Effect of Caffeic Acid and Chitosan on Spermatological Parameters and Pregnancy Rate in Tuj Breed Ram Semen

The aim of this study is to determine the effect of caffeic acid and chitosan (CS) added to the sperm diluent on post‐thaw spermatological parameters and pregnancy rates in laparoscopic artificial insemination (AI) procedures by utilizing their protective effects. Semen samples were collected from five Tuj rams and diluted using a TRIS‐based extender containing different concentrations of caffeic acid (0.018 mg, GA1; 0.009 mg, GA2), CS (0.005 mg, GB1; 0.01 mg, GB2), and a control (C) group without antioxidant supplementation. Post‐thaw evaluations included assessments of semen motility, live percent, percent abnormal, and hypo‐osmotic swelling test (HOST). Additionally, acrosomal integrity and mitochondrial membrane potential (MMP) were analyzed via flow cytometry. Each treatment group was subjected to laparoscopic intrauterine insemination using 10 estrus‐synchronized ewes during the breeding season. Following thawing, the groups supplemented with caffeic acid demonstrated significant improvements ( p < 0.05) in motility, live percent, and lower rates of morphological compared to other groups. Specifically, the GA1 group exhibited a significant positive effect on acrosomal integrity ( p < 0.05). However, the groups containing CS showed detrimental effects on MMP ( p < 0.05). Although a numerically higher pregnancy rate was observed in the GA1 group, the differences were not statistically significant ( p > 0.05). In conclusion, the study contributed novelty by subjecting these two antioxidants to detailed spermatological evaluations and assessing pregnancy outcomes following laparoscopic AI. Although there was no statistically significant difference in pregnancy rates, caffeic acid showed positive effects on sperm quality after dissolution in rams, while CS did not show any improvement compared to other groups.