Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation.

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.

Enhancing the quality of inferior oocytes of buffalo for in vitro embryo production: The impact of melatonin on maturation, SCNT, and epigenetic modifications.

Success of animal cloning is limited by oocyte quality, which is closely linked to reprogramming ability. The number of layers of cumulus cells is typically used to assess the quality of oocyte; a minimum of one-third of collected cumulus-oocyte complexes (COCs) are discarded as inferior oocytes because they have less cumulus cells. Melatonin, which has been recognised for its ability to sequester free radicals and perform multiple functions, has emerged as a potentially effective candidate for enhancing inferior oocytes quality and, consequently, embryo development competency. The current study investigates to improve the quality of inferior oocytes by supplementation of melatonin (10-9 M) during in vitro maturation (IVM) and subsequent cloned embryo production and its mechanism. The results indicate that melatonin supplementation significantly (p<0.05) enhances inferior oocytes maturation, reduces oxidative stress by reducing ROS levels, and improves mitochondrial function by boosting GSH levels. The melatonin treatment (10-9 M) enhances the expression of SOD, GPx1, GDF 9, BMP 15, ATPase 6, and ATPase 8 in inferior oocytes. Furthermore, melatonin treatment increases the total cell number in the treated groups, promoting cloned blastocyst formation rates derived from inferior oocytes. Furthermore, compared to the control, 10-9 M melatonin supplementation enhances H3K9ac acetylation and lowers H3K27me3 methylation in cloned blastocysts derived from inferior oocytes. In conclusion, 10-9 M melatonin supplementation during IVM increased inferior oocyte maturation and promoted cloned buffalo embryo development by lowering oxidative stress and promoting epigenetic alterations. These studies show that melatonin may improve the quality of poor oocytes and buffalo cloning.

Mito-Q supplementation of in vitro maturation or in vitro culture medium improves maturation of buffalo oocytes and developmental competence of cloned embryos by reducing ROS production.

Mito-Q is a well-known mitochondria-specific superoxide scavenger. To our knowledge, the effect of Mito-Q on buffalo oocyte maturation and developmental competency of cloned embryos has not been examined. To investigate the effects of Mito-Q on the in vitro maturation (IVM) of buffalo oocytes and the developmental competence of cloned embryos, different concentration of Mito-Q were supplemented with IVM (0, 0.1, 0.5, 1, 2 µM) and in vitro culture (IVC) medium (0, 0.1 µM). Supplementation of IVM medium with 0.1 µM Mito-Q significantly (P ≤ 0.05) increased the cumulus expansion, nuclear maturation, mitochondrial membrane potential (MMP) and antioxidants genes (GPX1 and SOD2) expression and effectively reduced ROS production leading to a significant improvement in the maturation rate of buffalo oocytes. Further, the supplementation of 0.1 µM Mito-Q in IVC medium promotes the cleavage and blastocyst rate significantly over the control. Mito-Q supplementation improves (P ≤ 0.05) MMP, antioxidant gene (GPX1) expression and reduced the ROS level and apoptosis related genes (caspase 9) expression in cloned blastocysts. In conclusion, the present study demonstrated that the supplementation of 0.1 µM Mito-Q in IVM and IVC media exerts a protective role against oxidative stress by reducing ROS production and improving MMP, fostering improved maturation of buffalo oocytes and enhanced developmental competence of cloned embryos. These findings contribute valuable insights into the optimization of assisted reproductive technologies protocols for buffalo breeding and potentially offer novel strategies to enhance reproductive outcomes in livestock species.

A new role of H89: Reduces capacitation-like changes through inhibition of cholesterol efflux, calcium influx, and proteins tyrosine phosphorylation during sperm cryopreservation in buffalo.

