Intraepididymal platelet-rich plasma improves semen cryoresistance via antioxidant, lipid and molecular modulation during the non-breeding season in rams.

This study aimed to investigate the effectiveness of intraepididymal platelet-rich plasma (PRP) administration in preventing cryopreservation-induced sperm damage in rams. Twelve adult rams were randomly assigned into two groups (n = 6) in the non-breeding season. Rams in the PRP group received 0.2 ml/per epididymis (150–200 × 10⁶ platelets) of PRP every 15 days for a total of six injections, while control group received the same volume of saline. Semen samples were collected biweekly and pooled within each group before undergoing standard cryopreservation procedures. Post-thaw analyses included morphological, functional, biochemical, and molecular assessments. Compared to the control, intraepididimal PRP significantly increased hypo-osmotic swelling (HOS) response, total and progressive motility, rapid sperm percentage, and kinetic parameters (VCL- curvilinear velocity, VSL- straight-line velocity, VAP- average path velocity), while reducing static and acrosome-damaged sperm in thawed semen. Intraepididimal PRP also enhanced catalase activity, cholesterol and myristic acid (C14:0) concentrations and the levels of steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase type 1 (HSD3β1), cation channel of sperm (CatSper1), platelet-derived growth factor (PDGF), platelet-derived growth factor receptor (PDGFR), and reduced malondialdehyde level in thawed semen. Intraepididimal PRP significantly increased the mRNA presence of CatSper2, CatSper3, CatSper4, transient receptor potential melastatin 3 (TRPM3) and transient receptor potential vanilloid 5 (TRPV5) ion channels, oar-miR-3958-3p and oar-miR-125b, and decreased bta-miR-22-3p and rno-miR-494 in thawed semen. Additionally, intraepididimal PRP significantly upregulated the protein expressions of CatSper3, HSD3β2 and PDGFA, and decreased protein expressions of vascular endothelial growth factor A (VEGFA) and transforming growth factor beta 1 (TGFβ1) in thawed semen. In conclusion, intraepididymal PRP administration improved cryoresistance in ram spermatozoa, likely due to its growth factors, lipids, and antioxidants. These effects enhanced post-thaw sperm quality by modulating oxidative stress, cholesterol, ion channels, microRNAs, and steroidogenic proteins, indicating PRP as a promising tool for improving semen cryotolerance in rams.