297 ADDITION OF GLUTATHIONE TO THAWING MEDIUM FOR BULL SPERMATOZOA IMPROVES THE IN VITRO EMBRYO PRODUCTION

Abstract

The cold shock of spermatozoa is associated with oxidative stress induced by reactive oxygen species (ROS) generation (Chatterjee et al. 2001 Mol. Reprod. Dev. 60, 498–506). Glutathione (l-�-glutamyl-l-cysteinylglycine; GSH) plays an important role as an intracellular defense mechanism against oxidative stress. The process of freezing is associated with a significant reduction in GSH content in novine spermatozoa (Bilodeau et al. 2000 Mol. Reprod. Dev. 55, 282–288). The main objective of this study was to evaluate the effect of GSH supplementation of the thawing extender on bull sperm used for in vitro embryo production. To examine the effect of GSH supplementation during the thawing process, spermatozoa from 6 different bulls were incubated without addition of GSH (control), and with addition of 1 mM or 5 mM GSH to the sperm-TALP medium, and maintained 30 min at 37�C in these media before assay. IVM and IVF were performed as described Coy et al. (2005 Reproduction 129, 19–26, 747–755). Oocytes were fertilized with frozen–thawed semen (106 total sperm/mL). After 18 h co-incubation, they were cultured in KSOM medium for 7 days. Embryos were examined for developmental stages at Day 2 (2–4 cells), Day 3 (8–16 cells), and Day 7 (morula–blastocysts). Finally, the total number of nuclei of blastocysts was determined by staining with Hoechst 33342 (10 mg mL-1; 20 min). The percentage of cleavage in the different embryo stages was higher in the 5 mM GSH group [n = 233, Day 2 (64.63%), Day 3 (56.92%), and Day 7 (31.28%)] than in control [n = 229, Day 2 (47.64%), Day 3 (42.16%), and Day 7 (22.32%)], and the 1 mM group maintained an intermediate position with [n = 246, Day 2 (54.88%), Day 3 (49.56%), and Day 7 (28.98%)]. The bull sperm affected significantly the cleavage parameters studied. The number of nuclei within the blastocysts ranged from 39.93 to 47.71, and no differences were observed between experimental groups. It is known that addition of GSH produces a reduction in ROS generation (Gadea et al. 2005 J. Androl. 26, 749–756). The results showed that in the treatment group there is a reduction in ROS generation in spermatozoa and an improvement in the cleavage rate and blastocyst formation when GSH was used. This study demonstrates that only a short period of incubation of the spermatozoa in an antioxidant could improve the spermatozoa functionality of producing more and better embryos. This work was supported by AGL2003-03144, BIOCARM 10BIO2005/01-6463 and Seneca 03018/PI/05.