Media switching at different time periods affects the reprogramming efficiency of buffalo fetal fibroblasts.

Many contrasting reports are available on generation of bovine induced pluripotent stem cells (iPSCs) employing different timelines and culture conditions which signifies reprogramming process varies between species and cell types. The present study determines an optimum time period required to re-initiate reprogramming events in buffalo fibroblasts after introduction of exogenous genes (OCT4, SOX2, KLF4 and c-MYC) by lentiviral vector. The reprogramming efficiency is cumulative result of many factors including culture conditions and addition of growth factors in culture media. In our study, we observed when stem cell culture conditions were provided Day 5 post-transduction, it results in maximum reprogramming efficiency in comparison when same conditions were provided too early or on later days. The putative iPSCs were expanded on feeder layer for 15 passages and found positive for alkaline phosphatase and pluripotency markers (OCT4, SOX2, KLF4, c-MYC, UTF, TELOMERASE, FOXD3, REX1, STAT3, NUCLEOSTAMIN and TRA1-81). Also, they produced embryoid bodies showing expression for ectodermal (NF68, MOBP), mesodermal (ASA, BMP4) and endodermal (GATA4, AFP) markers to confirm their pluripotent nature. Our results suggest that reprogramming is accompanied by time dependent events and providing stem cell culture conditions at definite time during reprogramming can help in generation of iPSCs with greater efficiency.

Cell synchronization by Rapamycin improves the developmental competence of buffalos (Bubalus bubalis) somatic cell nuclear transfer embryos.

This study mainly explored the effects of Rapamycin on the growth of the Buffalo ear fibroblast (BEF) and embryonic developmental competence of somatic cell nuclear transfer (SCNT). The results show that the appropriate concentration (1 µM) of Rapamycin could significantly improve the proportion of the G0/G1 phase in BEF cells treated at a certain time (72 hr). Simultaneously, the percentage of the G0/G1 phase also was significantly higher than the serum starvation and control group. This may be related to Rapamycin inhibiting the phosphorylation of mTOR and affecting the expression of cell cycle-related genes (CDK2, CDK4, P27, CycleD1, and CycleD3). Besides, compared with the control group and serum-starved group, Rapamycin significantly decreased BEF cell apoptosis by reducing ROS generation. Moreover, these results also indicated that the proportion of BEF cells with normal chromosome multiples treated by Rapamycin is significantly higher than that of the serum-starved group (p < .05). Finally, this study explored the effects of Rapamycin and serum starvation on the embryonic developmental competence of SCNT. The results show that Rapamycin significantly increased the rate of 8-cell and blastocyst, compared with the control group and serum starvation group (p < .05). To summarize, these results indicate that Rapamycin improved the embryonic development competence of SCNT, which may be related to Rapamycin increasing the percentage of G0/G1 phase and maintaining BEF cell quality.

Proteomic analysis demonstrates that parthenogenetically activated swamp buffalo embryos have dysregulated energy metabolism.

The comprehensive understanding of early embryo development is essential to optimize in vitro culture conditions. Protein expression landscape of parthenogenetically produced embryo remains unexplored. This study aimed to investigate the protein expression dynamics with a particular focus on energy metabolism throughout the early developmental stages of parthenogenetic buffalo embryos. For this purpose, we performed iTRAQ-based quantitative mass spectrometry and identified 280 proteins common in all stages. A total of 933 proteins were identified during the proteomics analysis. The data depicted that morula and blastocyst had distinct protein expression dynamics as compared to 2- to 16-cell-stage embryo. KEGG pathway analysis showed 23 proteins belonging to energy metabolism appeared in the data. Study of energy metabolism-related protein's expression pattern demonstrated that there was asynchrony in proteins related to glycolysis throughout the examined developmental stages. The expression pattern of pyruvate kinase mutase (PKM), an essential protein of glycolysis, indicated a slightly decreasing trend from 2-cell-stage embryo to blastocyst, and it was supported by expression of proteins involved in lactate production (LDHA and LDHB) suggesting the decreasing rate of aerobic glycolysis (Warburg Effect) at morula and blastocyst stage. The increased Warburg Effect is considered as the hallmark of proliferating cells or embryo at the blastocyst stage. Furthermore, the proteins involved in the citric acid cycle also showed down-regulation at the blastocyst stage, indicating a lesser role of oxidative phosphorylation at this stage. Therefore, it could be divulged from the study that there may be an irregular pattern of energy metabolism in early parthenogenetic embryos. Further studies are recommended to understand this phenomenon.

