000). Aneuploidy rate did not increase with ovarian response or gonadotropin doses. Also, the number of euploid embryos was inversely related to the amount of gonadotropins needed per oocyte obtained (ovarian sensitivity index). These results suggest that the number of euploid embryos available for embryo transfer increases as the number of oocytes obtained does. Considering the total number of euploid embryos seems more relevant than the aneuploidy rate.

New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization

PubMed Central

Cobo, Ana Cristina; Milán, Miguel; Al-Asmar, Nasser; García-Herrero, Sandra; Mir, Pere; Simón, Carlos

2014-01-01

The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2%) and pregnancy rates per transfer (range: 46.0–62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation. PMID:24877108

Sensitivity of embryos of chicken, domestic duck, and common eider duck to polychlorinated and non-halogenated aromatic hydrocarbons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brunstroem, B.

Embryos of chicken (Gallus domesticus), domestic duck (Anas platyrhynchos), and common eider duck (Somateria mollissima) were exposed in ovo to PCBs and to polycyclic aromatic hydrocarbons. Two coplanar PCBs, 3,3{prime},4,4{prime}-tetrachlorobiphenyl (PCB {number_sign}77) and 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB {number_sign}126), were considerably more lethal and potent as inducers of 7-ethoxyresorufin O-deethylase (EROD) in chicken embryos (Gallus domesticus) than in embryos of the other two species. In chicken embryos, these compounds caused edema and eye and beak deformities. An artificial mixture of 18 PAHs which all have been detected in environmental samples, was slightly more toxic to embryos of the domestic duck and the commonmore » eider duck than to chicken embryos. The most potent compound in the mixture was benzo(k)fluoranthene. When chicken embryo livers were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro, EROD was induced by very low concentrations and the EC{sub 50} value obtained was 5 {times} 10{sup {minus}12} M. Livers from embryos of eider ducks and domestic ducks were 2--4 orders of magnitude less responsive to TCDD than chicken embryo livers in terms of EROD induction in vitro.« less

Human chorionic gonadotropin (hCG) in the secretome of cultured embryos: hyperglycosylated hCG and hCG-free beta subunit are potential markers for infertility management and treatment.

PubMed

Butler, Stephen A; Luttoo, Jameel; Freire, Maísa O T; Abban, Thomas K; Borrelli, Paola T A; Iles, Ray K

2013-09-01

Human chorionic gonadotropin (hCG) is produced by trophoblast cells throughout pregnancy, and gene expression studies have indicated that hCG-beta subunit (hCGβ) expression is active at the 2 blastomere stage. Here, we investigated the qualitative hCG output of developing embryos in culture and hCG isoforms expressed in the secretome as a novel sensitive method for detecting hCG. Culture media was collected from the culture plates of 118 embryos in culture (including controls and embryos at different stages of culture) from 16 patients undergoing routine fertility treatment. The hCGβ was detectable in media from 2 pronuclear (2PN) stage embryos through to the blastocyst stage. The hCGβ was absent in 1PN and arrested embryos as well as all media controls. Prior to hatching, hyperglycosylated hCG (hCGh) was observed selectively in 3PN embryos, but after hatching, along with hCG, became the dominant hCG molecule observed. We have reported at the 2PN stage the earliest evidence of hCGβ expression in embryos. There is a suggestion this may be indicative of quality in early embryos, and hCGh seen at the pronuclear stage may suggest triploid abnormality. The dominance of hCG, and hCGh expression, seen after blastocyst hatching may be indicative of potential implantation success. Thus, hCG isoforms have potential roles as biomarkers of embryo viability for embryo/blastocyst transfer.

Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos

PubMed Central

Szabó, Katalin; Budai, Csilla; Losonczi, Eszter; Bernáth, Gergely; Csenki-Bakos, Zsolt; Urbányi, Béla; Pribenszky, Csaba; Horváth, Ákos; Cserepes, Judit

2017-01-01

Background Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. Methods In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. Results Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. Conclusions Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development. PMID:28166301

Association between amino acid turnover and chromosome aneuploidy during human preimplantation embryo development in vitro

PubMed Central

Picton, Helen M.; Elder, Kay; Houghton, Franchesca D.; Hawkhead, Judith A.; Rutherford, Anthony J.; Hogg, Jan E.; Leese, Henry J.; Harris, Sarah E.

2010-01-01

This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during development in vitro. One hundred and eighty-eight fresh and cryopreserved embryos from 59 patients (33.9 ± 0.6 years) were cultured for 2–5 days. The turnover of 18 amino acids was measured in spent media by high-performance liquid chromatography. Embryos were either fixed for interphase fluorescent in situ hybridization analysis of chromosomes 13, 18, 19, 21, X or Y, or were assayed for mitochondrial activity. Amino acid turnover was different (P < 0.05) between stage-matched fresh and cryopreserved embryos due to blastomere loss following warming. The proportion of embryos with aneuploid cells increased as cell division progressed from pronucleate- (23%) to late cleavage stages (50–70%). Asparagine, glycine and valine turnover was significantly different between uniformly genetically normal and uniformly abnormal embryos on Days 2–3 of culture. By Days 3–4, the profiles of serine, leucine and lysine differed between uniformly euploid versus aneuploid embryos. Gender significantly (P < 0.05) affected the metabolism of tryptophan, leucine and asparagine by cleavage-stage embryos. Pronucleate zygotes had a significantly higher proportion of active:inactive mitochondria compared with cleavage-stage embryos. Furthermore, mitochondrial activity was correlated (P < 0.05) with altered aspartate and glutamine turnover. These results demonstrate the association between the metabolism, cytogenetic composition and health of human embryos in vitro. PMID:20571076

PreImplantation factor (PIF) protects cultured embryos against oxidative stress: relevance for recurrent pregnancy loss (RPL) therapy.