It is a known fact that cryopreservation initiates premature capacitation in spermatozoa during the cryopreservation process. Protein tyrosine phosphorylation is a landmark of cascade reaction accountable for capacitation or capacitation-like changes in spermatozoa. Therefore, our hypothesis was to test an inhibitor (H89) that reversibly inhibits the cascade reaction responsible for capacitation during the cryopreservation process but does not hamper normal capacitation and fertilizing ability of sperm. For this, sixteen ejaculates were collected from Murrah buffalo bulls (n = 4). Each ejaculate was divided into four equal aliquots and diluted in an egg yolk-based semen dilutor supplemented with 0, 2, 10, and 30 µM concentrations of H89 and cryopreserved. Interestingly, H89 reduces cholesterol efflux from spermatozoa and protects spermatozoa from membrane damage during the cryopreservation process. H89 did not prevent lipid peroxidation of the sperm membrane. H89 reduced intracellular calcium concentration in spermatozoa in a dose-dependent manner, but tyrosine phosphorylation reduction was observed in the 2 and 10 µM H89 groups. The CTC assay revealed that the percentage of uncapacitated spermatozoa in different treatment groups increases in a dose-dependent manner. In the in vitro capacitation medium, the effect of H89 is abolished and spermatozoa underwent normal capacitation, but H89-treated spermatozoa attached to zona pellucida in large numbers compared to untreated spermatozoa. In conclusion, H89 does not only inhibit tyrosine phosphorylation of spermatozoa but it reduces cholesterol efflux and calcium influx, and ultimately reduces capacitation-like changes during the cryopreservation process.

Mitochondria-targeted antioxidant MitoQ ameliorates ROS production and improves cell viability in cryopreserved buffalo fibroblasts.

Cryopreservation commonly decreases the cellular functionality and post-thaw viability of cells. Reactive oxygen species (ROS) generated during cryopreservation degrade mitochondrial activity and promote the release of cytochrome C which activates caspases required for apoptosis. Antioxidants have the potential to improve the recovery efficiency of cells by reducing ROS production and maintaining mitochondrial membrane potential (MMP). The present study was conducted to explore the role of MitoQ, a derivative of coenzyme Q10 on cryopreserved fibroblasts derived from buffalo skin. To achieve our goal, buffalo skin fibroblasts were treated with varying concentrations of MitoQ (0, 0.1, 0.5, 1, 2, and 10 µM) for 24, 48, and 72 h. The MMP, ROS generation, cell viability was measured by flow cytometry. Furthermore, expression of genes related to mitochondrial oxidative stress (NRF2, GPX, and SOD), apoptosis (BAK and caspase 3) and cell proliferation (AKT) were also assessed. The results showed that over a period of 72 h lower concentrations of MitoQ (0.1-0.5 µM) decrease the ROS production, improves MMP and cell viability whilst the high concentration of MitoQ (2-10 µM) increased the oxidative damage to the cells. Taken together, our study provide important insights into the novel role of MitoQ in cryopreserved buffalo skin fibroblasts. In conclusion, we demonstrated the dose-dependent functional role of MitoQ on cryopreserved fibroblasts for improving post-thaw cell viability and cellular function.

Pluripotent Stem Cells for Livestock Health and Production.

Pluripotent stem cells (PSCs) have unlimited capacity for self-renewal and differentiation so that they can potentially produce any cell or tissue of animal's body. The PSCs derived from livestock represents a more appropriate model than a rodent for investigating human diseases due to their higher anatomical and physiological resemblance with human. Apart from that, livestock PSCs hold immense promises for innovative therapies, transgenic animal production and their biomedical interest. The realization of the full potential of PSCs, however, depends on the elucidation of the molecular mechanisms which play a critical role in the maintenance of pluripotency and reprogramming procedure remains poorly understood in livestock which in turn impedes the generation of true PSCs and their usage for clinical research. An in-depth understanding of pluripotency is extremely essential for improving health and welfare of livestock animals. Therefore, the present review focuses on the milestone achievements of PSCs in livestock animals and their potential application in health and production of livestock.

Sodium alginate potentiates antioxidants, cryoprotection and antibacterial activities of egg yolk extender during semen cryopreservation in buffalo.

The study was conducted to determine effects of sodium alginate on sperm during cryopreservation. Each ejaculate (n = 20) of five buffalo bulls (3-5 years) were divided into six equal fractions and diluted using egg yolk based extender supplemented with different concentrations of sodium alginate and cryopreserved. Frozen-thawed semen samples were evaluated using the CASA, hypo-osmotic swelling test, cervical mucus penetration capacity test, and chlortetracycline fluorescence assay (CTC). Phosphorylation of tyrosine containing proteins and malondialdehyde concentration of sperm membrane were evaluated using immunoblotting and thiobarbituric acid reactive substance assay respectively. The semen extender's anioxidative capacities were estimated by conducting 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays, metal chelating capacity by assessing ferrozine and antibacterial capacity using agar plate methods. Supplementation of sodium alginate in extender improved sperm longevity, plasma membrane integrity as well as capacity to transit through the cervical mucus. Supplementation of extender with sodium alginate minimises the phase transition of sperm membranes and phosphorylation of tyrosine containing proteins during cryopreservation. Malondialdehyde concentration of sperm was less in sodium alginate-treated sperm as compared with control samples. The results indicated that sodium alginate increased antioxidant capacity of semen extender. Supplementation with sodium alginate also improved the metal chelating capacity and antibacterial properties of the extender. In conclusion, supplementation of extender with sodium alginate enhances free radical scavenging, metal reduction and chelating capacities to protect sperm during cryopreservation.

Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions.

Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and lentiviral may cause insertional mutagenesis due to its integrating ability. Hence, alternate methods with safety concerns are needed to be investigated. Therefore, the present study was undertaken to reprogram buffalo fibroblasts using non-viral piggyBac (PB) transposon mediated transfer of six transcription factors. To generate buffalo iPS cells, fibroblasts were isolated from buffalo fetus at passage 2. The cells were co-electroporated with a PB transposon having CAGGS promoter driven cassette of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 transcription factors separated by self-cleaving 2A peptide and a helper plasmid pCMV-PB transposase. After 12-14 days post electroporation, fibroblast cells morphology was observed to change to round structures which formed loose aggregates of cells on day 18. Putative iPS cell colonies were propagated in feeder free system and characterized through expression of pluripotency markers such as alkaline phosphatase, SSEA-1, SSEA-4, SSEA-5, TRA-1-81, Oct4, Nanog and Sox2 and endogenous genes supported the stemness property of the generated cells. These cells differentiated in vitro to form embryoid bodies and were found to express three germ layers markers. In conclusion, generation of buffalo iPS cells using transposon system provides insights into viral-free iPS technology which will facilitate genetic modification of the buffalo genome and help in the production of transgenic animals using genetically modified iPS cells.

A novel combination of silane-coated silica colloid with hybrid RNA extraction protocol and RNA enrichment for downstream applications of spermatozoal RNA.

Spermatozoa are specialised cells with low RNA content as compared to somatic cells. The suitable sperm RNA extraction and enrichment protocols for downstream applications are available for human, cattle, stallion and mouse but not for buffalo spermatozoa. Therefore, the present work was conducted to find out suitable colloidal solution for sperm purification and appropriate protocol for sperm RNA extraction and enrichment/amplification of RNA. For purification, we used PVP-coated silica colloidal solution (PVP-Si), silane-coated silica colloidal solution (Silane-Si) and iodixanol. Sperm recovery rate, total sperm motility and progressive sperm motility were significantly improved after separation by Silane-Si and iodixanol compared to PVA-Si method. The combined guanidinium thiocyanate-phenol-chloroform (GTPC) with silica matrix (SM)-based RNA extraction yielded more quantity of RNA in compared to individual method. The hybrid of SM and GTPC into a single protocol yielded 360-450 ng RNA from 30 million buffalo spermatozoa. For the first time, we adopted new way to enrich sperm RNA that increased the RNA concentration 4-5 times that was sufficient for downstream applications. The linear amplification of sperm RNA increased RNA concentration around 27-45 times. In summary, Silane-Si colloid for sperm separation, hybrid SM and GTPC protocol for sperm RNA extraction followed by enrichment or amplification of RNA was found suitable for high-throughput analyses of buffalo sperm RNA.

Generation of Venus fluorochrome expressing transgenic handmade cloned buffalo embryos using Sleeping Beauty transposon.

The objective of this study was to optimise the electroporation conditions for efficient integration of Venus construct in buffalo fetal fibroblasts using Sleeping Beauty (SB) based transposition and to produce Venus expressing transgenic cloned embryos through handmade cloning (HMC) approach. Primary culture of buffalo fetal fibroblast cells was established and subsequently cultured cells were co-transfected with Venus and helper plasmid at different combinations of electroporation condition. In different combinations of voltage, time and plasmid dose, we observed that 300 V, single pulse for 10 ms in 2 mm cuvette and 1.5-2.0 µg transposons with 200-300 ng transposase dose was optimum for expressing Venus fluorescence in cells via electroporation. After electroporation, the cells were cultured for 2-3 days and then Venus expressing cells were picked with the help of a Pasteur pipette under the fluorescence microscope to enrich them through single cell culture method before using as donor cells for HMC. In vitro matured oocytes were reconstructed with either transfected or non-transfected buffalo somatic cells by electric fusion followed by activation. The reconstructed, activated embryos were cultured in 400 µL of Research Vitro Cleave medium supplemented with 1% fatty acid-free BSA in 4-well dish, covered with mineral oil and incubated in an incubator (5% CO2 in air) at 38.5 °C for 8 days and the developmental competence was observed. The percentage of cleaved, 4-8 and 8-16 cells stage embryos generated through Venus expressing cells were comparable with control, whereas, the morula (21.0 vs 53.0%) and blastocysts (10.5 vs 30.6%) produced through Venus expressing cells was found low as compared to control. These results indicate that fetal fibroblasts transfected with Venus could be used as donor cells for buffalo cloning and that Venus gene can be safely used as a marker of foreign gene in buffalo transgenesis.

