P. S. Yadav

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.

Seminal Plasma Proteome: Promising Biomarkers for Bull Fertility

The seminal plasma is a mixture of secretions from the testis, epididymis and male accessory sex glands. A number of seminal plasma proteins are associated with male fertility but most of these have not been studied in detail till now. Recently, proteomics has been used to show the differences in proteins profile in seminal plasma from high and low fertility bulls. For example, osteopontin, phospholipase A2, P25b, acidic seminal fluid proteins, α-l-fucosidase and cathepsin D are positively correlated with fertility of bulls and may act as useful fertility marker (s), while lipocalin-type prostaglandin D synthase, spermadhesin Z13, clusterin and ubiquitin are negatively correlated with fertility in bulls. Bovine seminal plasma proteins in seminal plasma act like a double-edged sword and showed a quadratic association with bull fertility. The physiological roles of the metalloproteinase-2 (TIMP-2), ecto-ADP-ribosyltransferase 5, nuclobindin, Niemann-Pick C2 and epididymal sperm-binding protein 1 and their relationship to bull fertility need further studies. This review summarizes the physiological functions of proteins of seminal plasma and their relation to bull fertility.

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.

Culture, characterization and differentiation of cells from buffalo (Bubalus bubalis) amnion

Stem cells present an important tool in livestock assisted reproduction and veterinary therapeutic field such as tissue engineering. We report for the first time isolation of pluripotent stem cell-like cells expressing pluripotency markers (alkaline phospahatase, OCT-4, NANOG and SOX-2) from the amnion of water buffalo (Bubalus bubalis). The cells showed no apparent abnormalities in their chromosomal profiles before and after cryopreservation. The cytochemical staining revealed that pluripotent cells were capable of undergoing directed differentiation in vitro into osteocytes. It could be inferred that amnion-derived pluripotent stem cell-like cells can be isolated, cultured for many passages and differentiated into mesoderm lineage, and may be an alternative source to mesenchymal stem cells. These cells can have applications in assisted reproduction, developmental biological and regenerative medicine.

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.

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

ABSTRACT 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.

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.

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.

Buffalo Embryonic, Fetal and Adult Stem Cells: Progress and Challenges

Since the generation of buffalo embryonic stem cells (ESCs) from the inner cell mass (ICM) of blastocysts in 2004 to till date, significant progress has been achieved in this species. Derivation and maintenance of ESCs from blastocysts generated by parthenogenesis, in vitro fertilization and nuclear transfer showed a steady progress towards finding the optimal conditions for the derivation of stable and highly competent buffalo ESCs. The molecular mechanisms for self-renewal of buffalo ESCs and the role of various growth factors involved therein as well as differentiation capacity were explored to a great extent. Although some reports showed the successful isolation and long-term culture of buffalo ESCs in vitro, but many studies were not able to establish suitable conditions for preventing spontaneous differentiation and senescence of ESCs. Hence a need to search alternative ways of deriving unipotent, pluripotent or multipotent stem cells in buffaloes always remained in trend. Therefore, in the present review, we analyse the available data on buffalo ESCs to have a better understanding about the strengths and weaknesses of the buffalo ESC has been analysed generation, which could help in the production of validated ESC lines in this species, besides the recent progress in buffalo fetal and adult tissue derived stem cells, which could be established as an alternative source of ESCs.