Abstract
BACKGROUND: Cryopreservation of spermatozoa involves reduction of temperature to a subzero level, leading to increased longevity. However, temperature reduction has a significant effect on sperm membranes. OBJECTIVE: To evaluate the impact of the rate of temperature drop during the first phase of freezing on subtle membrane changes in cryopreserved bull spermatozoa. MATERIALS AND METHODS: Thirty-two ejaculates from four bulls (eight ejaculates/bull) were collected using artificial vagina while keeping a 3 to 4 days gap between two collections. Diluted semen samples were equilibrated at 5°C for 4 hours. The samples were then placed in a pre-programmed semen freezer. The first phase of freezing, that is, 5°C till −10°C was subjected to three different temperature drop rates: accelerated (F1), moderate (F2), and slow (F3), at 20°C per min, 10°C per min and 5°C per min, respectively. After thawing, spermatozoa were assessed for percentage live, plasma, and acrosomal membrane integrity, along with the external appearance of phosphatidyl serine, indicating apoptosis. RESULTS: A significant difference (p<0.05) in viability, plasma membrane integrity (HOS test), and acrosome membrane integrity (PSA test) was observed between F3 and the other groups. However, the parameters did not significantly differ between F1 and F2. The annexin V-PI assay (AN/PI) categorized four types of sperm populations: non-apoptotic and viable (AN−/PI−), apoptotic and viable (AN+/PI−), non-apoptotic and non-viable (AN−/PI+), and apoptotic and non-viable (AN+/PI+). The proportion of spermatozoa with (AN−/PI−) and (AN+/PI+) differed significantly (p<0.05) between F3 and the other groups. The values for apoptotic and viable (AN+/PI−) and non-apoptotic and non-viable (AN−/PI+) sperm were not significantly different among all freezing categories. CONCLUSION: A slower temperature drop rate (freezing rate) during the first phase of freezing results in less damaging, subtle membrane changes.
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