Cryoprotectant Effect of Different Antioxidants Added to Bovine Sperm

Cryopreservation-thawing process of bovine sperm is associated with generation of elevated amounts of Reactive Oxygen Species (ROS). These ROS cause harmful effects on sperm. Therefore, the objective of this study was to evaluate the cryoprotectant potential effects of Cysteine (CYS), Caffeine (CAF), Ascorbic Acid (AA) and Rhus trilobata plant extract (RT), added to extender for cryopreservation of bull semen on post-thawed semen traits. Semen from six bulls (5 Angus, 1 Charolais) was obtained and each ejaculated was diluted to a final concentration of 40 x 10⁶ spermatozoa per mL and divided into five equal aliquots corresponding to each of the treatments: Control (CT), CYS (2 mM/ml), CAF (5 mM/ml), AA (4.5 mg/ml) y RT (16.6 µg/ml). Samples were packed in 0.5 mL straws and frozen in liquid nitrogen and then thawed for evaluation. Cellular viability (VIT) was assessed through nigrosin eosin smears. Hypo-osmotic swelling test (HOST) was used to assess the functional integrity of the sperm membrane. Computer-assisted sperm analysis (CASA) systems was used to measure motility parameters: spermatic motility (%, MT), progressive motility (%, MP) as well as spermatic kinematics: average path distance (DAP, µm), curved line distance (DCL, µm), straight line distance (DSL, µm), average path velocity (VAP, µm/s), straight line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), straightness (%, STR), linearity (%,LIN), wobble (%, WOB), amplitude of the lateral head displacement (ALH, µm) and frequency of head displacement (BCF, Hz). Data were analyzed using the MIXED procedure of SAS, with a model including treatment as fixed effect and bull as a random effect. Results indicated that the addition of RT extract to the extender for cryopreservation of bull semen preserves better the viability and integrity of the sperm membrane compared to the other treatments (P<0.05). The MT and MP were improved (P<0.05) whit RT treatment compared to the other groups. The characteristics of spermatic kinematics DAP, DSL, DCL, VAP, VSL, VCL and BCF were affected in a positive way (P<0.05) by the addition of RT and CAF treatments in comparison of CT, CYS and AA. In contrast, no statistical difference (P>0.05) was observed between the different treatments. In conclusion, our results indicate that the addition de RT to the extender for cryopreservation of bull semen allows better preservation of both, the cellular characteristics and the motility parameters of post-thawed bovine sperm.