43 Effect of resveratrol on cryopreservation of bull spermatozoa by conventional slow freezing or ultrarapid freezing

Resveratrol (RES) is a powerful antioxidant with free radical scavenger properties that result in a reduction in reactive oxygen species (ROS) generation and lipid peroxidation, these effects have been demonstrated in cryopreserved spermatozoa. This work evaluated the effect of RES (0 µM as control and 50 µM) on the cryopreservation of bull spermatozoa by conventional slow (CS) or ultrarapid (UR) freezing on post-thaw quality and fertility. Twenty-four ejaculates from four fertile bulls (six per bull) were collected using an artificial vagina and then diluted with TCG-EY (tris-citric acid-glucose + 6% egg yolk). Four treatments per ejaculation were conformed according to the freezing method and RES addition: CS-RES (n = 80), CS-0 (n = 80), UR-RES (n = 24), and UR-0 (n = 24). The CS freezing method was done by exposing the 0.25-mL sperm sample straws (plus 5% glycerol) to liquid nitrogen (NL₂) vapors, and the UR freezing was made using submerging 30-µL drops of samples (plus 100 mM sucrose) into NL₂. Initially, the kinetic parameters, plasma, and acrosome membranes integrity (IPIA), and oxidative stress were assessed using a CASA system (SCA®), PI/PNA-FICT double fluorescence test, and Cell-ROX Deep Red test. After that, IVF based on cleavage and blastocyst rates from the four treatments spermatozoa were assessed using 200 in vitro-matured bovine oocytes in each treatment. Eight IVF sessions were performed using two cryopreserved semen per bull. Sperm samples from all treatments were selected by Percoll^® (45/90%) before IVF. A factorial ANOVA and Tukey test were used to assess the interactions between the “freezing method” and “RES dose.” The results showed significant (P < 0.05) differences between bulls regarding post-thaw sperm quality. The CS freezing treatments yielded a better sperm cryoresponse than UR freezing treatments, irrespective of RES addition. After CS-RES treatment, the total and progressive motilities, and IPIA percentages were higher (P < 0.05) than after CS-0, UR-RES, and UR-0 treatments. In addition, the UR-RES treatment produced greater (P < 0.05) values of the beat cross frequency and lower values (P < 0.05) of oxidative stress than the UR-0 treatment. The CS-RES treatment produced a higher (P < 0.05) cleavage rate (%) than the CS-0 and UR-RES treatments (64.0 ± 3.40 vs 34.7 ± 3.44 and 42.0 ± 4.96, respectively). Likewise, the CS-RES treatment produced a higher (P < 0.05) blastocyst rate (%) than all treatments (CS-RES: 22.5 ± 3.11 vs CS-0: 10.2 ± 2.29; UR-RES: 8.0 ± 3.88; and UR-0: 4.0 ± 2.8). In conclusion, RES improved the motility, plasmatic and acrosomal membrane integrity, and in vitro fertility of spermatozoa after the freezing-thawing process. However, after ultrarapid freezing, RES reduced only oxidative stress.