Enhancing liquid-chilled storage and cryopreservation capacities of ram spermatozoa by supplementing the diluent with different additives

Abstract

Objective: In the present study, we determined efficiency of incorporating caffeine, melatonin or omega3 polyunsaturated fatty acid in the diluent on mitigating consequences of (a) liquid chilled and (b) cryostorage of ram spermatozoa. Methods: In the first experiment, ejaculates (n = 30) were collected from 5 adult rams and were pooled, diluted (1:10) with Triscitric acid (base diluent) and were split into 4 aliquots assigned for: control (untreated), caffeine (0.1 mM), melatonin (0.3 mM) or omega3 fatty acids (0.3 mM) (T0). The diluted specimens were stored at 4°C for 48 h, during which sperm physical and cytological properties were evaluated along with oxidative stress indices (T24, T48). In the second experiment, 15 ejaculates (3 per male) were pooled, diluted with glycerolized base diluent (4% glycerol, v/v) and were split corresponding to the same previous treatment groups before being processed for cryopreservation. Postthaw physical and kinematic sperm properties were assessed by a computerassisted sperm analysis system. Results: The results clarified superiority of both melatonin and omega3 supplementation on maintaining (p<0.05) sperm properties, while reducing (p<0.05) lipid peroxidase reaction and enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in preservation medium, compared to caffeine either during liquidchilled storage or cryopreservation of spermatozoa. Conclusion: Melatonin and omega3 are regarded efficient alternatives to caffeine when processing ram spermatozoa for application of artificial insemination or in vitro fertilization.