Effect of basic fibroblast growth factor (FGF2) on cumulus cell expansion, in vitro embryo production and gene expression in buffalo (Bubalus bubalis).

The present study was undertaken to evaluate the effect of different concentration of FGF2 viz. 5 ng (T1), 10 ng (T2), and 20 ng/mL (T3) on cumulus cell expansion, oocyte maturation, in vitro embryo production, total cell number (TCN) of the blastocyst, and expression of the FGF2 and FGFR2 transcripts in buffalo oocytes and the embryos. Results showed that the effect of FGF2 on the diameter of buffalo COC was significantly higher (P < 0.05) in the T1 group than the other groups at 24h of maturation. The maturation and cleavage rate of oocytes was significantly higher (P < 0.05) in the T3 group than the control, however, the values did not different (P> 0.05) from other groups. The effect of FGF2 on morula and blastocyst yield did not different (P > 0.05) between treatment groups. However, the TCN of the blastocyst was slightly higher (P > 0.05) in the T3 group than the control and other groups. In subsequent trials, the expression of the FGF2 transcript was higher (P < 0.05) in A-grade of oocytes than the C- and D-grade of oocytes, but the expression was not different (P> 0.05) from the B-grade of oocytes. While the FGFR2 expression was higher (P < 0.05) in cumulus cells than any grades of oocytes. The relative abundance of FGF2 and FGFR2 transcripts was significantly higher (P < 0.05) in the 2-cell stage of the embryo than the other stages of embryos. This study was further extended to characterize the FGF2 ligand-binding site in the D3 domain of the buffalo FGF2 receptor. Bioinformatics analysis showed that the bovine FGF2 ligand-binding site in the D3 domain of buffalo was different from the D3 domain of the cattle.

Activation of Wnt/ß-Catenin Signaling Pathway Enhances the Derivation of Buffalo (Bubalus bubalis) Embryonic Stem Cell-Like Cells.

Wnt/ß-Catenin signaling pathway plays an important role in maintaining self-renewal and pluripotency of human and mouse embryonic stem cells (ESCs). Activation of Wnt/ß-Catenin signaling pathway by glycogen synthase kinase-3 (GSK3) inhibitor, the Wnt signaling agonist, could maintain the pluripotency of human and mouse ESCs in the presence of serum. However, the role of signaling pathway in the derivation of buffalo ESCs remains unclear. In this study, we used GSK3 inhibitors (6-bromoindirubin-3'-oxime [BIO] and CHIR99021) and investigated the effect of Wnt/ß-Catenin activation on colony formation, proliferation, self-renewal, and pluripotency of Chinese swamp buffalo (buffalo) embryonic stem cell-like cells (ES-like cells), which were isolated from blastocysts. The results showed that buffalo ES-like cells displayed typical morphological characteristics of pluripotent stem cells: positive for alkaline phosphatase staining, expression of pluripotent markers, including OCT4, SOX2, SSEA-1, SSEA-4, LIN28, CH1, NANOG, and the proliferative markers, PCNA and C-MYC. Furthermore, activation of Wnt/ß-Catenin signaling pathway by GSK3 inhibitors could promote colony formation and proliferation of buffalo ES-like cells and maintain their undifferentiated state, and upregulate the expression levels of pluripotent-related genes and proliferation-related genes. These results indicated that Wnt/ß-Catenin signaling pathway plays an important role in the derivation and pluripotency of buffalo ES-like cells.

Expression pattern of GLUT 1, 5, 8 and citrate synthase transcripts in buffalo (Bubalus bubalis) preimplantation embryos produced in vitro and derived in vivo.