PubMed

Goodale, Lindsay F; Hayrabedran, Soren; Todorova, Krassimira; Roussev, Roumen; Ramu, Sivakumar; Stamatkin, Christopher; Coulam, Carolyn B; Barnea, Eytan R; Gilbert, Robert O

2017-05-16

Recurrent pregnancy loss (RPL) affects 2-3% of couples. Despite a detailed work-up, the etiology is frequently undefined, leading to non-targeted therapy. Viable embryos and placentae express PreImplantation Factor (PIF). Maternal circulating PIF regulates systemic immunity and reduces circulating natural killer cells cytotoxicity in RPL patients. PIF promotes singly cultured embryos' development while anti-PIF antibody abrogates it. RPL serum induced embryo toxicity is negated by PIF. We report that PIF rescues delayed embryo development caused by <3 kDa RPL serum fraction likely by reducing reactive oxygen species (ROS). We reveal that protein disulfide isomerase/thioredoxin (PDI/TRX) is a prime PIF target in the embryo, rendering it an important ROS scavenger. The 16F16-PDI/TRX inhibitor drastically reduced blastocyst development while exogenous PIF increased >2 fold the number of embryos reaching the blastocyst stage. Mechanistically, PDI-inhibitor preferentially binds covalently to oxidized PDI over its reduced form where PIF avidly binds. PIF by targeting PDI/TRX at a distinct site limits the inhibitor's pro-oxidative effects. The >3kDa RPL serum increased embryo demise by three-fold, an effect negated by PIF. However, embryo toxicity was not associated with the presence of putative anti-PIF antibodies. Collectively, PIF protects cultured embryos both against ROS, and higher molecular weight toxins. Using PIF for optimizing in vitro fertilization embryos development and reducing RPL is warranted.

The preparation of Drosophila embryos for live-imaging using the hanging drop protocol.

PubMed

Reed, Bruce H; McMillan, Stephanie C; Chaudhary, Roopali

2009-03-13

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

PubMed Central

Balbach, Sebastian Thomas; Esteves, Telma Cristina; Houghton, Franchesca Dawn; Siatkowski, Marcin; Pfeiffer, Martin Johannes; Tsurumi, Chizuko; Kanzler, Benoit; Fuellen, Georg; Boiani, Michele

2012-01-01

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development. PMID:22530006

γ-Oryzanol, tocol and mineral compositions in different grain fractions of giant embryo rice mutants.

PubMed

Jeng, Toong Long; Shih, Yi Ju; Ho, Pei Tzu; Lai, Chia Chi; Lin, Yu Wen; Wang, Chang Sheng; Sung, Jih Min

2012-05-01

Rice embryo is concentrated with lipid, protein and some bioactive chemicals. Two rice mutants IR64-GE and TNG71-GE (M7 generation) were characterised by an enlarged embryo compared with their wild types. In the present study, distributions of protein, lipid, total phenolics, γ-oryzanol, tocols and some essential minerals in these two giant embryo mutants and their respective normal embryo wild types IR64 and TNG71 were compared. The embryo dry weights of giant embryo mutants IR64-GE and TNG71-GE were 0.92 and 1.32 mg per seed respectively. These values were higher than those of their respective normal embryo genotypes (0.50 and 0.62 mg per seed). Large variations in protein, lipid, phenolic, γ-oryzanol, tocol and minerals levels were found between mutant and wild-type pairs. The brown rice of TNG71-GE had higher total γ-oryzanol (average of 24% increase) and total tocol (average of 75% increase) levels than TNG71, IR64 and IR64-GE. The embryo and bran parts of giant embryo mutant TNG71-GE were found to be good sources of vitamin E and γ-oryzanol. Therefore it could be used to produce high-value by-products from milled embryo and bran parts and as a genetic resource for rice improvement programmes. TNG71-GE can also be used as a nutrient-fortified rice cultivar. Copyright © 2011 Society of Chemical Industry.

Oxygen regulates amino acid turnover and carbohydrate uptake during the preimplantation period of mouse embryo development.

PubMed

Wale, Petra L; Gardner, David K

2012-07-01

Oxygen is a powerful regulator of preimplantation embryo development, affecting gene expression, the proteome, and energy metabolism. Even a transient exposure to atmospheric oxygen can have a negative impact on embryo development, which is greatest prior to compaction, and subsequent postcompaction culture at low oxygen cannot alleviate this damage. In spite of this evidence, the majority of human in vitro fertilization is still performed at atmospheric oxygen. One of the physiological parameters shown to be affected by the relative oxygen concentration, carbohydrate metabolism, is linked to the ability of the mammalian embryo to develop in culture and remain viable after transfer. The aim of this study was, therefore, to determine the effect of oxygen concentration on the ability of mouse embryos to utilize both amino acids and carbohydrates both before and after compaction. Metabolomic and fluorometric analysis of embryo culture media revealed that when embryos were exposed to atmospheric oxygen during the cleavage stages, they exhibited significantly greater amino acid utilization and pyruvate uptake than when cultured under 5% oxygen. In contrast, postcompaction embryos cultured in atmospheric oxygen showed significantly lower mean amino acid utilization and glucose uptake. These metabolic changes correlated with developmental compromise because embryos grown in atmospheric oxygen at all stages showed significantly lower blastocyst formation and proliferation. These findings confirm the need to consider both embryo development and metabolism in establishing optimal human embryo growth conditions and prognostic markers of viability, and further highlight the impact of oxygen on such vital parameters.

Insights on blastomere nuclearity.

PubMed

Gil, Mónica; D'Ommar, Gustavo; Póo, Maria E; Sosa, Anna; Piras, Marta; Piras, Romano; Rísquez, Francisco

2007-01-01

To analyze the results of our transferred embryos, especially those that "changed" their blastomere nuclearity from Multinucleated (MN) to Mono-nucleated during development. Pregnancies where at least one MN embryo was transferred were retrospectively evaluated and categorized in order to record and follow-up on the ones that were implanted. Embryos were classified as normal (when all blastomeres were mono-nucleated on day one and two of development), corrected (multinucleated embryos on day one that became mono-nucleated on day two) and non-corrected (multinucleated either on day one, on day two or both days). There were 633 transfer cycles analyzed. Thirty-three percent (206) had at least one embryo with a MN blastomere at a given stage of development. Pregnancy and implantation rates were 29.0% and 19.0% for the group of exclusively mono-nucleated embryo transfers, and 28.6% and 15.8% for the group with at least one MN embryo transferred. The pregnancy outcome for "corrected" and "non-corrected" embryos could be corroborated unequivocally in only 9 cases, with an outcome of 8 and 4 normal babies, respectively. Because the amount of data analyzed is not satisfactorily large, differences were not significantly different; however, a trend may exist showing that normal at term pregnancies obtained from corrected embryos are more likely to occur than those from non-corrected embryos. Nuclear observation on a daily basis should be one of the strategies used to select the best embryos for transferring, to improve implantation rates and avoid multiple pregnancies.