Epigenetic status of buffalo fibroblasts treated with sodium butyrate a chromatin remodeling agent.

The somatic cells having higher levels of DNA methylation and reducing it in donor cells before nuclear transfer (NT) by treating them with chemicals, may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Therefore, the present study was planned to understand mechanism of epigenetic changes in donor cells (buffalo fibroblasts) treated with different concentration of sodium butyrate (NaBu)-a chromatin remodeling agent. The cultured fibroblasts purity and lineage were confirmed by fibroblast specific protein and gene markers (Vimentin, Tubulin and Cytokeratin) at different passages using immuno-staining and qPCR respectively. The buffalo fibroblast cells were treated with 1, 3 and 5 mM of NaBu and observations were taken on their morphological changes, population doubling time and cell proliferation after 48 h of treatment. The epigenetic changes were observed using acetylation (H3K9ac) and methylation (H3K27me3) markers expression. The fibroblast cells derived from new born ear tissue started emerging and anchoring to cell culture flasks within 24 h and showed spindle-shaped morphology. The confluent culture was cryopreserved at different time interval. The post thaw culture behavior of the cryopreserved cells was also observed at different time interval and passages. The morphology of NaBu treated cells were changed with increase of dosages of NaBu treatment. The increase population doubling times and decreases the proliferation rate in the dose dependent manner. The NaBu treatment showed that the significantly increased the acetylation (H3K9ac) and methylation (H3K27me3) level over the control. Based on the observations of fibroblast characterization as well as epigenetic modifications of these cells after treatment with NaBu, results suggest that the cells may provide a useful approach for better epigenetic reprogramming in SCNT embryos.

Estimation of endogenous levels of osteopontin, total antioxidant capacity and malondialdehyde in seminal plasma: Application for fertility assessment in buffalo (Bubalus bubalis) bulls.

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high-fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high-fertile bulls was found to be higher (p < .05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p < .05) in high-fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r = .678, p = 0.013) and TAC (r = .648, p = .042) were found to be positively correlated with bull fertility and the level of MDA (r = -.718, p = .019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds.

Differences in developmental competence and gene expression profiles between buffalo (Bubalus bubalis) preimplantation embryos cultured in three different embryo culture media.

The objective of this study was to compare effects of in vitro culture systems on embryonic development and expression patterns of developmentally important genes in preimplantation buffalo embryos. After IVM/IVF presumptive zygotes were cultured in one of three systems: undefined TCM-199, mCR2aa medium supplemented with 10 % FBS and defined PVA-myo-inositol-phosphate-EGF medium. No (P > 0.05) differences at 2-cell, 4-cell and 8-cell to 16- cell stages were observed among the three cultured media used, however, increased (P < 0.05) blastocyst yield, cell number and hatching rate were found in defined medium compared to undefined media. The expression patterns of genes implicated in embryo metabolism (GLUT-1), anti-apoptosis (BCL-2), imprinting (IGF-2R), DNA methylation (DNMT-3A) and maternal recognition of pregnancy (IFNT) were increased (P < 0.05) in hatched blastocysts derived from defined medium compared to undefined media. In conclusion, serum-free, defined medium improved developmental competence of in vitro cultured buffalo embryos. Whether these differences in morphological development and gene expression have long-term effects on buffalo calves born after embryo transfer remains unknown. However, it is possible that early adaptations of the preimplantation embryo to its environment persist during fetal and post-natal development.

A comparative study on expression profile of developmentally important genes during pre-implantation stages in buffalo hand-made cloned embryos derived from adult fibroblasts and amniotic fluid derived stem cells.

