Evaluación de crioprotectores alternativos, glicerol y antioxidantes en la congelación del eyaculado equino

Abstract

Nowadays freezing horse semen is a technique widely demanded by the global equine industry. In this way it is possible to store and preserve the genetic material of highly valuable horses, either by their morphology, genetics or athletic achievements. Artificial Insemination (IA) also has been facilitated by the elimination of the ban imposed by the studbooks of different breeds. The new international regulation has facilitated the international trade of semen of elite horses. In spite of the advantages offered by the use of frozen semen, this technique has also several drawbacks. The equine ejaculate shows great individual variability in the response to freezing nd thawing. The origin of this variation is genetic related to breeding criteria not taking in account sperm quality, Thereby the technique has a level of development that can be considered suboptimal. The main source of damage affecting equine sperm during freezing and thawing is osmotic shock. The present Thesis pursued the design and development of extenders for freezing stallion semen. Also the protective effects of glycerol versus dimethylformamide (DMF) and the combination of both agents were compared. The incorporation of butylated hydroxytoluene in the freezing medium was evaluated as well as the addition of BAPTA-AM as intracellular calcium chelating agent. Finally, in the last paper, we investigate the presence of TNF? and its receptors in equine sperm. The used of fresh and frozen semen lysates allowed to evaluate if cryopreservation induces changes in the expression of TNF? and its receptors. Cáceres® extender, designed and developed in our laboratory, increased sperm survival and reduced variability among stallions. These results show a correlation between reduced osmotic shock and the combination of these cryoprotectants in the freezing extender. The results also showed a high mitochondrial membrane potential, which may ,as well, be attributed to the osmotic shock reduction achieved with our medium. It is known that a major cause of osmotic shock for equine sperm during the freezing and thawing process, is the cryoprotective agent itself. Therefore, in our second paper we compared glycerol against DMF and the combination of both. The best values of sperm motility and velocity were obtained with a combination of both cryoprotectants or when using 4% DMF as a single agent. Best results in terms of membrane integrity and mitochondrial membrane potential were obtained when 4% DMF was the only cryoprotectant. These results confirm that the use of the amide reduces the osmotic shock. It is important the fact that six stallions, of the 7 used in this research were considered bad freezers. Group in which there is greater sensitivity of their ejaculates to the osmotic shock are the poor freezers, hence explaining the best response using DMF as the unique cryoprotectant. Lethal and sublethal damage suffered by the sperm during cryopreservation relate to lipid peroxidation of the plasma membrane. Therefore, in our third paper the effect of the addition of fat-soluble antioxidant BHT in the freezing medium was investigated. The results showed no significant improvement in any of the post-thawing parameters evaluated by incorporating this agent at a concentration of 1 mM in the extender. In the fourth paper we studied the effect of the incorporation in the freezing extender of intracellular calcium chelator BAPTA-AM and the effect of different media for thawing The results obtained in the first part of the work, showed no effect of the addition of BAPTA-AM on semen quality. The results obtained in the second part of the study showed an improvement of the kinetic parameters and sperm motility as well as the percentage of living sperm, after changing to Tyrode´s media and only in samples incorporating BAPTA-AM. Finally, we studied the presence of tumor necrosis factor alpha (TNF?) and its receptors (TNF-R1, TNF-R2) in the equine sperm. In fresh semen samples were detected three forms of TNF, while in frozen-thawed sperm only was detected the soluble form and a reduction of the intensity of the band corresponding to the active form of TNF?. In the case of the receptors, only TNF-R2 was detected, being the first time that the presence of this receptor appears in the equine sperm.