Anomalous oxygen consumption in porcine somatic cell nuclear transfer embryos.

PubMed

Sugimura, Satoshi; Yokoo, Masaki; Yamanaka, Ken-ichi; Kawahara, Manabu; Moriyasu, Satoru; Wakai, Takuya; Nagai, Takashi; Abe, Hiroyuki; Sato, Eimei

2010-08-01

Oxygen consumption reflects overall metabolic activity of mammalian embryos. We measured oxygen consumption in individual porcine somatic cell nuclear transfer (SCNT) and in vitro-fertilized (IVF) embryos by modified scanning electrochemical microscopy. Oxygen consumption in IVF embryos rapidly increased at day 5 of the blastocyst stage (D5BL). IVF embryos that consumed >0.81 x 10(14)/mol sec(-1) of oxygen at D5BL exhibited significantly higher hatching and hatched rates at D7BL, whereas D5BL SCNT embryos using porcine fetal fibroblasts did not show an increase in oxygen consumption until D7BL. The numbers of inner cell mass and trophectoderm (TE) cells and incidence of apoptosis did not significantly differ between IVF and SCNT embryos at D5BL. At D7BL, a significant lower number of TE cell and higher incidence of apoptosis were observed in SCNT than in IVF embryos; this significantly correlated with their oxygen consumption at D5BL. Use of cumulus cells as donor cells neutralized the low oxygen consumption in SCNT embryos at D5BL, regardless of the difference between the recipient cytoplasm and donor nucleus. Some of SCNT embryos at D7BL were retrieved the hatching completion and were improved the number of TE cell and apoptosis incidence by using cumulus cells. Thus, anomalous oxygen consumption in porcine SCNT embryos at D5BL could be sign of limited hatchability, which may be responsible for the low TE cell number and high apoptosis incidence.

Simple and efficient production of embryonic stem cell-embryo chimeras by coculture.

PubMed Central

Wood, S A; Pascoe, W S; Schmidt, C; Kemler, R; Evans, M J; Allen, N D

1993-01-01

A method for the production of embryonic stem (ES) cell-embryo chimeras was developed that involves the simple coculture of eight-cell embryos on a lawn of ES cells. After coculture, the embryos with ES cells attached are transferred to normal embryo culture medium and allowed to develop to the blastocyst stage before reimplantation into foster mothers. Although the ES cells initially attach to the outside of the embryos, they primarily colonize the inner cell mass and its derivatives. This method results in the efficient production of chimeras with high levels of chimerism including the germ line. As embryos are handled en masse and manipulative steps are minimal, this method should greatly reduce the time and effort required to produce chimeric mice. Images Fig. 1 Fig. 2 PMID:8506303

Maize embryogenesis.

PubMed

Fontanet, Pilar; Vicient, Carlos M

2008-01-01

Plant embryo development is a complex process that includes several coordinated events. Maize mature embryos consist of a well-differentiated embryonic axis surrounded by a single massive cotyledon called scutellum. Mature embryo axis also includes lateral roots and several developed leaves. In contrast to Arabidopsis, in which the orientation of cell divisions are perfectly established, only the first planes of cell division are predictable in maize embryos. These distinctive characteristics joined to the availability of a large collection of embryo mutants, well-developed molecular biology and tissue culture tools, an established genetics and its economical importance make maize a good model plant for grass embryogenesis. Here, we describe basic concepts and techniques necessary for studying maize embryo development: how to grow maize in greenhouses and basic techniques for in vitro embryo culture, somatic embryogenesis and in situ hybridization.

Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers.

PubMed

Lopes-da-Costa, L; Chagas e Silva, J; Deloche, M C; Jeanguyot, N; Humblot, P; Horta, A E M

2011-08-01

The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size at transfer (whole or demi embryo) on Day 7 of the estrous cycle (Day 0 = estrus) and P4 supplementation between Days 7 to 19 through an intravaginal device (yes or no) on plasma P4 and PSPB concentrations and on embryo measurements. Plasma P4 concentrations were measured by RIA on Days 0, 7, 14, 19, 21, 25, 35, 42, 49, 56 and 63 of pregnancy and, PSPB concentrations were measured by ELISA on Days 7, 21, 25, 35, 42, 49, 56 and 63. The presence of an embryonic vesicle was detected on Day 25, embryonic/fetal movements and heartbeat were evaluated on Days 42 and 63 and embryo measurements [crown-rump length (CRL) and width at mid body] were obtained on Day 42 through ultrasonography. In non-supplemented pregnancies, Day 42 whole embryos had higher (P < 0.05) CRL and width than demi embryos, but the difference averaged only 1 to 2 mm. In P4 supplemented pregnancies, whole and demi embryos attained a similar size on Day 42 of pregnancy. Embryo size at transfer, early exogenous P4 supplementation and their interactions had no effects (P > 0.05) on plasma P4 concentrations. However, the post-hoc LSD evaluation showed that plasma P4 concentrations on Day 25 were higher (P < 0.001) in whole than in demi embryo derived pregnancies and, that exogenous P4 supplementation increased (P < 0.05) plasma P4 concentrations on Day 19 of pregnancy. The plasma PSPB detection rate on Days 7 to 63 of pregnancy was similar in pregnancies resulting from the transfer of whole and demi embryos. From a total of 93 recipients remaining pregnant until Day 63, plasma PSPB was constantly undetectable on Day 7, was detected in 4% of Day 21 samples, 41% of Day 25, 95% of Day 35, 96% of Day 42, 99% of Day 49 and in 100% of samples of Days 56 and 63. Concentrations of PSPB increased (P < 0.05) from Days 21 to 42 and from Days 56 to 63, with a plateau between Days 42 to 56. Demi embryo pregnancies had higher (P < 0.05) plasma PSPB concentrations on Days 35 and 42 than whole embryo pregnancies. Progesterone supplementation had a positive effect (P < 0.01) on PSPB concentrations from Days 35 to 63. Concentrations of PSPB were similar in non-supplemented whole and demi embryo pregnancies from Days 7 to Day 63. In contrast, in supplemented recipients, demi embryo pregnancies had higher (P < 0.05) PSPB concentrations on Days 25 to 42 than whole embryo pregnancies. No significant correlation was found between P4 and PSPB concentrations or between the concentrations of these hormones and embryonic measurements on Day 42. In conclusion, demi embryos experienced a compensatory growth until Day 42 of pregnancy, attaining a similar size to that of whole embryos and originating conceptuses producing similar plasma PSPB concentrations to those of whole embryo derived conceptuses. Embryonic growth and conceptus secretion of PSPB were positively stimulated by early pregnancy exogenous P4 treatment. Copyright © 2011 Elsevier Inc. All rights reserved.