Abnormal gene expression in somatic cell nuclear transfer embryos due to aberrant epigenetic modifications of the donor nucleus may account for much of the observed diminished viability and developmental abnormalities. The present study compared the developmentally important gene expression pattern at 4-cell, 8- to 16-cell, morula, and blastocyst stages of buffalo nuclear transfer (NT) embryos from adult fibroblasts (AFs) and amniotic fluid stem cells (AFSCs). In vitro fertilized embryos were used as control embryos. Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), growth factor signaling, and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), oxidative stress (MnSOD), metabolism (GLUT1) regulation were observed in cloned embryos. The expression of transcripts in AFSC-NT embryos more closely followed that of the in vitro fertilized embryos compared with AF-NT embryos. It is concluded that AFSCs with a relatively undifferentiated genome may serve as suitable donors which could be reprogrammed more efficiently to reactivate expression of early embryonic genes in buffalo NT.

Phenotypical and functional characteristics of mesenchymal stem cells derived from equine umbilical cord blood.

Mesenchymal stem cells (MSCs) offer promise as therapeutic aid in the repair of tendon and ligament injuries in race horses. Fetal adnexa is considered as an ideal source of MSCs due to many advantages, including non-invasive nature of isolation procedures and availability of large tissue mass for harvesting the cells. However, MSCs isolated from equine fetal adnexa have not been fully characterized due to lack of species-specific markers. Therefore, this study was carried out to isolate MSCs from equine umbilical cord blood (UCB) and characterize them using cross-reactive markers. The plastic-adherent cells could be isolated from 13 out of 20 (65 %) UCB samples. The UCB derived cells proliferated till passage 20 with average cell doubling time of 46.40 ± 2.86 h. These cells expressed mesenchymal surface markers but did not express haematopoietic/leucocytic markers by RT-PCR and immunocytochemistry. The phenotypic expression of CD29, CD44, CD73 and CD90 was shown by 96.36 ± 1.28, 93.40 ± 0.70, 73.23 ± 1.29 and 46.75 ± 3.95 % cells, respectively in flow cytometry, whereas, reactivity against the haematopoietic antigens CD34 and CD45 was observed only in 2.4 ± 0.20 and 0.1 ± 0.0 % of cells, respectively. Osteogenic and chondrogenic differentiation could be achieved using established methods, whereas the optimum adipogenic differentiation was achieved after supplementing media with 15 % rabbit serum and 20 ng/ml of recombinant human insulin. In this study, we optimized methodology for isolation, cultural characterization, differentiation and immunophenotyping of MSCs from equine UCB. Protocols and markers used in this study can be employed for unequivocal characterization of equine MSCs.

A comparative study on efficiency of adult fibroblasts and amniotic fluid-derived stem cells as donor cells for production of hand-made cloned buffalo (Bubalus bubalis) embryos.

The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical "S" shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10-15 and 8-12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.

Assessment of sperm damages during different stages of cryopreservation in water buffalo by fluorescent probes.

The present study was designed to investigate the sperm damages occurring in acrosome, plasma membrane, mitochondrial activity, and DNA of fresh, equilibrated and frozen-thawed buffalo semen by fluorescent probes. The stability of sperm acrosome and plasma membrane stability, mitochondrial activity and DNA status were assessed by fluorescein conjugated lectin Pisum sativum agglutinin, Annexin-V/propidium iodide, JC-1 and TUNEL assay, respectively, under the fluorescent microscope. The damages percentage of acrosome integrity was significantly increased during equilibration and freezing-thawing process. The stability of sperm plasma membrane is dependent on stability of phosphatidylserine (PS) on the inner leaflet of plasma membrane. The frozen-thawed sperm showed externalization of PS leading to significant increase in apoptotic, early necrotic and necrotic changes and lowered high mitochondrial membrane potential as compared with the fresh sperm but all these parameters were not affected during equilibration. However, the DNA integrity was not affected during equilibration and freezing-thawing procedure. In conclusion, the present study revealed that plasma membrane and mitochondria of buffalo sperm are more susceptible to damage during cryopreservation. Furthermore, the use of fluorescent probes to evaluate integrity of plasma and acrosome membranes, as well as mitochondrial membrane potential and DNA status increased the accuracy of semen analyses.

Amnion Epithelial Cells of Buffalo (Bubalus Bubalis) Term Placenta Expressed Embryonic Stem Cells Markers and Differentiated into Cells of Neurogenic Lineage In Vitro.