In vitro-produced (IVP) embryos are reported to be developmentally lesser competent than in vivo-derived (IVD) embryos and supposed to differ in the expression of genes related with glucose metabolism. So, the present study was conducted to analyse the expression pattern of GLUT 1, 5, 8 and citrate synthase (CS) in oocytes and embryos produced in vivo or in vitro in buffalo. IVD embryos were obtained from 18 superovulated buffaloes. IVP embryos were obtained from slaughterhouse-derived oocytes subsequently subjected to in vitro fertilization and culture. Total RNA was isolated from different stages of oocytes (immature and in vitro matured) and embryos (8-16 cell to blastocysts of IVP embryos and morula to blastocysts of IVD embryos). Results demonstrated that the expression of GLUT1, GLUT 8 increased from 8 to 16 cells to blastocyst and was significantly (p < .05) higher in IVP embryos. Expression of both genes was (p < .05) higher in IVD than in IVP blastocysts; though GLUT5 transcripts were not detected at 8- to 16-cell stage IVP embryos, significantly (p < .05) higher transcripts were found at morula and blastocyst stages irrespective of embryo source with significantly (p < .05) higher expression in IVD embryos compared to IVP embryos. No significant difference was observed in citrate synthase expression in embryos at morula stage irrespective of the embryo source while significantly (p < .05) higher transcript level was observed at blastocyst stage with no difference between in vivo and in vitro embryos. It can be concluded that expression of GLUTs and CS is upregulated with progression of embryonic stage and expression is higher in in vivo embryos than in vitro counter parts; thus, it can be said that in vivo-produced embryos are metabolically superior to in vitro embryos.

The domesticated buffalo - An emerging model for experimental and therapeutic use of extraembryonic tissues.

Large animals play important roles as model animals for biomedical sciences and translational research. The water buffalo (Bubalus bubalis) is an economically important, multipurpose livestock species. Important assisted reproduction techniques, such as in vitro fertilization, cryo-conservation of sperm and embryos, embryo transfer, somatic cell nuclear transfer, genetic engineering, and genome editing have been successfully applied to buffaloes. Recently, detailed whole genome data and transcriptome maps have been generated. In addition, rapid progress has been made in stem cell biology of the buffalo. Apart from embryonic stem cells, bubaline extra-embryonic stem cells have gained particular interest. The multipotency of non-embryonic stem cells has been revealed, and their utility in basic and applied research is currently investigated. In particular, success achieved in bubaline extra-embryonic stem cells may have important roles in experimental biology and therapeutic regenerative medicine. Progress in other farm animals in assisted reproduction techniques, stem cell biology and genetic engineering, which could be of importance for buffalo, will also be briefly summarized.

Developmental potential of buffalo embryos cultured in serum free culture system.

The presence of serum in embryo culture medium has been implicated for increased embryo's sensitivity to cryopreservation, compromised viability, abnormal embryo and fetal development. Hence, designing a serum free culture system is indispensable. The present study aims to compare the efficiency of the serum and granulosa cells monolayer free commercial culture system (SFCS) with the conventional serum supplemented co-culture system (SSCS) and optimized culture system (OCS). Generally, SFCS is designed explicitly for bovine oocyte maturation and embryo culture (SF-IVM and SF-IVC), and SSCS (based on M199, SS-IVM, and SS-IVC) is utilized for buffalo in vitro embryo production. However, OCS is a newly designed culture system in which oocyte maturation is performed in serum supplemented maturation medium, and the subsequent embryos are co-cultured with granulosa cells in serum free culture medium. To evaluate the effect of serum on buffalo embryo production, buffalo oocytes, and their subsequent embryos were cultured in SSCS, SFCS, and OCS, simultaneously. The percentage of cleaved embryos cultured in SSCS and OCS was approximately 4% higher as compared to SFCS. However, OCS significantly showed the maximum proportion of embryos that developed to the blastocyst stage (7d) and hatched (6d) as compared to the SFCS and SSCS. Additionally, OCS promoted the expression of developmentally important genes (BCL2-L1 and VEGF-A), cell number, and cryo-survival ability of blastocysts in comparison with SSCS. Taken together, OCS is more suitable for the oocyte maturation and culture of buffalo embryos. However, to design the serum free culture system, it is recommended to find suitable serum alternatives for in vitro oocyte maturation.