Glutamine supplementation enhances development of in vitro-produced porcine embryos and increases leucine consumption from the medium.

PubMed

Chen, Paula R; Redel, Bethany K; Spate, Lee D; Ji, Tieming; Salazar, Shirley Rojas; Prather, Randall S

2018-05-31

Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with +Gln or +Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P < 0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism were corrected when embryos were cultured with +Gln or +Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with +Max and increased consumption of leucine by embryos cultured with +Gln or +Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with +Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with +Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.

Survival of mouse embryos after vitrification depending on the cooling rate of the cryoprotectant solution.

PubMed

Hredzák, R; Ostró, A; Zdilová, Viera; Maracek, I; Kacmárik, J

2006-03-01

The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196 degrees C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitro culturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitro without freezing in a culturing medium in an environment consisting of 95% air and 5% CO2. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitro culturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival.

Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development1[W

PubMed Central

Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

2007-01-01

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159

Culture of bovine embryos on a polydimethylsiloxane (PDMS) microwell plate.

PubMed

Akagi, Satoshi; Hosoe, Misa; Matsukawa, Kazutsugu; Ichikawa, Akihiko; Tanikawa, Tamio; Takahashi, Seiya

2010-08-01

We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.

Influence of "Solcoseryl" during culture on the sex-dependent repair of bovine demi-embryos.

PubMed

Tominaga, K; Yoneda, K; Utsumi, K

1996-03-01

The purpose of this experiment was to determine the effect of culture conditions on the development of split embryos after bisection and on the sex ratio of resultant bovine demi-embryos. Embryos that had developed into blastocysts on days 6 1/2 to 7 or on days 7 1/2 to 8 from oocytes matured and fertilized in vitro were bisected in BMOC-3 medium supplemented with 33% calf serum. The medium also contained 0%, 0.1% or 1.0% Solcoseryl, a deproteinized hemodialysate product from calf blood. The demi-embryos were first cultured for 4 hours in the same medium in which they had been bisected and then co-cultured with cumulus cells in TCM199 supplemented with 1% calf serum for an additional 20 hr. The rate of production of good to excellent quality demi-embryos obtained from days 6 1/2 to 7 blastocysts was higher than from those on days 7 1/2 to 8. The rate was also significantly improved when blastocysts were bisected in medium containing 0.1% or 1.0% Solcoseryl, compared to the medium without Solcoseryl. Male embryos seemed to recover more rapidly than female embryos, as assessed by morphological quality at 4 hr, although the quality of female embryos had improved by 24 hr. The percentage of males after culture was higher in the medium without Solcoseryl than in its presence. Thus, addition of Solcoseryl at either 0.1% or 1.0% to BMOC-3 medium seemed to improve the production efficiency of good quality demi-embryos, but did not influence the sex ratio. It appears as if female demi-embryos required more time than male embryos to be repaired after bisection.

The Effect of Vitrification and in vitro Culture on the Adenosine Triphosphate Content and Mitochondrial Distribution of Mouse Pre-Implantation Embryos

PubMed Central

Amoushahi, Mahboobeh; Salehnia, Mojdeh; HosseinKhani, Saman

2013-01-01

Background: The mitochondria are an important source of adenosine triphosphate (ATP) production in pre-implantation embryo. Therefore, the objective of this study was to investigate the effect of vitrification and in vitro culture of mouse embryos on their mitochondrial distribution and ATP content. Methods: The embryos at 2-PN, 4-cell and blastocyst stages were collected from the oviduct of stimulated pregnant mice and uterine horns. Then, the embryos were vitrified with the cryotop method using ethylene glycol and dimethylsulphoxide. After evaluating the survival rates of vitrified embryos, their development to hatching stages were assessed. The ATP content of collected in vivo and in vitro embryos at different stages was measured by luciferin-luciferase bioluminescence assay. The distribution of mitochondria was studied using Mito-tracker green staining under a fluorescent microscope. Results: The survival rates of vitrified embryos at 2-PN, 4-cell and early blastocyst stages were 84.3, 87.87 and 89.89%, respectively. The hatching rates in previous developmental stages in vitrified group were 57.44, 66.73 and 70.89% and in non-vitrified group were 66.32, 73.25 and 75.89%, respectively (P>0.05). The ATP content of in vivo or in vitro collected embryos was not significantly different in both vitrified and non-vitrified groups (P>0.05). Mitochondrial distribution of vitrified and non-vitrified 2-PN embryos was similar, but some clampings or large aggregation of mitochondria within the vitrified 4-cell embryos was prominent. Conclusions: Vitrification method did not affect the mouse embryo ATP content. Also, the cellular stress was not induced by this procedure and the safety of vitrification was shown. PMID:23748889

Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.

PubMed

Ferreira, A R; Machado, G M; Diesel, T O; Carvalho, J O; Rumpf, R; Melo, E O; Dode, M A N; Franco, M M

2010-07-01

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8- to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. (c) 2010 Wiley-Liss, Inc.

Natural Selection of Human Embryos: Decidualizing Endometrial Stromal Cells Serve as Sensors of Embryo Quality upon Implantation

PubMed Central

Teklenburg, Gijs; Salker, Madhuri; Molokhia, Mariam; Lavery, Stuart; Trew, Geoffrey; Aojanepong, Tepchongchit; Mardon, Helen J.; Lokugamage, Amali U.; Rai, Raj; Landles, Christian; Roelen, Bernard A. J.; Quenby, Siobhan; Kuijk, Ewart W.; Kavelaars, Annemieke; Heijnen, Cobi J.; Regan, Lesley; Brosens, Jan J.; Macklon, Nick S.