Aim of the present study was the isolation, culture, and characterization of amniotic membrane-derived epithelial cells (AE) from term placenta collected postpartum in buffalo. We found that cultured cells were of polygonal in shape, resistance to trypsin digestion and expressed cytokeratin-18 indicating that they were of epithelial origin. These cells have negative expression of mesenchymal stem cell markers (CD29, CD44, and CD105) and positive for pluripotency marker (OCT4) genes indicated that cultured cells were not contaminated with mesenchymal stem cells. Immunofluorescence staining with pluripotent stem cell surface markers, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 indicated that these cells may retain pluripotent stem cell characteristics even after long period of differentiation. Differentiation potential of these cells was determined by their potential to differentiate into cells of neurogenic lineages using retinoic acid. In conclusion, we demonstrate that AE cells expressed pluripotent stem cell markers and have propensity to differentiate into cells of neurogenic lineage upon directed differentiation in vitro.

Differential developmental competence and gene expression patterns in buffalo (Bubalus bubalis) nuclear transfer embryos reconstructed with fetal fibroblasts and amnion mesenchymal stem cells.

The developmental ability and gene expression pattern at 8- to 16-cell and blastocyst stages of buffalo (Bubalus bubalis) nuclear transfer (NT) embryos from fetal fibroblasts (FFs), amnion mesenchymal stem cells (AMSCs) and in vitro fertilized (IVF) embryos were compared in the present studies. The in vitro expanded buffalo FFs showed a typical "S" shape growth curve with a doubling time of 41.4 h and stained positive for vimentin. The in vitro cultured undifferentiated AMSCs showed a doubling time of 39.5 h and stained positive for alkaline phosphatase, and these cells also showed expression of pluripotency markers (OCT 4, SOX 2, NANOG), and mesenchymal stem cell markers (CD29, CD44) and were negative for haematopoietic marker (CD34) genes at different passages. Further, when AMSCs were exposed to corresponding induction conditions, these cells differentiated into adipogenic, chondrogenic and osteogenic lineages which were confirmed through oil red O, alcian blue and alizarin staining, respectively. Donor cells at 3-4 passage were employed for NT. The cleavage rate was significantly (P < 0.05) higher in IVF than in FF-NT and AMSC-NT embryos (82.6 ± 8.2 vs. 64.6 ± 1.3 and 72.3 ± 2.2 %, respectively). However, blastocyst rates in IVF and AMSC-NT embryos (30.6 ± 2.7 and 28.9 ± 3.1 %) did not differ and were significantly (P < 0.05) higher than FF-NT (19.5 ± 1.8 %). Total cell number did not show significant (P > 0.05) differences between IVF and AMSC-NT embryos (186.7 ± 4.2, 171.2 ± 3.8, respectively) but were significantly (P < 0.05) higher than that from FF-NT (151.3 ± 4.1). Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), growth factor signaling and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), metabolism (GLUT1) and oxidative stress (MnSOD) regulation were observed in cloned embryos. The transcripts or expression patterns in AMSC-NT embryos more closely followed that of the in vitro derived embryos compared with FF-NT embryos. The results demonstrate that multipotent amnion MSCs have a greater potential as donor cells than FFs in achieving enhanced production of cloned buffalo embryos.

Liposome-based semen extender is suitable alternative to egg yolk-based extender for cryopreservation of buffalo (Bubalus bubalis) semen.

Demand for alternative of egg yolk in freezing extenders have increased in recent years due to variability in egg yolk composition, risk of microbial contamination and presence of steroid hormones. The alternative to egg yolk-based extender (EY) can be soya lecithin-based extender (SL) and liposome-based extender (LP). However, the efficacy of SL is still a matter of debate. Few studies have been performed on the effect of LP but to date evaluation of buffalo semen cryopreserved in LP has not been studied. Therefore, this study was designed to compare SL and LP with conventional EY for evaluation of post-thaw quality of buffalo semen. Results showed that total, progressive and rapid sperm motility were found significantly higher (P<0.05) in LP among these extenders. In vitro assessment of post-thaw sperm longevity has also resulted in better maintenance of sperm kinetics and motility in LP in comparison to other extenders. Furthermore, sperm cryopreserved in LP travelled significantly more (P<0.05) distance in cervical mucus as compared to SL and EY. Therefore, it can be concluded that the LP is more efficient than SL and EY for the cryopreservation of buffalo semen.