Parthenogenetic activation of buffalo (Bubalus bubalis) oocytes: comparison of different activation reagents and different media on their developmental competence and quantitative expression of developmentally regulated genes.

This study was carried out to compare the efficacy of different methods to activate buffalo A + B and C + D quality oocytes parthenogenetically and to study the in vitro developmental competence of oocytes and expression of some important genes at the different developmental stages of parthenotes. The percentage of A + B oocytes (62.16 ± 5.06%, range 53.8-71.3%) was significantly higher (P < 0.001) compared with that of C + D oocytes (37.8 ± 5.00%, range 28.6-46.1%) retrieved from slaughterhouse buffalo ovaries. Among all combinations, ethanol activation followed by culture in research vitro cleave medium gave the highest cleavage and blastocyst yields for both A + B and C + D grade oocytes. Total cell numbers, inner cell mass/trophectoderm ratio and apoptotic index of A + B group blastocysts were significantly different (P < 0.05) from their C + D counterpart. To determine the status of expression patterns of developmentally regulated genes, the expression of cumulus-oocyte complexes, fertilization, developmental competence and apoptotic-related genes were also studied in parthenogenetically produced buffalo embryos at different stages, and indicated that the differential expression patterns of the above genes had a role in early embryonic development.

Effects of treatment with a microRNA mimic or inhibitor on the developmental competence, quality, epigenetic status and gene expression of buffalo (Bubalus bubalis) somatic cell nuclear transfer embryos.

Expression levels of 13 microRNAs (miRNAs) were compared between buffalo blastocysts produced by somatic cell nuclear transfer through hand-made cloning and IVF to improve cloning efficiency. Expression of miR-22, miR-145, miR-374a and miR-30c was higher, whereas that of miR-29b, miR-101, miR-302b, miR-34a, miR-21 and miR-25 was lower, in nuclear transferred (NT) than IVF embryos; the expression of miR-200b, miR-26a and miR-128 was similar between the two groups. Based on these, miR-145, which is involved in the regulation of pluripotency, was selected for further investigation of NT embryos. miR-145 expression was lowest at the 2-cell stage, increased through the 4-cell stage and was highest at the 8-cell or morula stage in a pattern that was similar between NT and IVF embryos. miR-145 expression was higher in NT than IVF embryos at all stages examined. Treatment of reconstructed embryos 1h after electrofusion with an inhibitor of miR-145 for 1h decreased the apoptotic index and increased the blastocyst rate, total cell number, ratio of cells in the inner cell mass to trophectoderm, global levels of acetylation of histone 3 at lysine 18 and expression of Krueppel-like factor 4 (KLF4), octamer-binding transcription factor 4 (OCT4) and SRY (sex determining region Y)-box 2 (SOX2) in blastocysts. Treatment with an miR-145 mimic had the opposite effects. In conclusion, treatment of NT embryos with an miR-145 inhibitor improves the developmental competence and quality, and increases histone acetylation and expression of pluripotency-related genes.

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

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.

Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2'-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos.

We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50nM) and 5azadC (7.5nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.

Comparative Expression Analysis of Gametogenesis-Associated Genes in Foetal and Adult Bubaline (Bubalus bubalis) Ovaries and Testes.

This study was conducted to identify and analyse the expression of gametogenesis-associated genes and proteins in foetal and adult buffalo gonads of both the sexes. Relative quantification of the genes was determined by qPCR and Western blotting. Immunohistochemistry was also performed for various gametogenesis-associated proteins in foetal and adult gonads of both the sexes. We observed significantly (p < 0.05) increased expression of primordial germ cell-specific, meiotic as well as genes associated with oocyte maturation and development in foetal ovaries as compared to the adult ones. However, significantly (p < 0.05) increased expression of proteins associated with oocyte maturation like GDF9 and ZP4 was found in adult ovaries, indicating temporal regulation of mRNA translation during oogenesis. Meiotic genes showed significantly (p < 0.05) increased expression in adult testes as compared to foetal testes and ovaries, indicating onset of meiosis at a later stage in spermatogenesis. In general, the expression of primordial germ cell-associated as well as meiotic genes was higher in adult testes, indicating the increased biological activity in the organ. Immunohistochemistry revealed localized expression of gametogenesis-associated proteins in ovarian follicles and seminiferous tubules of testes, while the surrounding somatic tissues were devoid of these proteins. The study gives an understanding of the sequential and temporal events of gene expression as well as mRNA translation during male and female gametogenesis. It could also be concluded that follicles and seminiferous tubules are the functional units of the female and male gonads, respectively, and their function could be enhanced by appropriate chemical and genetic intervention of the somatic tissue immediately surrounding them. This assumes importance in the context that buffalo attains sexual maturity at an older age of 2-3 years and have smaller ovaries with lesser number of primordial follicles in comparison with cattle, which is suggested to be the main reason of their poor breeding performance.