2010-01-01

Background Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown. Methodology/Principal Findings We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1β, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo. Conclusions Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies. PMID:20422011

Birth of correctly genotyped calves after multiplex marker detection from bovine embryo microblade biopsies.

PubMed

Peippo, Jaana; Viitala, Sirja; Virta, Jouni; Räty, Mervi; Tammiranta, Niina; Lamminen, Terttu; Aro, Johanna; Myllymäki, Hannu; Vilkki, Johanna

2007-11-01

We report a method for multiplex genotyping of bovine embryo microblade biopsies. We have tested the reliability of the method and the viability of the embryos in vitro and in vivo. Two polymorphic gene markers (GHR F279Y and PRLR S18N) associated with milk production traits and one marker for sex diagnosis (ZFX/ZFY) were genotyped simultaneously with a method that combines nested PCR and allelic discrimination. To test the accuracy of genotyping, in the first experiment the genotypes of 134 biopsies from in vitro produced embryos were compared to genotypes determined from the corresponding embryos after biopsy. The method proved to be highly accurate as only in three cases (two for PRLR S18N and one for GHR F279Y) out of 395 genotypes the genotype was in disagreement between the two samples. The viability of similarly biopsied embryos was tested in parallel: after 24-hr culture 94.6% of embryos recovered in vitro. In the second experiment, a total of 150 in vivo-produced embryos were biopsied on Day 7 and genotyped. After the genotyping results were obtained on Day 8, female embryos were selected for transfer. From a total of 57 selected embryos 43 were transferred individually and 14 as pairs. After single embryo transfers, 19 recipients became pregnant and after embryo transfers in pairs one became pregnant. The success of genotyping was tested with the genotypes of donors and bulls and also from the hair samples of born calves. All calves were females and of the same genotypes determined from the biopsy. (c) 2007 Wiley-Liss, Inc.

Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm, and testa

PubMed Central

Winkelmann, Traud; Ratjens, Svenja; Bartsch, Melanie; Rode, Christina; Niehaus, Karsten; Bednarz, Hanna

2015-01-01

Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified. Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos. PMID:26300898

Developmental kinetics of pig embryos by parthenogenetic activation or by handmade cloning.

PubMed

Li, J; Li, R; Liu, Y; Villemoes, K; Purup, S; Callesen, H

2013-10-01

The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed. © 2013 Blackwell Verlag GmbH.

Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos.

PubMed

Dos Santos-Neto, P C; Cuadro, F; Barrera, N; Crispo, M; Menchaca, A

2017-10-01

The objective was to evaluate pregnancy outcomes and birth rate of in vivo derived vs. in vitro produced ovine embryos submitted to different cryopreservation methods. A total of 197 in vivo and 240 in vitro produced embryos were cryopreserved either by conventional freezing, or by vitrification with Cryotop or Spatula MVD methods on Day 6 after insemination/fertilization. After thawing/warming and transfer, embryo survival rate on Day 30 of gestation was affected by the source of the embryos (in vivo 53.3%, in vitro 20.8%; P < 0.05) and by the method of cryopreservation (conventional freezing 26.5%, Cryotop 52.0%, Spatula MVD 22.2%; P < 0.05). For in vivo derived embryos, survival rate after embryo transfer was 45.6% for conventional freezing, 67.1% for Cryotop, and 40.4% for Spatula MVD. For in vitro produced embryos, survival rate was 7.3% for conventional freezing, 38.7% for Cryotop, and 11.4% for Spatula MVD. Fetal loss from Day 30 to birth showed a tendency to be greater for in vitro (15.0%) rather than for in vivo produced embryos (5.7%), and was not affected by the cryopreservation method. Gestation length, weight at birth and lamb survival rate after birth were not affected by the source of the embryo, the cryopreservation method or stage of development (average: 150.5 ± 1.8 days; 4232.8 ± 102.8 g; 85.4%; respectively). This study demonstrates that embryo survival and birth rate of both in vivo and in vitro produced ovine embryos are improved by vitrification with the minimum volume Cryotop method. Copyright © 2017 Elsevier Inc. All rights reserved.

Parental genetic material and oxygen concentration affect hatch dynamics of mouse embryo in vitro.

PubMed

Zhan, Shaoquan; Cao, Shanbo; Du, Hongzi; Sun, Yuan; Li, Li; Ding, Chenhui; Zheng, Haiyan; Huang, Junjiu

2018-04-21

Hatching is crucial for mammalian embryo implantation, since difficulties during this process can lead to implantation failure, ectopic pregnancy and consequent infertility. Despite years of intensive researches, how internal and external factors affecting embryo hatch are still largely unclear. The effects of parental genetic material and oxygen concentration on hatch process were examined. Fertilized and parthenogenetic mouse preimplantation embryos were cultured in vitro under 5 and 20% oxygen for 120 h. Zona pellucida drilling by Peizo micromanipulation were performed to resemble the breach by sperm penetration. Firstly, parthenogenetic embryos had similarly high blastocyst developmental efficiency as fertilized embryos, but significantly higher hatch ratio than fertilized embryos in both O 2 concentrations. 5% O 2 reduced the hatch rate of fertilized embryos from 58.2 to 23.8%, but increased that of parthenogenetic embryos from 81.2 to 90.8% significantly. Analogously, 5% O 2 decreased the ratio of Oct4-positive cells in fertilized blastocysts, whereas increased that in parthenogenetic blastocysts. Additionally, 5% O 2 increased the total embryonic cell number in both fertilized and parthegenetic embryos, when compared to 20% O 2 , and the total cell number of fertilized embryos was also higher than that of parthegenetic embryos, despite O 2 concentration. Real-time PCR revealed that the expression of key genes involving in MAPK pathway and superoxide dismutase family might contribute to preimplantation development and consequent blastocyst hatch in vitro. Finally, we showed that fertilized and parthenogenetic embryos have diverse hatch dynamics in vitro, although the zona pellucida integrity is not the main reason for their mechanistic differences. Both parental genetic material and O 2 concentration, as the representative of intrinsic and extrinsic factors respectively, have significant impacts on mouse preimplantation development and subsequent hatch dynamics, probably by regulating the gene expression involving in MAPK pathway and superoxide dismutase family to control embryonic cell proliferation and allocation of ICM cells.

