Cryoprotection of Humanin-like Peptides in Seminal Plasma for Ejaculated Spermatozoa of Crossbred Bulls

BACKGROUND: Cryopreservation process negatively affects spermatozoa functions. Humanin, a small polypeptide encoded in the mitochondrial genome, is well known for its role in cell survival. OBJECTIVE: To quantify the endogenous levels of humanin in seminal plasma of crossbred Frieswal bulls and to study its role in cryoprotection. The presence of humanin in bull spermatozoa was also investigated. MATERIALS AND METHODS: A total of 40 semen samples were separated into two groups based on the initial progressive motility (IPM): Good (IPM ≥70%) and Poor ( IPM ≤50%) groups; and/or based on the post-thaw motility (PTM): Freezable (PTM≥50%) and Non-freezable (PTM < 50%) groups. Humanin concentration in seminal plasma (SP-HN) was quantified using ELISA. RESULTS: SP-HN concentration ranged from undetectable to 67.6 pg/mL with a median level of 35.2 pg/mL. SP-HN level was significantly higher in the good quality semen group than in the poor quality semen group (p<0.001), and also significantly higher in the freezable group than in the non-freezable group (p<0.001). SP-HN level was positively correlated with initial progressive motility, post-thaw semen motility, viability, acrosome intactness and plasma membrane integrity, but negatively correlated the level of reactive oxygen species and malondialdehyde content. Immunochemical localization showed the presence of humanin in the proximal region of the middle piece of spermatozoa. CONCLUSION: Endogenous humanin level had significant correlation with semen quality and might protect sperm cells against freeze-induced oxidative stress.