Addition of a motility stimulant improves kinetic parameters in post-thawed cholesterol-loaded stallion semen

Abstract

The freezing process exposes sperm to thermal stress, resulting in structural damage that may compromise their functionality. This study aimed to assess the impact of including cholesterol bound to cyclodextrin (CLC) before cryopreservation and of adding a motility stimulant (FertTalp) (Parrish et al. Theriogenology. 1986; 25:591-600) after thawing, on sperm kinetics, intracellular hydrogen peroxide production, mitochondrial potential and plasma membrane destabilization in equine semen. Twelve Mangalarga Marchador stallions were used to collect two ejaculates each, subsequently divided into four experimental groups: G1 (no CLC), G2 (1 mg CLC), G3 (1.5 mg CLC) and G4 (2 mg CLC). In Experiment I, the semen was cryopreserved according to Papa et al. (Animal Reproduction Science. 2008; 107:293-301), and sperm kinetics, mitochondrial potential and membrane destabilization were evaluated after thawing at 37°C. In Experiment II, the same parameters were evaluated after addition of FertTalp after thawing (20% v/v), as well as the evaluation of intracellular hydrogen peroxide production and a thermo-resistance test (TTR) to verify semen longevity for 120 minutes. Statistical analysis was performed using the F test, Tukey, Friedman and Conover, significance 5%. The results of Experiment I indicated that group G3 (1.5 mg CLC) showed higher total motility (TM) and progressive motility (PM) than group G1 (without CLC) (P<0.05), suggesting that this concentration of cholesterol is efficient in maintaining the kinetic quality of sperm after thawing. There were no significant differences between the groups regarding mitochondrial potential and plasma membrane destabilization. In Experiment II, addition of FertTalp post-thawing resulted in a significant increase in sperm velocity parameters (VCL, VSL, VAP) after 80 minutes of thawing, when compared to groups without FertTalp. The addition of the stimulant also favoured plasma membrane integrity and reduced intracellular hydrogen peroxide production, indicating that FertTalp was beneficial in preserving cell functionality during the thawing process. Data from the TTR showed that, over time (0 to 120 minutes), groups with FertTalp addition maintained higher motility and superior sperm velocity parameters when compared to other groups. These results suggest that the addition of CLC at a concentration of 1.5 mg before cryopreservation can improve sperm quality after thawing, especially concerning the motility. Furthermore, the addition of FertTalp as a post-thaw motility stimulant provides additional benefits, especially in preserving velocity and cellular integrity over time. In conclusion, the addition of CLC is beneficial for improving sperm motility after cryopreservation, and the use of FertTalp provides additional advantages in maintaining sperm quality during thawing and subsequent incubation, promoting greater sperm longevity and viability.