Proteomics Analysis Reveals that Warburg Effect along with Modification in Lipid Metabolism Improves In Vitro Embryo Development under Low Oxygen.

The molecular mechanism regulating embryo development under reduced oxygen tension remains elusive. This study aimed to identify the molecular mechanism impacting embryo development under low oxygen conditions. Buffalo embryos were cultured under 5% or 20% oxygen and were evaluated according to their morphological parameters related to embryo development. The protein profiles of these embryos were compared using iTRAQ-based quantitative proteomics. Physiological O2 (5%) significantly promoted blastocyst yield, hatching rate, embryo quality and cell count as compared to atmospheric O2 (20%). The embryos in the 5% O2 group had an improved hatching rate of cryopreserved blastocysts post-warming (p < 0.05). Comparative proteome profiles of hatched blastocysts cultured under 5% vs. 20% O2 levels identified 43 differentially expressed proteins (DEPs). Functional analysis indicated that DEPs were mainly associated with glycolysis, fatty acid degradation, inositol phosphate metabolism and terpenoid backbone synthesis. Our results suggest that embryos under physiological oxygen had greater developmental potential due to the pronounced Warburg Effect (aerobic glycolysis). Moreover, our proteomic data suggested that higher lipid degradation, an elevated cholesterol level and a higher unsaturated to saturated fatty acid ratio might be involved in the better cryo-survival ability reported in embryos cultured under low oxygen. These data provide new information on the early embryo protein repertoire and general molecular mechanisms of embryo development under varying oxygen levels.

Maternal transcription profiles at different stages for the development of early embryo in buffalo.

Maternal mRNAs deposited in the egg during oogenesis are critical during the development of early embryo, before the activation of the embryonic genome. However, there is little known about the dynamic expression of maternally expressed genes in mammals. In this study, we made buffalo parthenogenesis as our research model to analyse maternal transcription profiles of pre-implantation embryo in buffalo using RNA sequencing. In total, 3,567 unique genes were detected to be differentially expressed among all constant stages during early embryo development (FPKM > 0). Interestingly, a total of 10,442 new genes were discovered in this study, and gene ontology analysis of the new differentially expressed genes indicated that the new genes have a wide cellular localization and are involved in many molecular functions and biological processes. Moreover, we identified eight clusters that were only highly expressed in a particular developmental stage and enriched a number of GO terms and KEGG pathways that were related to specific stage. Furthermore, we identified 1,530 hub genes (or key members) from the maternally expressed gene networks, and these hub genes were involved in 11 stage-specific modules. After visualization using Cytoscape 3.2.1 software, we obtained complex interaction network of hub genes, indicating the highly efficient cooperation between genes during the development in buffalo embryos. Further research of these genes will greatly deepen our understanding of embryo development in buffalo. Collectively, this research lays the foundation for future studies on the maternal genome function, buffalo nuclear transfer and parthenogenetic embryonic stem cells.

Embryo transfer in buffalo (Bubalus bubalis).

The use of assisted reproductive technologies, such as superovulation and in vivo embryo production and in vitro embryo production (IVEP), has increased rapidly in recent years and is now applied worldwide for genetic improvement in beef and dairy buffaloes. Although in vivo embryo production has been shown to be feasible in buffalo, low efficiency and limited commercial application has been documented. These results could be associated with low antral follicle populations, high levels of follicular atresia and/or failures of the oocyte to enter the oviduct after superovulation. Additionally, IVEP technology has been shown to be an important tool for multiplying genetic material from donors of superior merit, and promising results have been achieved with the use of ovum pick-up (OPU) along with IVEP in buffalo. However, several factors appear to be critical for successful OPU/IVEP, including circulating levels of anti-Müllerian hormone, antral follicle populations, sizes of the follicles available for the OPU, reproductive seasonality, semen (sire) used for IVEP, donor category and farm. Furthermore, technologies applied to control follicular wave emergence and ovulation at predetermined times, without the need for estrus detection in recipients, has facilitated management and improved the efficiency of embryo transfer programs in buffalo herds. Conclusively, with the considerable evidence of poor results with in vivo embryo production in buffaloes, the association of OPU with IVEP represents a new alternative for the exploitation of buffalo genetics.

