225 DOES THE SUPPLEMENTATION OF THE BOAR SEMEN EXTENDER WITH CARNOSINE, L-HISTIDINE, AND TAURINE PRESERVE SPERM FUNCTION DURING STORAGE?

Abstract

High levels of reactive oxygen species (ROS: superoxide, hydroxyl, hydrogen peroxide, nitric oxide, peroxynitrile) endanger sperm motility, viability, and function by interaction with membrane lipids, proteins, and nuclear and mitochondrial DNA (Sikka 2004 J. Androl. 25, 5–18). ROS generation has a significant negative effect on the fertilization rate after IVF, and so measurement of ROS levels in semen specimens before IVF may be useful in predicting the IVF outcome (Agarwal et al. 2005 Fertil. Steril. 84, 228–231). Several compounds of the antioxidant systems have been identified in the epididymal environment, spermatozoa, and seminal plasma. The antioxidants carnosine, L-histidine (Ducci et al. 2006 Pol. J. Vet. Sci. 9, 159–163), and taurine (Van der Horst and Grooten 1966 Biochim. Biophys. Acta. 117, 495–497) have been detected in boar semen and added to the extender in freezing procedures in several species. The main objective of this study was to evaluate the effect of carnosine, L-histidine, and taurine supplementation of the extender on boar sperm functionality as measured by sperm motility during computer-assisted semen analysis (CASA) and by IVF ability using mature oocytes, as previously described (Selles et al. 2003 Reprod. Domest. Anim. 38, 66–72). The sperm-rich fraction from mature fertile boars was diluted with isothermal Beltsville thawing solution (BTS) extender. Diluted semen was placed at 15°C and centrifuged at 800g for 10 min. The semen pellet was resuspended with BTS supplemented by 5 mm of carnosine, L-histidine, or taurine or not supplemented (control) to provide 75 × 10⁶ spermatozoa mL^–1 and stored at 15°C for 24 h (IVF assay), or 48 or 120 h (for CASA assay). We observed that the motility parameters were affected by storage time and that the addition of taurine increased the motility at 48 h of storage. Alternately, the addition of L-histidine to the extender reduced significantly the motility parameters after 120 h. The results showed that the addition of L-histidine induced a significant (P ≤ 0.01) decrease of the penetration rate (L-histidine 75.8% v. control 89.9%) and the number of sperm per oocyte penetrated (L-histidine 3.1 v. control 4.1). The rate of male pronuclear formation was not affected by the addition of antioxidants to the extender (over 85% in all cases). The addition of carnosine and taurine had no effect on the IVF parameters. In conclusion the antioxidants carnosine, taurine, and L-histidine affect sperm functionality differently, and further studies are necessary to elucidate what changes in sperm function take place during storage and the mechanisms by which these antioxidants exert their effects.

This work was supported by Italian-Spanish research project HI2005-0165 and AGL2006-03495.