INTRODUCTION

Cryopreservation of sperm is an important tool to conserve genetic material and maintain genetic diversity in wild and domestic species (^{Lermen et al., 2009}). However, it is known that the freeze-thaw processes of semen used in most mammalian species cause the death of a large number of cells (^{Watson, 2000}).

In sheep, although a relatively high percentage (40-60%) of sperm retain their motility after thawing, only about 20-30% remain biologically intact (^{Salamon y Maxwell, 2000}). This is because during the refrigeration and cryopreservation process there is an increase in the levels of reactive oxygen species (ROS), which produce biophysical and biochemical changes in the sperm membrane (^{Chatterjee et al., 2001}; ^{Kankofer et al., 2005}) and even damage to its DNA (^{Aitken, 1999}; ^{Agarwal et al., 2003}), affecting viability and its fertilizing capacity. In recent years, cryopreservation protocols have been substantially improved with the addition of compounds with antioxidant properties, positively impacting the improvement of the quality of thawed semen (^{Peña et al., 2005}; ^{Thuwanut et al., 2008}; ^{Domínguez-Rebolledo et al., 2010}; ^{Mata-Campuzano et al., 2015}).

Antioxidants play an important role in the protection of sperm cells, against the harmful effects produced by ROS (^{Halliwell, 1997}). The function of an antioxidant is based on donating electrons to other molecules that have one or more lost electrons (ROS), and thus preventing alterations in the molecules of lipids, proteins and sperm DNA (^{Tremellen et al., 2008}).

The antioxidant Trolox is a water-soluble vitamin E analogue and has been used in a wide variety of cellular systems to avoid the effects of ROS (^{Halliwel, 1994}; ^{Michielset al., 1994}). It has been observed that the addition of this antioxidant in the freezing medium improves the quality of the thawed pork sperm (^{Peña et al., 2005}); as well as the sperm viability of thawed semen in sheep of the Churra breed (^{Mata-Campuzano et al., 2015}). In sperm samples from the Iberian deer epididymis (postmortem), it was shown that this antioxidant is capable of decreasing the amounts of ROS and lipoperoxidation. In addition to protecting acrosomes and sperm DNA (^{Domínguez-Rebolledo et al., 2010}). ^{Thuwanut et al. (2008}) observed that the addition of Trolox to the freezing diluent improved motility and viability in cat epididymis sperm in defrosting.

The antioxidant Crocina (glycosyl ester of crocetin), is a water-soluble carotenoid, found in the yellow pigment of saffron (Crocussativus). It has been shown that the addition of this antioxidant in the semen incubation medium improves the quality of the thawed sperm of the Iberian deer (^{Domínguez-Rebolledo et al., 2010}) and bull (^{Sapanidou et al., 2014}). In semen of roosters, it improves viability, motility, mitochondrial activity and reduces lipoperoxication to defrosting (^{Mehdipour et al., 2019}).

Therefore, the objective of the present study was to evaluate the effect of the addition of the antioxidants Trolox and Crocina to the freezing medium, on the post-cryopreservation sperm characteristics in sheep of the Pelibuey breed.

MATERIAL AND METHODS

RESULTS AND DISCUSSION

At 0 and 6 h of incubation, none of the parameters evaluated showed differences (P> 0.05) between treatments (Table 1). These results differ from those obtained by ^{Thuwanut et al. (2008}) with cat epididymal sperm supplemented with the antioxidant Trolox at 1mM; since MT, MP and sperm viability improved defrosting. Although they are different species, the difference in results could be attributed to the fact that sperm from cat epididymis by castration, have never been in contact with the secretions of the glands that form the seminal plasma, which is where the greatest antioxidant defense is found (^{Chen et al., 2003}). Therefore, there are differences in freezing between sperm from ejaculates and epididymis (^{Gilmore et al., 1998}), which affect sensitivity to cooling, such as resistance to freezing (^{Schmehl et al., 1986}). In this sense, Trolox could have acted directly and without any competition on the sperm of the epididymis, similar to that observed by ^{Domínguez-Rebolledo et al. (2007)}, with seminal sheep plasma on thawed semen.

On the other hand, in synergy with the antioxidants present in the seminal plasma, the seminal samples used in this study could be altered, causing their effect to diminish. Likewise, it has been reported that supplementation of the diluent with Trolox at 1mM, depending on the type of additive used in the diluent, may or may not improve the viability of the frozen semen of sheep of the Churra breed (^{Mata-Campuzano et al., 2015}); that is, when the diluent was made from soy lecithin, the antioxidant Trolox had a greater viability in the thawed samples; while when a tris-based diluent was used, such as the one used in this study (Triladyl^®), the viability in the sperm did not improve. Soy contains isoflavones, which act as antioxidants; which could have exerted some protection on the sperm during the freeze-thaw process or in combination with Trolox, producing a synergy that helped to enhance the beneficial effects on the sperm. With the addition of the antioxidant Crocina, greater viability, motility, mitochondrial activity and reduction of lipoperoxidation have been reported, in rooster sperm samples to defrost (^{Mehdipour et al., 2019}).

Through the incubation time (0 to 6 hours), the MT behaved similarly in T1 and T2, but not in the TT (P <0.01); on the contrary, the integrity of acrosomes in all three treatments was similar (P>0.05). MP, viability, mitochondrial activity and Host decreased over time in a similar way in the three treatments (table 1). The results are similar to those reported by Domínguez-Rebolledo et al. (2010) in sperm samples of epididymis thawed from the Iberian deer, where MT and acrosome integrity were maintained during incubation at 37 °C with the antioxidant Trolox and Crocina at 1mM. Also, ^{Sapanidou et al. (2014}) observed that the motility, viability and integrity of acrosomes in bovine sperm were maintained during incubation at 37 ° C with 1mM Crocina. In view, of the results after 6 h of incubation where the T1 and T2 maintained the MT and the integrity of the acrosomes, it can be said that the antioxidants tested are efficient and recommended for incorporation into the diluent prior to freezing; in such a way, that the seminal dose will be in force in the genital tract, after insemination until the moment close to ovulation.

CONCLUSION

The addition of the antioxidants Trolox and Crocina to 1mM in the freezing diluent, allows to maintain the total motility and integrity of the acrosomes during 6 h of incubation at 37 °C.