Effects of melatonin and Single Layer Centrifugation on cryosurvival of ram semen samples

Abstract: The composition of the ram sperm membrane renders it vulnerable to injury during cryopreservation. Production of reactive oxygen species, which are by-products of metabolism, increases during freezing and thawing, causing damage to sperm membranes and acrosomes as well as affecting mitochondrial activity. This results in a poor post-thaw yield of viable, morphologically normal spermatozoa for insemination. In the present study, we investigated the effect of melatonin addition and centrifugation through a single layer of a species-specific colloid (Ovicoll) on the quality of frozen/thawed ram semen samples. Ejaculates from autochthonous Jezersko-Solčava rams were available for this study (n = 20). Each ejaculate was split and melatonin (1 mM) was added to the Tris-based egg-yolk-extender of one part prior to freezing. Sperm motility, viability and morphology were analysed 0, 3 and 6 hours after thawing. Membrane integrity was evaluated by HOST (hypoosmotic swelling test). Melatonin addition significantly improved sperm motility (p < 0.05), progressive motility (p < 0.01), membrane integrity (p < 0.001) and normal morphology (p < 0.001). These parameters are indicators of functional spermatozoa, suggesting that they might be able to reach and fertilize the oocyte. However, melatonin did not improve recovery rate, sperm viability and acrosome integrity. In conclusion, melatonin addition can help to protect ram spermatozoa against cryodamage, possibly by neutralisation of reactive oxygen species.