Effect of cumulus cells of cumulus-oocyte complexes on in vitro maturation, embryonic developmental and expression pattern of apoptotic genes after in vitro fertilization in water buffalo (Bubalus bubalis).

In the present study, the potential of different grades of cumulus-oocyte complexes (COCs) for in vitro maturation (IVM) and embryonic development was assessed. Further, the association of the expression pattern of anti-apoptotic Mcl-1 and pro-apoptotic Bax genes in embryonic development was analyzed. Abattoir derived oocytes were graded into grade A and B based on surrounding cumulus rings. Out of 1050 ovaries, a total number of 770 and 1360, were of grade A and B COCs, respectively, were aspirated. After IVM, grade A COCs had a significantly higher number of polar bodies (92.04 ± 0.60%) as compared to grade B (85.88 ± 0.46%). On IVF and embryo culture, grade A COCs produced the significantly higher rate of cleavage and blastocyst (90.44 ± 0.71% and 41.55 ± 0.96%) as compared to grade B COCs (79.77 ± 0.76% and 30.44 ± 0.96%). The transcriptional analysis of apoptotic genes expression by Real-time PCR revealed a significantly higher expression of Mcl-1 gene in embryos of grade A as compared to grade B, whereas, the relative expression of Bax gene was down-regulated in grade A than grade B embryos. Thus it was concluded that the pattern of apoptotic genes expression in early-stage embryos can be used as a marker gene to predict the developmental competence of COCs.

A new three-dimensional glass scaffold increases the in vitro maturation efficiency of buffalo (Bubalus bubalis) oocyte via remodelling the extracellular matrix and cell connection of cumulus cells.

At present, many three-dimensional (3D) culture systems have been reported, improving the oocyte quality of in vitro maturation (IVM), yet the mechanism still needs to be further explored. Here we examined the effects of a new self-made 3D glass scaffold on buffalo oocyte maturation; meanwhile, the underlying mechanism on buffalo oocyte maturation was also detected. Compared to the two-dimensional (2D) glass dish culture, results revealed that the 3D culture can improve the first polar body rate of oocytes, subsequent cleavage and blastocysts rate of parthenogenetic activation embryos (p < .05). The extracellular matrix-related proteins COL1A1, COL2A1, COL3A1, FN and cell connection-related proteins N-cadherin, E-cadherin, GJA1 were found higher in cumulus cells of 3D culture. Moreover, in cumulus cells, proteins of the PI3K/AKT pathway reported being regulated by FN and E-cadherin including PI3K P85 and p-AKT were also higher in 3D culture. Furthermore, proapoptosis proteins P53, BAX, caspase-3 were lower in both cumulus cells and oocytes in 3D culture, while proteins PCNA and BCL2 showed the opposite result. Results also showed that the apoptosis was inhibited, and the proliferation was enhanced in cumulus cells of 3D culture. Finally, the cumulus expansion-related genes HAS2, CD44, HMMR, PTX3, PTGS2 were found higher in cumulus cells of 3D culture. Taken together, the 3D culture could promote oocyte maturation by regulating proteins correlated with the ECM, cell connection and PI3K/AKT pathway, inhibiting the apoptosis of cumulus cells and oocytes, enhancing the proliferation of cumulus cells and the cumulus expansion.

Effects of laser zona thinning and artificial blastocoel collapse on the cryosurviving and hatching of buffalo (Bubalus bulalis) blastocysts of different ages.

