Effect of platelet rich plasma lysate and fibroblast growth factor 2 on sperm motility in stallions
Type of DegreeMaster's Thesis
General Veterinary Medicine
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Semen extenders are continually tested to improve sperm motility, longevity, and, consequently, improve fertility. Growth factors (GFs) modulate cell function, which could be advantageous to sperm by improving motility. In humans and mice, fibroblast growth factor 2 (FGF2) improved sperm motility. Platelet rich plasma (PRP), which is rich in growth factors (GFs) including FGF2, reduced post-mating inflammatory response within the uterus when infused 24 hours before or after artificial insemination. The effect of PRP on sperm has not been determined. In this study, PRP lysate was used instead of PRP since it is more purified and highly concentrated in GFs. The objective was to evaluate the effect of adding either pooled PRP lysate (PRPL) or recombinant equine FGF2 (reFGF2) at varying concentrations to sperm extended in commercial semen extender (INRA 96) in the stallion. Eight aliquots were tested using concentrations of 1, 2.5, 5, and 10% of PRPL containing 1 IU/ml of heparin, and 0.1, 1, 10, and 100 ng/ml of reFGF2 and compared to control groups with and without 1 IU/ml of heparin. Heparin use was based on a pilot study to prevent gel formation from PRPL reaction with semen extender, precluding motility analysis. Motility parameters were evaluated with samples standardized to 50 million sperm/ml using computer-assisted semen analysis (CASA) at hours 0, 0.5, 1, 1.5, 6, and 24 after treatment. For both PRPL and reFGF2 treatments, there were no differences (p = 0.99) in motility among groups at any time point, with ranges from 60 to 66% at hour 0 and 46 to 49% at hour 24. Interestingly, results revealed that concentrations of PRPL > 5% induced sperm agglutination via head-to-head attachment (HHA), starting at hour 1, whereas PRPL at concentrations below 2.5% did not induce HHA nor affect sperm motility. In addition, HHA was objectively detected by decreases in the total number of cells counted per field (total cells) and estimated concentration measured by CASA, assuming that only free sperm were counted, whereas sperm entrapped by HHA were not counted by CASA. Decreases in total sperm number and concentration were different (p < 0.0001) for 5 and 10% PRP groups, being more pronounced for 10% PRPL group, suggesting a dose-dependent characteristic of HHA induced by PRPL. One important finding was that PRPL induced HHA but did not kill sperm. Although no motility improvement was observed, results suggest that the direct addition of PRPL in semen extender at doses below 5% could be used without substantially affecting sperm motility. However, further research on uterine inflammatory response is needed to test whether this would have a beneficial effect in the endometrium.