Subtle membrane changes in cryopreserved bull spermatozoa when modified temperature drop rates are used during the...

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.

To evaluate the impact of the rate of temperature drop during the first phase of freezing on subtle membrane changes in cryopreserved bull spermatozoa.

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 degree C for 4 hours. The samples were then placed in a pre-programmed semen freezer. The first phase of freezing, that is, 5 degree C till -10 degree C was subjected to three different temperature drop rates: accelerated (F₁), moderate (F₂), and slow (F₃), at 20 degree C per min, 10 degree C per min and 5 degree 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.

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.

A slower temperature drop rate (freezing rate) during the first phase of freezing results in less damaging, subtle membrane changes. Doi.org/10.54680/fr24410110312.