The objective of this study was to determine the mechanism by which RU 486 (mifepristone) protects sperm to undergo premature capacitation during cryopreservation. For this, semen ejaculate (n = 20) was divided into four equal fractions and diluted using egg yolk-based extender supplemented with different concentrations of RU 486 (0, 5, 10 and 20 µM) and cryopreserved. We found that RU 486 did not impair the post-thaw sperm kinetics and motility but prevented cholesterol efflux, calcium influx, and protected CatSper channels during cryopreservation. The RU 486 protected sperm from premature capacitation which was confirmed by intracellular calcium level, expression of tyrosine phosphorylated proteins (75 and 80 kDa) and CTC (chlortetracycline) assay. Furthermore, antioxidant ability of RU 486 was reflected by the ferric reducing ability, lower production of sperm malondialdehyde and intracellular reactive oxygen species. Also, we demonstrated that RU 486 treated sperm underwent normal capacitation, zona pellucida binding and zygote cleavage indicating normal fertilizing ability of sperm. In conclusion, we report a new role of RU 486 in protecting buffalo sperm from premature capacitation during cryopreservation.

Somatic cell nuclear transfer (SCNT) technology provides an opportunity to multiply superior animals that could speed up dissemination of favorable genes into the population. In the present study, we attempted to reproduce a superior breeding bull of Murrah buffalo, the best dairy breed of buffalo, using donor cells that were established from tail-skin biopsy and seminal plasma. We studied several parameters such as cell cycle stages, histone modifications (H3K9ac and H3K27me3) and expression of developmental genes in donor cells to determine their SCNT reprogramming potentials. We successfully produced the cloned bull from an embryo that was produced from the skin-derived cell. Growth, blood hematology, plasma biochemistries, and reproductive organs of the produced cloned bull were found normal. Subsequently, the bull was employed for semen production. Semen parameters such as CASA (Computer Assisted Semen Analysis) variables and in vitro fertilizing ability of sperms of the cloned bull were found similar to non-cloned bulls, including the donor bull. At present, we have 12 live healthy progenies that were produced using artificial insemination of frozen semen of the cloned bull, which indicate that the cloned bull is fertile and can be utilized in the buffalo breeding schemes. Taken together, we demonstrate that SCNT can be used to reproduce superior buffalo bulls.

Semen contains epithelial cells that can be cultured in vitro . For somatic cell nuclear transfer applications, it is essential to know whether clone(s) produced from semen-derived epithelial cells (SedECs) are healthy and reproductively competent. In this study, the semen and fertility profile of a cloned bull (C1) that was produced from a SedEC were compared with its donor (D1) and with two cloned bulls (C2, C3) that were produced from commonly used skin-derived fibroblast cells (SkdFCs). We observed variations in some fresh semen parameters (ejaculated volume and mass motility), frozen-thawed sperm parameters (plasma membrane integrity, and computer-assisted semen analysis (CASA) indices), but values are within the normal expected range. There was no difference in sperm concentration of ejaculated semen and frozen-thawed semen parameters which include sperm motility, percentage of live and normal morphology sperm, and distance traveled through oestrus mucus. Following in vitro fertilization (IVF) experiments, zygotes from C1 had higher ( P < 0 . 05 ) cleavage rates (81%) than C2, C3, and D1 (71%, 67%, and 75%, respectively); however, blastocyst development per cleaved embryo and quality of produced blastocysts did not differ. The conception rate of C1 was 46% (7/15) and C2 was 50% (8/15) following artificial insemination with frozen-thawed semen. Established pregnancies resulted in births of 7 and 6 progenies sired by C1 and C2, respectively, and all calves show no signs of phenotypical abnormalities. These results showed that semen from a cloned bull derived from SedECs is equivalent to semen from its donor bull and bulls cloned from SkdFCs.