Expression of microRNAs in bovine and human pre-implantation embryo culture media

PubMed Central

Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

2014-01-01

MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

Chronic effects of silver exposure on ion levels, survival, and silver distribution within developing rainbow trout (Oncorhynchus mykiss) embryos.

PubMed

Guadagnolo, C M; Brauner, C J; Wood, C M

2001-03-01

Rainbow trout embryos were chronically exposed to silver (as AgNO3) in moderately hard water (120 mg CaCO3/L, 0.70 mM Cl-, 1.3 mg/L dissolved organic matter. 12.3+/-0.1 degrees C) at nominal concentrations of 0.1, 1, and 10 microg/L (measured = 0.117+/-0.008, 1.22+/-0.16, and 13.51+/-1.58 microg/L, respectively) to investigate the effects on mortality, ionoregulation, and silver uptake and distribution of the embryo. Mortalities in the low concentrations (0.1 and 1.2 microg/L) were not significantly different from controls throughout embryonic development (days 1-32 postfertilization). Mortalities of embryos in the 13.5-microg/L treatment reached 56% by day 32 postfertilization (33% when accounting for control mortality), by which time more than 50% of surviving embryos had hatched. Accumulation of silver in whole embryos of 1.2- and 13.5-microg/L treatments reached the highest concentrations of 0.13 and 0.24 microg/g total silver, respectively, by day 32, but whole embryo silver burden was not correlated with mortality. Silver concentrations in different compartments of the whole embryo (chorion, dissected embryo, and yolk) were greatest just before hatch and were higher in the chorion for all experimental treatments. Up to 85% of total whole embryo silver content was bound to the chorion, which acts as a protective barrier during silver exposure. Whole embryo Na+ concentration in the 13.5-microg/L treatment was significantly reduced relative to controls from days 23 to 32 postfertilization, and levels in the embryo were reduced by 40% at day 32 postfertilization, indicating that silver toxicity in the whole embryo is associated with an ion regulatory disturbance that is similar to the acute effect of AgNO3 in juvenile and adult trout.

Turtle embryos move to optimal thermal environments within the egg.

PubMed

Zhao, Bo; Li, Teng; Shine, Richard; Du, Wei-Guo

2013-08-23

A recent study demonstrated that the embryos of soft-shelled turtles can reposition themselves within their eggs to exploit locally warm conditions. In this paper, we ask whether turtle embryos actively seek out optimal thermal environments for their development, as do post-hatching individuals. Specifically, (i) do reptile embryos move away from dangerously high temperatures as well as towards warm temperatures? and (ii) is such embryonic movement due to active thermoregulation, or (more simply) to passive embryonic repositioning caused by local heat-induced changes in viscosity of fluids within the egg? Our experiments with an emydid turtle (Chinemys reevesii) show that embryos avoid dangerously high temperatures by moving to cooler regions of the egg. The repositioning of embryos is an active rather than passive process: live embryos move towards a heat source, whereas dead ones do not. Overall, our results suggest that behavioural thermoregulation by turtle embryos is genuinely analogous to the thermoregulatory behaviour exhibited by post-hatching ectotherms.

Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation.

PubMed

Zenker, Jennifer; White, Melanie D; Gasnier, Maxime; Alvarez, Yanina D; Lim, Hui Yi Grace; Bissiere, Stephanie; Biro, Maté; Plachta, Nicolas

2018-04-19

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Human endometrial cell coculture reduces the endocrine disruptor toxicity on mouse embryo development

PubMed Central

2012-01-01

Backgrounds Previous studies suggested that endocrine disruptors (ED) are toxic on preimplantation embryos and inhibit development of embryos in vitro culture. However, information about the toxicity of endocrine disruptors on preimplantation development of embryo in human reproductive environment is lacking. Methods Bisphenol A (BPA) and Aroclor 1254 (polychlorinated biphenyls) were used as endocrine disruptors in this study. Mouse 2-cell embryos were cultured in medium alone or vehicle or co-cultured with human endometrial epithelial layers in increasing ED concentrations. Results At 72 hours the percentage of normal blastocyst were decreased by ED in a dose-dependent manner while the co-culture system significantly enhanced the rate and reduced the toxicity of endocrine disruptors on the embryonic development in vitro. Conclusions In conclusion, although EDs have the toxic effect on embryo development, the co-culture with human endometrial cell reduced the preimplantation embryo from it thereby making human reproductive environment protective to preimplantation embryo from the toxicity of endocrine disruptors. PMID:22546201

Effect of Medium Salt Concentration on Differentiation and Maturation of Somatic Embryos of Cassava (Manihot esculenta Crantz)

PubMed Central

GROLL, J.; MYCOCK, D. J.; GRAY, V. M.

2002-01-01

Culture of cassava somatic embryos on media with an altered macro‐ and micro‐nutrient salt concentration affected embryo development and germination capability. In the tests, quarter‐, half‐, full‐ or double‐strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half‐ or full‐strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full‐strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half‐ and full‐strength MS medium yielded 8·3 and 8·6 germinants g–1 NEC tissue, respectively. For this important but often disregarded culture factor, either half‐ or full‐strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination. PMID:12099540

Persons and their bodies: how we should think about human embryos.

PubMed

McLachlan, Hugh V

2002-01-01

The status of human embryos is discussed particularly in the light of the claim by Fox, in Health Care Analysis 8 that it would be useful to think of them in terms of cyborg metaphors. It is argued that we should consider human embryos for what they are--partially formed human bodies--rather than for what they are like in some respects (and unlike in others)--cyborgs. However to settle the issue of the status of the embryo is not to answer the moral questions which arise concerning how embryos should be treated. Since persons rather than bodies have rights, embryos do not have rights. However, whether or not embryos have rights, people can have duties concerning them. Furthermore, the persons whose fully developed bodies embryos will, might (or might have) become can have rights. Contrary to what is often assumed, it is not merely persons who have (or have had) living, developed human bodies who have moral rights: so it is argued in this paper.

Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.