The objectives of this study were to investigate whether blastocoel collapse before vitrification induced by laser improves the cryo-survivability of buffalo in-vitro-fertilized (IVF) blastocysts and whether laser assisted hatching (LAH) promotes hatchability of fresh and frozen-thawed IVF blastocysts. The expanded blastocysts were harvested on Days 6-9 and randomly allocated into five groups as follows: (1) blastocysts were vitrified and thawed without any treatment; (2) blastocysts were vitrified after 15-20 µm zona pellucida (ZP) thinning opposite to the inner cell mass, and blastocoels were also blotted in order to outflow the blastocoelic fluid before vitrification; (3) ZP thinning was made immediately after thawing; (4) fresh blastocysts underwent LAH; and (5) as a control, fresh blastocysts without treatment. Results of the present study showed that the cryosurvival rates of vitrified Day 8 and Day 9 blastocysts in Group 2 were significantly (P < 0.01) higher in Group 2 than Group 1. The hatching rates of Day 8 and Day 9 blastocysts in Group 2 and Group 3 were also significantly (P < 0.01) higher compared with Group 1. Moreover, the hatching rate of Day 9 blastocysts in Group 4 was notably (P < 0.05) higher than Group 5. In conclusion, LAH promotes the hatching rates of Day 9 fresh and Days 8-9 vitrified blastocysts, and artificial blastocoel collapse before vitrification improves the cryosurvival rate of Days 8-9 IVF buffalo blastocysts.

Importance of lipid metabolism on oocyte maturation and early embryo development: Can we apply what we know to buffalo?

The knowledge about the biological events that regulate lipid metabolism in oocytes and embryos in buffalo is scarce. Lipogenesis, lipolysis, transport and oxidation of fatty acids (FAs) occur in gametes and embryonic cells of all mammalian species, as an intrinsic component of energy metabolism. In oocytes and cumulus cells, degradation of lipids is responsible for the production of ATP that is essential for the metabolic processes that lead to oocyte maturation in in vivo and in vitro culture conditions. Similarly, throughout embryo development, blastomeres have the capacity to use exogenous and/or endogenous lipid reserves to serve as an energy source necessary for early embryonic development. In addition, supplementation of culture media with L-carnitine to promote lipid metabolism during in vitro oocyte maturation and early embryonic development leads to an improved embryo quality. The limited scientific evidence available in buffalo indicates there is relatively greater oocyte lipid content as compared with many other species that undergoes a dynamic distribution during folliculogenesis and follicle maturation and that has a positive effect on oocyte maturation and embryo development when there is L-carnitine supplementation of the media. Advances in the understanding of the biological peculiarities of lipid metabolism, and the consequences of its alteration on the quality of buffalo gametes and embryos, therefore, are necessary to design specific culture media and laboratory procedures as a strategy to increase in vitro-derived embryo production rates.

Reducing the cytoplasmic volume during hand-made cloning adversely affects the developmental competence and quality, and alters relative abundance of mRNA transcripts and epigenetic status of buffalo (Bubalus bubalis) embryos.

Hand-made cloning (HMC) is a method of choice for somatic cell nuclear transfer (SCNT). There is 20% to 50% of cytoplasm lost during manual enucleation of oocytes with HMC. To compensate, two enucleated demicytoplasts, instead of one, are fused with each donor cell, which leads to cytoplasm pooling from two different demicytoplasts. In this study, effects of using one, instead of two demicytoplasts (controls) was examined, for production of embryos using HMC. Use of one demicytoplast decreased blastocyst development (12.7 ±â€¯1.98% compared with 47.6 ±â€¯3.49%, P < 0.001), total cell number (TCN, 167.6 ± 14.66 compared with 335.9 ± 58.96, P < 0.01), apoptotic index (2.11 ± 0.38 compared with 3.43±0.38, P < 0.05) but did not significantly alter inner cell mass:trophectoderm cell number ratio (0.17 ± 0.01 compared with 0.19 ± 0.02) and the global content of H3K9ac and H3K27me3 of blastocysts, compared to controls. There were gene expression alterations in pluripotency- (SOX2 and NANOG but not OCT4), epigenetic- (DNMT1 but not DNMT3a and HDAC1), apoptosis- (CASPASE3 but not BCL-2 and BAX), trophectoderm- (CDX2), development- (G6PD but not GLUT1) and cell cycle check point control-related related genes (P53) compared with controls. Transfer of cloned blastocysts from one demicytoplast (n = 8) to recipients resulted in a live calf birth that after 12 days died whereas, with transfer of control blastocysts (n = 14) there was birth of a healthy calf. In conclusion, use of one, instead of two demicytoplasts for HMC, compromises in vitro developmental competence, and alters expression of several important genes affecting embryo development.

