Platelet-rich plasma strategy against freezing damage in ram spermatozoa: its effect on miRNA, ion channels, growth factors, lipids and oxidative stress.

CONTEXT: Platelet-rich plasma (PRP) is a plasma component containing high concentrations of platelets, growth factors, antioxidants and proliferative properties.

AIM: To mitigate the negative effects of cryopreservation on ram semen by utilising PRP.

METHODS

Semen was collected from six rams twice a week for 3 weeks during the breeding season. Pooling was performed by dilution with tris + egg yolk diluent. Pooling was divided into three equal parts and re-diluted with diluents containing control (0% PRP), 5% PRP and 10% PRP. Sperms were frozen in an automatic freezing device and stored in liquid nitrogen. After thawing, spermatological, flow-cytometric, oxidative stress, cholesterol, fatty acid, ELISA, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses were performed.

KEY

RESULTS

Compared with the control group, the 5% PRP group exhibited a significant increase in progressive motility, viability and cholesterol ratios, glutathione-peroxidase activity, CATSPER1 (Cation Channel of Sperm), CATSPER3, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) levels and KCNJ11 (Potassium Channel, Inwardly Rectifying, Subfamily J, Member 11), HSA-MIR-181A, HSA-MIR-150 and HSA-MIR-374 transcripts. Apoptotic protein, malondialdehyde and HSA-MIR-410, OAR-MIR-10B, BTA-MIR-22-3P and RNO-MIR-494 transcripts were decreased in 5% PRP group compared with control. PRP supplementation at 10% increased dead sperm and heptadecenoic acid ratios, VEGF and PDGF levels and HSA-MIR-410, PPY-MIR-16, CFA-MIR-199, HSA-MIR-181A, HSA-MIR-150, OAR-MIR-127, HSA-LET-7A and HSA-MIR-374 transcripts as well as CATSPER3, HSD3β2, PDGFB and VEGFA proteins compared with the control. PRP supplementation at 10% significantly decreased plasma membrane integrity, insulin-like growth factor 1 (IGF1) level and CATSPER3 and KCNJ11 transcripts compared with the control.

CONCLUSIONS

The addition of 5% PRP before cryopreservation has beneficial effects on the functional and molecular properties of frozen-thawed ram spermatozoa, whereas the addition of 10% PRP has negative effects.

IMPLICATIONS: Five per cent PRP should be added to ram semen diluents.