Humanin as a Molecular Indicator of Semen Quality and Cryotolerance in Crossbred Cattle

The present study aimed to assess the presence of humanin in the sperm of crossbred cattle bulls using immunocytochemistry and to evaluate its potential as a semen quality marker. For this, a total of 49 ejaculates were collected from three Vrindavani crossbred bulls and were evaluated for volume, individual progressive motility (IPM), abnormality, and sperm concentration. The ejaculates were grouped into four categories, that is normozoospermia, oligozoospermia, asthenozoospermia and oligoasthenozoospermia, based on sperm concentration and IPM. Furthermore, normozoospermic ejaculates were cryopreserved and retrospectively subdivided into two groups (good-freezable and poor-freezable) based on post-thaw motility (PTM). Immunofluorescence assays revealed humanin in the neck and upper midpiece regions of sperm. Humanin levels in seminal plasma were significantly higher (p < 0.05) in normozoospermic individuals than in other groups. Humanin levels in seminal plasma were positively and significantly correlated with sperm concentration and individual progressive motility (p < 0.05). Out of 28 normozoospermia ejaculates, 19 ejaculates (67.86%) were categorized as good freezable, and nine ejaculates (32.14%) were grouped as poor freezable. Humanin level in the seminal plasma of good freezable ejaculates was significantly (p < 0.05) higher than that of poor freezable ejaculates. In conclusion, our research demonstrated, the presence of the humanin peptide in the sperm and seminal plasma of Vrindavani bulls. Additionally, the role of humanin as a potential semen-quality marker in crossbred bull semen was established to identify high-quality, freezeable ejaculates.