Isolation and culture of epithelial cells from stored buffalo semen and their use for the production of cloned embryos.

The aim of the present study was to isolate somatic cells from semen, a non-invasive source of donor somatic cells, for somatic cell nuclear transfer (SCNT) experiments. The study had two parts: (1) isolation and culture of somatic cells from semen, which was stored at 4°C; and (2) investigating the SCNT competence of semen-derived somatic cells. We successfully cultured somatic cells from freshly ejaculated semen, which was stored for different times (0, 4, 12, 24, 72 and 144h after semen collection) at 4°C, using a Percoll gradient method. Up to 24h storage, 100% cell attachment rates were observed; cell attachment rates of 66% were observed for the 72 and 144h storage groups. The attached cells observed in all groups examined were proliferated (100%). Cultured cells exhibited epithelial cell morphology and culture characteristics, which was further confirmed by positive expression of cytokeratin 18, an epithelial cell-type marker. We compared the SCNT competence of semen-derived epithelial cells and skin-derived fibroblasts. The cleavage rate, blastocyst production rate, total number of cells in blastocysts and the apoptotic index of blastocysts were similar for embryos produced from semen-derived epithelial cells and skin-derived fibroblasts, indicating that semen-derived epithelial cells can serve as donors for SCNT experiments. In conclusion, we demonstrate a method to culture epithelial cells from stored semen, which can be used to produce cloned embryos of breeding bulls, including remote bulls.

Comparative analysis of buffalo (Bubalus bubalis) non-transgenic and transgenic embryos containing human insulin gene, produced by SCNT.

Somatic cell nuclear transfer (SCNT), using transgenic donor cells, is a highly efficient method for producing transgenic embryos. We compared the developmental competence, quality and gene expression of transgenic embryos produced by Hand-made cloning from buffalo fetal fibroblasts (BFFs) containing human insulin gene, with non-transgenic embryos produced from BFFs (Controls). The expression vector (pAcISUBC), constructed by inserting human insulin gene between DNA fragments containing mammary gland-specific buffalo ß-lactoglobulin (buBLG) promoter and terminator buBLG 3'UTR regions into pAcGFP-N1 vector, was used for obtaining the 11 kb insert for transfection of BFFs by nucleofection. Presence of the transgene in embryos was confirmed by examining GFP expression by RT-PCR and immunofluorescence. The blastocyst rate was lower (P < 0.05) for transgenic embryos than for controls (35.7 ±â€¯1.8% vs 48.7 ±â€¯2.4%). The apoptotic index was higher (P < 0.05) for transgenic than for control blastocysts which, in turn, was higher (P < 0.05) than for IVF counterparts (6.9 ±â€¯0.9, 3.8 ±â€¯0.5 and 1.8 ±â€¯0.3, respectively). The total cell number was similar for transgenic and non-transgenic blastocysts (143.2 ±â€¯17.0 and 137.2 ±â€¯7.6, respectively). The expression level of pro-apoptotic genes BAX and BID but not that of CASP3 and CASP9, and cell cycle check point control-related gene P53 was higher (P < 0.05), and that of development- (IGF-1R and G6PD) and pluripotency-related gene NANOG was lower (P < 0.05) in transgenic than in control embryos. The expression level of epigenetic-related genes DNMT1, DNMT3a and HDAC1 and pluripotency-related gene OCT4 was similar in the two groups. The expression level of BAX, BID, CASP9, P53, DNMT1 and DNMT3a was higher (P < 0.05) and that of OCT4, NANOG IGF-1R and G6PD was lower (P < 0.05) in cloned transgenic than in IVF blastocysts whereas, that of CASP3 and HDAC1 was similar between the two groups. In conclusion, these results suggest that transgenic embryos produced by SCNT have lower developmental competence and quality, and altered gene expression compared to non-transgenic embryos.