PubMed

Bolton, Helen; Graham, Sarah J L; Van der Aa, Niels; Kumar, Parveen; Theunis, Koen; Fernandez Gallardo, Elia; Voet, Thierry; Zernicka-Goetz, Magdalena

2016-03-29

Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic.

Turtle embryos move to optimal thermal environments within the egg

PubMed Central

Zhao, Bo; Li, Teng; Shine, Richard; Du, Wei-Guo

2013-01-01

A recent study demonstrated that the embryos of soft-shelled turtles can reposition themselves within their eggs to exploit locally warm conditions. In this paper, we ask whether turtle embryos actively seek out optimal thermal environments for their development, as do post-hatching individuals. Specifically, (i) do reptile embryos move away from dangerously high temperatures as well as towards warm temperatures? and (ii) is such embryonic movement due to active thermoregulation, or (more simply) to passive embryonic repositioning caused by local heat-induced changes in viscosity of fluids within the egg? Our experiments with an emydid turtle (Chinemys reevesii) show that embryos avoid dangerously high temperatures by moving to cooler regions of the egg. The repositioning of embryos is an active rather than passive process: live embryos move towards a heat source, whereas dead ones do not. Overall, our results suggest that behavioural thermoregulation by turtle embryos is genuinely analogous to the thermoregulatory behaviour exhibited by post-hatching ectotherms. PMID:23760168

TERRESTRIAL PLANET FORMATION AROUND THE CIRCUMBINARY HABITABLE ZONE: INWARD MIGRATION IN THE PLANETESIMAL SWARM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gong Yanxiang; Zhou Jilin; Xie Jiwei, E-mail: yxgong@nju.edu.cn, E-mail: zhoujl@nju.edu.cn

2013-01-20

According to the core accretion theory, circumbinary embryos can form only beyond a critical semimajor axis (CSMA). However, due to the relatively high density of solid materials in the inner disk, a significant amount of small planetesimals must exist in the inner zone when embryos form outside this CSMA. Thus, embryo migration induced by the planetesimal swarm is possible after gas disk depletion. Through numerical simulations, we found that (1) the scattering-driven inward migration of embryos is robust and planets can form in the habitable zone if we adopt a mass distribution of an MMSN-like disk; (2) the total massmore » of the planetesimals in the inner region and continuous embryo-embryo scattering are two key factors that cause significant embryo migrations; and (3) the scattering-driven migration of embryos is a natural water-delivery mechanism. We propose that planet detections should focus on the close binary with its habitable zone near CSMA.« less

Birth of normal infants after transfer of embryos that were twice vitrified/warmed at cleavage stages: report of two cases.

PubMed

Valle, Marcello; Guimarães, Fernando; Cavagnoli, Melissa; Sampaio, Marcos; Geber, Selmo

2012-12-01

The role of cryopreservation in assisted reproductive technology programs has increased within the last years allowing the transfer of a limited number of embryos and the storage of the remaining for future use. The reduction in the number of transferred embryos decreases the frequency of multiple pregnancy rates and of ovarian hyperstimulation syndrome while the cumulative pregnancy rate can be maximized. Moreover, as not all embryos will survive the warming process more cleavage stage embryos are warmed to improve selection for transfer. Therefore, surplus good quality cleavage stage embryos and/or blastocysts must be re-vitrified for further transfer to achieve pregnancy. To our knowledge, there have been no reports demonstrating that human embryos can be successfully vitrified/warmed twice at the cleavage stage. Thus we report two successful pregnancies and deliveries of healthy babies after transfer of embryos that were twice vitrified/warmed at 2-4 cells stage. Copyright © 2012 Elsevier Inc. All rights reserved.

Refrigeration of rainbow trout gametes and embryos.

PubMed

Babiak, Igor; Dabrowski, Konrad

2003-12-01

Prolonged access to early embryos composed of undifferentiated, totipotent blastomeres is desirable in situations when multiple collections of gametes are not possible. The objective of the present study is to examine whether the refrigeration of rainbow trout Oncorhynchus mykiss gametes and early embryos would be a suitable, reliable, and efficient tool for prolonging the availability of early developmental stages up to the advanced blastula stage. The study was conducted continuously during fall, winter, and spring spawning seasons. In all, more than 500 experimental variants were performed involving individual samples from 26 females and 33 males derived from three strains. These strains represented three possible circumstances. In optimal one, gametes from good quality donors were obtained soon after ovulation. In the two non-optimal sources, either donors were of poor genetic quality or gametes were collected from a distant location and transported as unfertilized gametes. A highly significant effect of variability of individual sample quality on efficiency of gamete and embryo refrigeration was revealed. The source of gametes significantly affected viability of refrigerated oocytes and embryos, but not spermatozoa. On average, oocytes from optimal source retained full fertilization viability for seven days of chilled storage, significantly longer than from non-optimal sources. Spermatozoa, regardless of storage method, retained full fertilization ability for the first week of storage. Refrigeration of embryos at 1.4+/-0.4 degrees C significantly slowed the development. Two- week-old embryos were still in blastula stage. Average survival rate of embryos refrigerated for 10 days and then transferred to regular incubation temperatures of 9-14 degrees C was 92% in optimal and 51 and 71% in non-optimal source variants. No effect of gamete and embryo refrigeration on the occurrence of developmental abnormalities was observed. Cumulative refrigeration of oocytes and embryos resulted in an average embryo survival rate of 71% in optimal source variants after 17 days of refrigeration (7 days oocytes+10 days embryos). The study shows that both gamete and embryo refrigeration can be successfully used as an efficient tool for prolonging availability of rainbow trout embryos in early developmental stages. Copyright 2003 Wiley-Liss, Inc.

Cholinomimetic teratogens. IV. Effects of the genotype for muscular dystrophy in chickens.

PubMed

Landauer, W; Clark, E M; Larner, M M

1976-12-01

Embryos of a family of chickens homozygous for muscular dystrophy (md/md) reacted with a higher incidence of malformations to treatment with carbachol than did White Leghorn embryos. The same difference in response of embryos from the two stocks occurred after treatment with sulfanilamide. Embryos of reciprocal crosses between these two stocks differed greatly, however, in their response to carbachol, F1 embryos from dystrophic hens producing a much higher incidence of malformations than did those from Leghorn hens. In contrast, both F1 sibs reacted similarly to sulfanilamide, the teratogenic effects being intermediate between those of embryos from the parent stocks.

