Novel Antioxidant Humanin Analogue Restores Bull Semen Cryosurvivability

Abstract

The current study was intended to determine the effects of humanin analogue (HNG) supplementation on semen freezability and oxidative stress parameters of cryopreserved bull spermatozoa. A total of 24 semen ejaculates, from Frieswal breeding bulls, were selected for the experiment. Samples with ≥70% progressive motility, ≥500 million spermatozoa/mL, and >2 mL volume were utilized for further processing, supplementation, and cryopreservation. Each sample was divided into four equal groups and the experiment consisted of three treatment groups and one control group. In the control group, the spermatozoa were frozen in an extender without HNG, while in the other three groups, HNG supplementation was done at a concentration of 2, 10, and 20 μM, respectively. The fortification of HNG at a concentration of 10 and 20 µM significantly ($p<0.05$) improved the percent post-thaw motility and kinetic variables as assessed by CASA, viability, acrosome- and plasma membrane-integrity as compared to 2 µM and control group. The supplementation showed significant ($p<0.05$) controlled oxidative stress as exhibited by a reduction in lipid peroxidation and reactive oxygen species levels. The supplemented groups also exhibited significant ($p<0.05$) improvements in total antioxidant levels, increased mean percentage of spermatozoa exhibiting high mitochondrial membrane potential, DNA integrity, acrosome-intact noncapacitated spermatozoa, and zona binding ability. In conclusion, the supplementation of humanin analogue at 10 and 20 µM significantly improved the post-thaw semen quality, functional parameters, and zona binding ability and controlled the oxidative stress in cryopreserved spermatozoa. Thus, the supplementation of its analogue may potentially prove to be a valuable approach for the improvement of semen freezability in crossbred Frieswal bulls.