Addition of various antioxidants in semen extender is one of the vital strategies being applied in reproductive biology for attaining functionally/structurally integral sperms and hence an appropriate conception rate. It is well established that chilling of buffalo semen results in decreased semen quality which is highly associated with decreased antioxidant activity and higher ROS production. Furthermore, buffalo bull spermatozoa are more susceptible to oxidative damage as compared to cattle bull spermatozoa. It is believed that this difference is due to higher contents of polyunsaturated phospholipids present in plasma membrane of buffalo bull spermatozoa. Freezing process accelerates the production of ROS molecules which may decrease the viability of buffalo bull spermatozoa during storage. Therefore, supplementation of antioxidants in semen extender is required to decrease the ROS-mediated damages to buffalo spermatozoa. The present study had, hence, been designed to monitor the effects of trehalose and L-Cysteine on the semen quality, antioxidant enzyme activity and fertility of Nili Ravi Buffalo bulls.
Semen samples (n= 20) from 4 buffalo bulls were diluted in Tris-citric acid based extender having different concentrations of trehalose (0.0, 15, 30, 45, and 60 mM) and frozen in French straws. At post dilution, profile of sperm catalase (U/mL) was higher (P < 0.05) in extenders containing 15, 30 and 45 mM of trehalose as compared to control. While profiles of superoxide dismutase (U/mL) and total glutathione (μM) were higher (P < 0.05) in extenders containing 15 and 30 mM of trehalose as compared to control. At pre freezing, sperm catalase, superoxide dismutase and total glutathione profiles were higher (P < 0.05) in all the treatment groups as compared to control. At post thawing, the profiles of catalase and total glutathione
were higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment groups and control. Whereas, profile of superoxide dismutase was higher (P < 0.05) in extenders containing 30, 45 and 60 mM of trehalose as compared to control and 15 mM group. Post thaw total sperm motility (%) was higher (P < 0.05) in extender containing 30 mM trehalose as compared to control and 15 and 60 mM groups. While sperm progressive motility (%), rapid velocity (%), average path velocity (μm/s), straight linear velocity (μm/s), curvilinear velocity (μm/s), plasma membrane (structural and functional, %), acrosome (%) and DNA (%) integrity were higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment groups and control. The fertility rates (61% vs. 43%) were higher (P < 0.05) in buffaloes inseminated with semen doses cryopreserved in extender containing 30 mM of trehalose than the control. It is concluded that addition of 30 mM trehalose in extender improves the semen antioxidant enzymes activity, post thaw quality, and fertility in Nili Ravi buffaloes.
Similarly Semen samples from 4 buffalo bulls were diluted in Tris-citric acid based extender having different concentrations of L-cysteine (0.0, 0.5, 1.0, 2.0, and 3.0 mM) and frozen into 0.5 ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase/reductase)] were significantly higher (P< 0.05) at pre freezing and post thawing in extender containing 2.0 mM L-Cysteine as compared to other groups. Post thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity(μm s-1), straight line velocity (μm s-1), curvilinear velocity (μm s-1), beat cross frequency (Hz), viable sperm with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with addition of 2.0 mM L-cysteine as compared to other groups (P < 0.05). The fertility rates (59 vs. 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mM of L-cysteine than the control. In conclusion, addition of 2.0 mM L-cysteine in extender improved the
SUMMARY
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antioxidant enzymes profile, post thaw quality and in vivo fertility of Nili Ravi buffalo bull spermatozoa.
Conclusion
It was concluded that addition of 30mM Trehalose and 2.0mM L-Cysteine in semen extender has significantly improved semen antioxidant enzymes activity, post thaw quality and fertility in Nili Ravi buffaloes.