Label-free characterization of vitrification-induced morphology changes in single-cell embryos with full-field optical coherence tomography

NASA Astrophysics Data System (ADS)

Zarnescu, Livia; Leung, Michael C.; Abeyta, Michael; Sudkamp, Helge; Baer, Thomas; Behr, Barry; Ellerbee, Audrey K.

2015-09-01

Vitrification is an increasingly popular method of embryo cryopreservation that is used in assisted reproductive technology. Although vitrification has high post-thaw survival rates compared to other freezing techniques, its long-term effects on embryo development are still poorly understood. We demonstrate an application of full-field optical coherence tomography (FF-OCT) to visualize the effects of vitrification on live single-cell (2 pronuclear) mouse embryos without harmful labels. Using FF-OCT, we observed that vitrification causes a significant increase in the aggregation of structures within the embryo cytoplasm, consistent with reports in literature based on fluorescence techniques. We quantify the degree of aggregation with an objective metric, the cytoplasmic aggregation (CA) score, and observe a high degree of correlation between the CA scores of FF-OCT images of embryos and of fluorescence images of their mitochondria. Our results indicate that FF-OCT shows promise as a label-free assessment of the effects of vitrification on embryo mitochondria distribution. The CA score provides a quantitative metric to describe the degree to which embryos have been affected by vitrification and could aid clinicians in selecting embryos for transfer.

Cryotop vitrification as compared to conventional slow freezing for human embryos at the cleavage stage: survival and outcomes.

PubMed

Lin, Tseng-Kai; Su, Jin-Tsung; Lee, Fa-Kung; Lin, Yu-Ru; Lo, Hsiao-Ching

2010-09-01

This study was conducted to compare the efficacy of cryotop vitrification of human cleavage-stage embryos to that of conventional slow freezing of these embryos with respect to survival. A second objective was to compare the two cryopreservation techniques with respect to outcomes for a cohort of women. Cleavage-stage embryos from 102 patients were cryopreserved either by vitrification (57 patients) or by traditional slow freezing (45 patients). After thawing, rates of embryo survival, implantation, and clinical pregnancy were determined. Survival of embryos was significantly higher with the vitrification procedure as compared to traditional slow freezing [287/298 (96.3%) vs. 294/446 (65.9%); p < 0.05). Rates of implantation and clinical pregnancy were also significantly higher using vitrification procedure as compared to the slow freezing procedure (24.3% vs. 7.1% and 35.6% vs. 15.6% respectively, p < 0.05). As compared to conventional slow freezing, cryopreservation of human cleavage-stage embryo using vitrification results in higher rates of embryo survival, implantation, and clinical pregnancy. Vitrification therefore represents the superior cryopreservation technique for cleavage-stage embryos. Copyright © 2010 Taiwan Association of Obstetric & Gynecology. Published by Elsevier B.V. All rights reserved.

Limited importance of pre-embryo pronuclear morphology (zygote score) in assisted reproduction outcome in the absence of embryo cryopreservation.

PubMed

Nicoli, Alessia; Valli, Barbara; Di Girolamo, Roberta; Di Tommaso, Barbara; Gallinelli, Andrea; La Sala, Giovanni B

2007-10-01

To investigate the hypothesis that Z-score criteria represent a reliable predictor of implantation rate and pregnancy outcome in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, excluding the possibility of embryo selection before the embryo transfer. Retrospective clinical study. Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova (ASMN), Reggio Emilia, Italy. We analyzed 393 pregnancies obtained by IVF or ICSI cycles. Morphologic evaluations of Z-score in pre-embryos obtained from IVF or ICSI cycles. Evaluations of Z-scores, implantation rate, and clinical pregnancy outcome. We did not find any statistically significant correlation between the Z-score of 1032 embryos transferred in 393 embryo transfers and the implantation rate or the pregnancy outcome. In particular, the best Z-score identified (Z1, 7.2%) did not seem to correlate with embryo implantation rate or pregnancy outcomes any better than those with worse scores (Z2, 6.9% and Z3, 85.9%). Our results seem to confirm that Z-score alone cannot be considered a better tool than standard morphologic criteria for identifying, controlling, or selecting embryos with a better chance of successful ongoing pregnancy.

EmbryoMiner: A new framework for interactive knowledge discovery in large-scale cell tracking data of developing embryos.

PubMed

Schott, Benjamin; Traub, Manuel; Schlagenhauf, Cornelia; Takamiya, Masanari; Antritter, Thomas; Bartschat, Andreas; Löffler, Katharina; Blessing, Denis; Otte, Jens C; Kobitski, Andrei Y; Nienhaus, G Ulrich; Strähle, Uwe; Mikut, Ralf; Stegmaier, Johannes

2018-04-01

State-of-the-art light-sheet and confocal microscopes allow recording of entire embryos in 3D and over time (3D+t) for many hours. Fluorescently labeled structures can be segmented and tracked automatically in these terabyte-scale 3D+t images, resulting in thousands of cell migration trajectories that provide detailed insights to large-scale tissue reorganization at the cellular level. Here we present EmbryoMiner, a new interactive open-source framework suitable for in-depth analyses and comparisons of entire embryos, including an extensive set of trajectory features. Starting at the whole-embryo level, the framework can be used to iteratively focus on a region of interest within the embryo, to investigate and test specific trajectory-based hypotheses and to extract quantitative features from the isolated trajectories. Thus, the new framework provides a valuable new way to quantitatively compare corresponding anatomical regions in different embryos that were manually selected based on biological prior knowledge. As a proof of concept, we analyzed 3D+t light-sheet microscopy images of zebrafish embryos, showcasing potential user applications that can be performed using the new framework.

Viability of bovine demi- and quarter-embryos after transfer.

PubMed

Bredbacka, P; Huhtinen, M; Aalto, J; Rainio, V

1992-07-01

The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6/13) for heifers and 33.3% (4/12) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8/13) for heifers and 8.3% (1/12) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

PubMed

Liu, C; Liu, Y; Larsen, K; Hou, Y P; Callesen, H

2018-01-01

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Yan; Liu, Chunying; Lu, Wenwen

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysismore » revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.« less