Preselección del sexo en caballos: Identificación de factores limitantesSex preselection in horses: identification of limiting factors

Abstract

Flow cytometric sex sorting is a valuable technique to use on equine reproduction. Nevertheless, the effects that it causes on the spermatozoon are not negligible. While staining with H33342 for 90 minutes affects total and progressive motility, and other kinematic parameters, the sorting procedure itself mostly damages the plasmalemma. These damages cause an increase in the permeability of the plasmatic membrane. Apart from inducing premature cell death, these membranary alterations are probably related to an increase in the oxidative stress. Indeed, total ROS production, genomic oxidation and fragmentation were increased in sex sorted semen. Also, an increase in the subpopulation of spermatozoa with lower mitochondrial membrane potential was also registered. In this technology, oxidative stress apparently induces DNA damage and cell death. Moreover, approximately the same results were obtained after 20h refrigeration of stained samples, prior to sorting. This provides a new option for the sorting procedure, allowing shipping of refrigerated stallion semen to the insemination centre. Finally, binding to porcine oocytes was approximately equal between sorted and unsorted samples. However, acrosomal reactivity was increased in sorted stallion spermatozoa, and tyrosine phosphorylation patterns were also altered after sorting. Both data account for an advanced status of membrane destabilization, possibly related to capacitation. The plasmalemma appears to be the most affected organelle after the sorting procedure. Destabilization of the membrane hastens premature death, probably through increased oxidative stress and genomic damage. Prevention of this damage should be considered, as it could increase the lifetime of sorted cells.