Early cleavage of handmade cloned buffalo (Bubalus bubalis) embryos is an indicator of their developmental competence and quality.

Following IVF, embryos which cleave early have been shown to have higher developmental competence and quality than those that cleave relatively later across many species. We investigated the effect of time of cleavage on the developmental competence, quality, epigenetic status and gene expression in buffalo embryos produced by handmade cloning (HMC). Following classification of embryos as early cleaving (EC) or late cleaving (LC) based on whether they had cleaved or not at 24 h post in vitro culture, 54% (164/303) were found to be EC and the rest to be LC. The blastocyst rate (58.1 ± 3.4 vs 36.9 ± 1.6%, p < 0.01) and the total cell number (285.5 ± 41.9 vs 141.4 ± 36.1, p < 0.05) were higher, whereas the apoptotic index (3.6 ± 0.6 vs 12.2 ± 1.7, p < 0.01) and the global level of H3K9ac and H3K27me3 were lower (p < 0.05) in the blastocysts produced from EC than in those produced from LC embryos. The relative transcript level of CASPASE3, CASPASE7, DNMT1, DNMT3a and CDX2 was higher (p < 0.05) and that of SOX2 was lower (p < 0.05) in blastocysts produced from LC than in those produced from EC embryos, whereas the expression level of CASPASE6, P53, P21, HDAC1, OCT4 and NANOG was not significantly different between the two groups. These results show that (i) following HMC, blastocysts produced from embryos that cleave early differ from those produced from late cleaving embryos in terms of epigenetic status and expression level of many important apoptosis-, pluripotency-, trophectoderm- and epigenetics-related genes, and (ii) EC embryos are superior to LC embryos in view of their higher developmental competence and quality.

Production of wild buffalo (Bubalus arnee) embryos by interspecies somatic cell nuclear transfer using domestic buffalo (Bubalus bubalis) oocytes.

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin-18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open-pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re-expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified-warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.

Effect of TGF-ß1 superfamily members on survival of buffalo (Bubalus bubalis) embryonic stem-like cells.

This study examined the effects of supplementation of ES-like cell culture medium with bone morphogenetic protein (BMP)-4 (0, 10, 20 or 100 ng/ml) or Noggin (250, 500 or 750 ng/ml) or TGF-ß1 (0, 0.1, 1 or 10 ng/ml) or SB431542 (0, 10, 25 or 50 µm), an inhibitor of TGF-ß1 signalling, on survival, colony area and expression level of pluripotency genes in buffalo ES-like cells at passage 40-80, under different culture conditions. BMP-4 supplementation significantly reduced (p < 0.05) colony survival rate, percentage increase in colony area and relative mRNA abundance of OCT4, whereas that of NANOG and SOX-2 was increased significantly (p < 0.05). Noggin supplementation did not affect the colony survival rate and percentage increase in colony area in the presence of FGF-2 and LIF. In the presence of FGF-2 alone, it significantly reduced (p < 0.05) the relative mRNA abundance of OCT4 and SOX-2 and increased (p < 0.05) that of NANOG. Supplementation with TGF-ß1 at 1.0 ng/ml but not at other concentrations increased colony survival rate but had no effect on percentage increase in colony area at any concentration. Supplementation with SB-431542 decreased (p < 0.05) colony survival rate at 50 µm but not at other concentrations. The percentage increase in colony area was lower (p < 0.05) with 10 µm SB-431542 than that in the controls, whereas at higher concentrations of 25 or 50 µm, SB-431542 decreased (p < 0.05) the colony size instead of increasing it. In conclusion, these results suggest that BMP-4 induces differentiation in buffalo ES-like cells, whereas TGF-ß/activin/nodal pathway may not be playing a crucial role in maintaining pluripotency in these cells.

Buffalo (Bubalus bubalis) ES cell-like cells are capable of in vitro skeletal myogenic differentiation.