Treatment of Buffalo (Bubalus bubalis) Somatic Cell Nuclear Transfer Embryos with MicroRNA-29b Mimic Improves Their Quality, Reduces DNA Methylation, and Changes Gene Expression Without Affecting Their Developmental Competence.

microRNA-29b (miR-29b) plays an important role in controlling DNA methylation in cells. We investigated its role during early embryonic development in buffalo embryos produced by somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). miR-29b expression was highest at the 2-cell stage, decreased (p < 0.001) at the 4-cell stage, and remained low thereafter at the 8-cell, morula, and blastocyst stages, showing a similar pattern in cloned and IVF embryos. Treatment of reconstructed embryos with miR-29b mimic for 1 hour after 1 hour of electrofusion increased (p < 0.05) the total cell number and decreased (p < 0.05) the levels of apoptosis and DNA methylation compared with controls. It also increased (p < 0.05) the ratio of inner cell mass:trophectoderm cell numbers of blastocysts compared with controls to the levels observed in IVF blastocysts. However, the blastocyst rate was not affected by treatment with miR-29b mimic (29.0% ± 2.0% vs. 27.0% ± 2.0% for controls). The treatment decreased (p < 0.001) the expression of epigenetic-related genes, DNMT3A and DNMT3B, but not DNMT1, and increased (p < 0.05) that of pluripotency- (NANOG, OCT4, and SOX2) and development-related genes (FGF4 and GLUT1) in blastocysts compared with controls. Our results suggest that miR-29b mimic treatment of reconstructed embryos improves the quality, reduces the level of apoptosis and DNA methylation, and changes gene expression in SCNT blastocysts without affecting the blastocyst rate.

Brain-derived neurotrophic factor (BDNF) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development.

Brain-derived neurotrophic factor (BDNF) has been discovered and characterized for several decades, yet its expression pattern in non-neuronal tissues like ovary and potential mechanism during oocyte maturation are still poorly understood. Thus the present study was devised to determine the expression pattern and mechanism of BDNF during buffalo oocyte maturation. The results revealed that BDNF was presented at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. BDNF's receptor p75 was detected in granulosa cells, cumulus cells, oocytes, and early embryos, while another receptor neurotrophic tyrosine kinase receptor, type2 (NTRK2) was only identified in granulosa cells and cumulus cells. To determine the effect of BDNF on oocyte maturation and early embryo development, different concentrations (0, 1, 10, 100 ng/mL) of BDNF were added into the in vitro maturation media, respectively. It was divulged that 10 ng/mL BDNF promoted the in vitro maturation rate of buffalo oocytes and the blastocysts rate of embryos cultured in vitro (P < 0.05). Then through using NTRK2 inhibitor K-252a, we found BDNF and its receptor NTRK2 in cumulus cells played an essential role during oocyte maturation. Moreover, to further investigate the underlying mechanism by which BDNF enhances oocyte maturation, RT-qPCR was performed. 10 ng/mL BDNF treatment could decrease the expression level of apoptosis-related genes CCASP9, FAS, up-regulate the expression level of receptor gene NTRK2, cell proliferation-related genes CCNB1, PCNA, gap junction-related genes GJA4, GJA1 as well as cumulus cells expansion-related genes HAS2, PTX3 and TNFAIP6 (P < 0.05). Altogether, our results showed for the first time that BDNF was expressed throughout buffalo ovarian follicle development, oocyte maturation and early embryogenesis. Furthermore, BDNF treatment could improve the efficiency of buffalo oocyte maturation through regulating genes expression in cumulus cells and then promote early embryo development.