Cell and Tissue Journal

Abstract Aim: The purpose of this study was to evaluate the effects of sub-lethal osmotic stress (325, 350, 375 and 400 mOsm) during cryopreservation in commercially diluter medium (Bioexcell) on Holstein mail cow sperm qualitative characters after freezing-thawing. Material and Methods: Semen was collected from four Holstein mail cows using artificial vagina two times for a week. All of obtained semen mixed together and then were divided into five equal parts. Each part was freeze-melted according to the experimental treatments consisting of different osmotic stresses. 300 mOsm treatment medium was applied as control group. Sperms motility and progressive motility were assessed by computer assessment semen analysis. Also sperms viability, membrane integrity, mitochondria activity and membrane lipid peroxidation of frozen-thawed semen were assessed using Eosin-Nigrosin, hypo osmotic swelling test, Hankok, Rhodamin 123 and TBA procedures, respectively. Results: Results showed that 375 mOsm osmotic stress treatment had the most significant improvement of motility%, progressive motility and viability in comparison with other treatments. Also mitochondria activity in 350 and 375 mOsm osmotic pressures treatments was significantly higher than other experimental treatments (p < 0.05). Different osmotic stresses treatments did not show significant effect on sperm morphology and membrane lipid proxidation. Conclusion: It is seemed that mild and sub-lethal osmotic stresses induction in cow sperm freezing diluters can significantly improved some of sperm qualitative characters such as their motility and viability.