When buffalo embryonic stem (ES) cell-like cells that expressed surface markers SSEA-4, TRA-1-60, TRA-1-81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX-1 and NUCLEOSTEMIN as confirmed by RT-PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three-dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF-68 and NESTIN (ectodermal lineage), BMP-4 and α-skeletal actin (mesodermal lineage), and α-fetoprotein, GATA-4 and HNF-4 (endodermal lineage). When these EBs were cultured on gelatin-coated dishes, they spontaneously differentiated to several cell types such as epithelial- and neuron-like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10(-8) M or 10(-7) M retinoic acid for 25 days, ES cells could be directed to form muscle cell-like cells, the identity of which was confirmed by expression of α-actinin by immunofluorescence and of MYF-5, MYOD and MYOGENIN genes by RT-PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell-like cells to undergo directed differentiation to cells of skeletal myogenic lineage.

Cryopreservation of zona-free cloned buffalo (Bubalus Bubalis) embryos: slow freezing vs open-pulled straw vitrification.

This study was carried out to compare the post-thaw cryosurvival rate and the level of apoptosis in vitro produced zona-free cloned buffalo blastocysts subjected to slow freezing or vitrification in open-pulled straws (OPS). Zona-free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re-expansion rate following post-thaw culture for 22-24 h. The post-thaw re-expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen-thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non-cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non-cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona-free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.

Effect of physiologically relevant heat shock on development, apoptosis and expression of some genes in buffalo (Bubalus bubalis) embryos produced in vitro.

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8-cell, 16-cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8-16-cell and blastocyst stages, relative mRNA abundance of stress-related genes HSP 70.1 and HSP 70.2 and pro-apoptotic genes CASPASE-3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress-related gene HSF1. Expression level of anti-apoptotic genes BCL-XL and MCL-1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8-16-cell and blastocyst stages. Among the genes related to embryonic development, at 8-16-cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR-1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress-, apoptosis- and development-related genes.

In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell.

PURPOSE: Spermatogonial stem cells (SSCs) have the unique ability both to self-renew and to produce progeny that undergo differentiation to spermatozoa. The present study has been carried out to develop a method to purify and enrich the pure populations of spermatogonial stem cell like cells in buffalo. METHODS: The spermatogonial cells were isolated from testes of 3-7 month old buffalo calves and disaggregated by double enzymatic digestion. Mixed population of isolated cells were then plated on Datura stramonium agglutinin (DSA) lectin coated dishes for attachment of Sertoli cells. The desired cells were obtained from suspension medium after 18 h of incubation and then loaded on discontinuous density gradient using percoll (20-65 %) and different types of spermatogonia cells were obtained at interface of each layer. These cells were cultured in vitro. RESULTS: Spermatogonial cells isolated have spherical outline and two or three eccentrically placed nucleoli, created a colony after proliferation during first week or immediately after passage. After 7-10 days of culture, the resulted developed colonies of spermatogonial cells expressed the spermatogonial specific genes like Plzf and VASA; and other pluripotency related markers viz. alkaline phosphtase, DBA, CD9, CD90, SSEA-1, OCT-4, NANOG and REX-1. CONCLUSION: Our results show that the isolated putative spermatogonial stem cells exhibit the expression of pluripotency related and spermatogonial specific genes. This study may help to establish a long term culture system for buffalo spermatogonia.

Presence of nitric oxide synthase immunoreactivity and mRNA in buffalo (Bubalus bubalis) oocytes and embryos.

This study examined the presence of immunoreactivity and mRNA for different nitric oxide synthase (NOS) isoforms in immature and in vitro matured oocytes and in embryos at two-, four- and eight-cell, and morula and blastocyst stages in buffalo. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to in vitro maturation in TCM-199 + 10% FBS + 5 µg/ml pFSH + 1 µg/ml estradiol-17ß + 0.81 mm sodium pyruvate + 10% buffalo follicular fluid + 50 µg/ml gentamycin sulphate for 24 h in a CO(2) incubator (5% CO(2) in air) at 38.5°C. Following in vitro fertilization carried out by incubating them with 2-4 million spermatozoa/ml for 18 h, the presumed zygotes were cultured in mCR2aa medium containing 0.6% BSA and 10% FBS for up to 8 days post insemination. Immunofluorescence staining of NOS using antibodies that cross-reacted either with all the NOS isoforms i.e., universal (uNOS) or specifically with inducible (iNOS) or endothelial (eNOS) isoforms revealed that NOS was present in oocytes and embryos at all the stages examined. Examination of the semi-quantitative expression of NOS genes by RT-PCR revealed that the iNOS, eNOS and nNOS mRNA was present in the immature and mature oocytes and in all the embryonic stages examined. In conclusion, it was demonstrated in the present study that immunoreactivity and mRNA for different NOS isoforms was present in buffalo oocytes and pre-implantation stage embryos.

