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

İbrahim Güngör , Gaffari Türk, Aslıhan Çakır Cihangiroğlu, Gözde Arkalı, AHMET TEKTEMUR , Görkem Kırmızıkaya Özmen, Nida Badıllı, Tutku Acısu, Mustafa Bulan, Seyma Ozer Kaya, Mustafa Sönmez, Seyfettin Gür, Abdurrauf Yüce, Okkes Yilmaz, Erdoğan Memili, Zafer Çambay

Abstract

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 three 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, 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, CATSPER3, VEGF and PDGF levels and KCNJ11, 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 to control. 10% PRP supplementation 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 to control. 10% PRP supplementation significantly decreased plasma membrane integrity, IGF1 level and CATSPER3 and KCNJ11 transcripts compared to control. Conclusions: The addition of 5% PRP before cryopreservation has beneficial effects on the functional and molecular properties of frozen-thawed ram spermatozoa, while the addition of 10% PRP has negative effects. Implications: 5% PRP should be added to ram semen diluents.

RD25064  Accepted 03 September 2025

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