We conducted this study to determine the potential cryopreservative effects of different hesperidin (vitamin P; H) doses on ram semen after freeze-thawing. Semen samples were obtained from Sönmez rams using an artificial vagina. The samples were divided into six groups: control, 10, 50, 100, 250, and 500 µg/mL H (C, H10, H50, H100, H250, and H500, respectively). At the end of the study, sperm motility and kinetic parameters, acrosome integrity (AI), mitochondrial membrane potential (MMP), viability, lipid peroxidation levels (LPL), chromatin damage, oxidant parameters, and antioxidant parameters were assayed. None of the doses of H added to the semen extender showed any enhancing effects on progressive motility compared to C (p > 0.05). In fact, H500 had negative effects (p < 0.05). Moreover, AI was the highest at the H10 dose, while LPL values were the lowest at the same dose (p < 0.05). The doses of H10 and H50 added to the Tris extender medium showed positive effects on sperm cell chromatin damage. Consequently, we can say that H doses used in this study are not effective on semen progressive motility, but the H10 dose is effective on AI and chromatin damage by reducing LPL.

Baicalein (B) has potential antioxidant properties, but it has not been tested as a ram semen extender. This study aimed to assess the impact of B on various sperm parameters and determine its potential influence on semen quality after the freeze-thawing process. During the breeding season, ejaculates were obtained from four rams with the aid of an artificial vagina. The collected mixed semen samples were divided into four groups: control (C; 0), B0.5 (0.5 mM), B1 (1 mM), and B2 (2 mM). After semen extension, the samples were loaded into 0.25 mL straws and stored for 2 h at 4°C prior to freezing in liquid nitrogen vapor and thawed in a water bath at 37°C. Among the groups, B0.5 demonstrated the highest progressive motility results, while B1 and B2 exhibited reduced motility (p < 0.05). In terms of high mitochondrial membrane potential, plasma membrane and acrosome integrity, and viability, B0.5 showed significantly superior outcomes to the other B groups (p < 0.05), although it was not significantly better than C. B1 displayed the highest plasma membrane integrity levels (p < 0.05). Notably, B2 displayed the lowest total antioxidant status levels among the groups (p < 0.05). The findings of this study suggested that the in vitro spermatological characteristics of ram spermatozoa such as progressive motility and chromatin integrity can be protected from the freeze-thawing process by using the 0.5 mM dose of baicalein as a semen extender. The treatment of sperm freezing might benefit from further in-depth research on the role of B in the improvement of cryoinjury and its underlying processes.