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

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.

Oxygen concentration and cysteamine supplementation during in vitro production of buffalo (Bubalus bubalis) embryos affect mRNA expression of BCL-2, BCL-XL, MCL-1, BAX and BID.

This study examined the effects of O(2) concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 µm, respectively; IVM, IVF and IVC carried out in 20% O(2)), on blastocyst rate and relative mRNA abundance of some apoptosis-related genes measured by real-time qPCR in immature and in vitro-matured buffalo oocytes and in embryos at 2-, 4-, 8- to 16-cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL-positive cells was significantly lower (p < 0.05) under 5% O(2) than that under 20% O(2). The mRNA expression of anti-apoptotic genes BCL-2 and MCL-1 was significantly higher (p < 0.05) and that of pro-apoptotic genes BAX and BID was lower (p < 0.05) under 5% O(2) than that under 20% O(2) concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL-XL and MCL-1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O(2) groups and in cysteamine supplemented vs controls. At the 8- to 16-cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL-2 and MCL-1 was highest under 5% O(2) concentration and that of BAX and BID was highest (p < 0.05) under 20% O(2) concentration. These results suggest that one of the mechanisms through which beneficial effects of low O(2) concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti-apoptotic and a decrease in the expression of pro-apoptotic genes.

Derivation of buffalo embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells.

PURPOSE: The aim of the present study is to compare the ability of homologous and heterologous embryonic fibroblast feeder layers to support isolation and proliferation of buffalo ES-like cells generated from hatched and expanded blastocysts produced by in vitro fertilization and characterization of derived cells through expression of pluripotent markers. METHODS: Embryonic stem cells were derived from hatched and expanded blastocysts through intact blastocyst culture and enzymatic method respectively and compared for proliferation rate on homologous (buffalo) and heterologous feeder layers (goat and sheep). RESULTS: A total of 69 hatched and 83 expanded blastocysts were used for isolation of inner cell masses which were seeded on buffalo, goat and sheep embryonic feeder layers. Following seeding, attachment rate, primary colony formation rate and survival to maximum number of passages were observed to be higher on homologous feeder layers. CONCLUSIONS: Upon comparison of different feeder layer cells for derivation and maintenance of buffalo ES-like cells from hatched and expanded blastocysts, buffalo embryonic fibroblast cells were able to provide a better environment for maintaining pluripotency in culture conditions.

Buffalo (Bubalus bubalis) embryonic stem cell-like cells and preimplantation embryos exhibit comparable expression of pluripotency-related antigens.

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin-C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2- to 4-cell, 8- to 16-cell, morula and blastocyst stages strongly expressed stage-specific embryonic antigen (SSEA)-4 but lacked expressions of SSEA-1 and SSEA-3. Putative ES cells also expressed tumour rejection antigen (TRA)-1-60, TRA-1-81 and Oct4. Whereas in all early embryonic stages, TRA-1-60 was observed only in the periplasmic space, and TRA-1-81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency-related surface antigen phenotype, which resembles that of the ICM.

Activation of zona-free buffalo (Bubalus bubalis) oocytes by chemical or electrical stimulation, and subsequent parthenogenetic embryo development.

Parthenogenetic activation using zona-free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona-free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6-dimethylaminopurine (6-DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona-free oocytes when compared to zona-intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona-free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6-DMAP resulted in the highest blastocyst rate.

Assessment of DNA damage during in vitro development of buffalo (Bubalus bubalis) embryos: effect of cysteamine.

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 µM, respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8- to 16-cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8- to 16- (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2-cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8- to 16-cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2-cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H2O2 caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.

Successful transplantation of transfected enriched buffalo (Bubalus bubalis) spermatogonial stem cells to homologous recipients.

Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ±â€¯4.56%, P < 0.05) for enriched SSCs in comparison to fugene HD (6.7 ±â€¯0.25%) and lipofectamine 3000 (15.57 ±â€¯0.74%). The transfected enriched SSCs were transplanted into buffalo males under the ultrasound guidance and testis was removed by castration after 7-8 weeks of transplantation. Persistence and localization of donor cells within recipient seminiferous tubules was confirmed using fluorescent microscopy. Further confirmation was done by flow cytometric evaluation of GFP expressing cells among those isolated from two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated for the first time, generation of buffalo transfected enriched SSCs and their successful homologous transplantation in buffaloes. This study represents the first step towards genetic modifications in buffaloes using SSC transplantation technique.