Alpha-lipoic Acid-loaded Nanoliposomes Improve the Freezability and Post-thaw Quality of Buffalo Spermatozoa

Abstract

Buffalo spermatozoa are highly susceptible to cryopreservation-induced damage, which can compromise post-thaw semen quality. This study evaluated the cryoprotective effect of alpha-lipoic acid-loaded nanoliposomes (ALAN) supplemented in a Tris–egg yolk–fructose extender. Alpha-lipoic acid was encapsulated in nanoliposomes using the thin-film hydration technique. Twelve ejaculates collected from four healthy Murrah buffalo bulls were pooled, divided into six aliquots, and supplemented with ALAN at 0, 50, 100, 150, 175, or 200 µg/mL before cryopreservation. The untreated extender served as the control, and all samples underwent the same freezing and thawing conditions. After thawing, progressive motility, viability, morphological abnormalities, plasma-membrane integrity, and acrosome integrity were evaluated. ALAN supplementation significantly affected all assessed sperm-quality parameters (p < 0.05). The ALAN150 group showed the highest progressive motility (61.67 ± 3.07%), viability (70.83 ± 3.00%), and plasma-membrane integrity (58.17 ± 2.36%), together with the lowest proportion of abnormal spermatozoa (8.83 ± 0.48%). The greatest acrosome integrity was recorded in the ALAN200 group (69.17 ± 1.62%). Across the evaluated outcomes, 150 µg/mL ALAN produced the most consistent overall improvement in post-thaw sperm quality. These findings indicate that nanoliposomal delivery of alpha-lipoic acid may reduce cryopreservation-associated damage in buffalo spermatozoa. Supplementation at 150 µg/mL may therefore be suitable for further validation as an extender additive for buffalo-semen cryopreservation.