Mitochondrial derived peptide humanin improved semen quality, semen freezability, antioxidant status and in-vitro fertility in crossbred bull

The sperm quality, freezability, and fertility of crossbred bulls exhibit significant unpredictability, which consequently impacts the breeding program. The present study documents the supplementation of humanin, a mitochondria-derived peptide, on crossbred bull's sperm quality, freezability, antioxidant status and in-vitro fertility. For this objective a total of 24 ejaculates, 8 from each of the three Vrindavani crossbred bulls, were selected based on sperm progressive motility (≥ 70%) and concentration (≥ 500 million). The selected fresh semen samples were divided into four aliquots and diluted with Tris-Fructose-Egg-Yolk-Glycerol (TFEGY) extender supplemented with various concentrations of humanin peptide; control (Group-1) without supplementation; Group II with 2.5 µM; Group III supplemented with 5 µM and Group IV supplemented with 7.5 µM. Semen quality parameters, sperm kinematics, sperm plasma membrane integrity, mitochondrial membrane potential, sperm cryo-capacitation status, ejaculate freezability rate, antioxidant status and in-vitro fertility assays were done. At the pre-freeze stage, there was no significant effect on the percentage of individual progressive motility in any group. Supplementation with 5 µM humanin resulted in an 87.5% ejaculate recovery rate after cryopreservation. At the post-thaw stage, sperm quality parameters, sperm kinematics, sperm plasma membrane integrity, mitochondrial membrane potential, sperm cryo-capacitation status, antioxidant status and in-vitro fertility were significantly improved in Group III (5 µM humanin supplementation). In conclusion, our results demonstrate that humanin supplementation in crossbred bull semen enhances semen freezability and post-thaw ejaculate recovery rate.