New Tools for in Vitro Handling and Selection of Cryopreserved Sperm Samples in Equines

Abstract

Cryopreserved sperm (SPZ) are used widely in domestic animals for assisted reproduction techniques (ART). However, cryopreservation procedures negatively affect SPZ quality, causing changes at the structural and molecular levels. Therefore, using cryopreserved SPZ for ART can decrease the efficiency of these techniques as well as the quality of the embryos obtained, being necessary to optimize the handling of this type of sample. Particularly in equines, ICSI is the most used technique for low-quality equine semen samples where SPZ is selected based on their motility and morphology. Physiologically, the oviduct epithelial cells (OEC) are involved in the selection of a population of SPZ suitable for oocyte fertilization, which is released from the oviduct due to the capacitation process. This work aimed to evaluate different conditions for the in vitro manipulation and incubation of equine cryopreserved SPZ and to establish an in vitro OEC culture to select an SPZ population with fertilizing capacity suitable for its use in ART in this species.Regarding the handling and the effect on the motility of post-thawed equine cryopreserved SPZ samples, we used non-capacitating Whitten´s medium, less effect on motility was observed through the time (120 min) when SPZ were incubated at concentrations of 30 mill/ml compared to lower concentrated samples (CASA, p<0.05). Also, the motility decreased by half with centrifugations at 200g longer than 1 min (p<0.05). On the other hand, we have established a culture model of OECs in vitro, in which we demonstrated the expression of epithelial markers (E-cadherin and cytokeratin) by both RT-PCR and immunofluorescence (IF). When we performed SPZ-OEC cocultures, we observed that the attached SPZs were motile and presented intact acrosome (PSA-FITC, IF), suggesting a selection by the oviductal model. Then, the co-cultures were incubated in capacitating conditions (capacitating Whitten´s medium) and the released SPZ population was recovered. Under these conditions, a greater number of live sperm (Hoechst258 assessed by IF), capacitated (activation of PKA and phosphorylation on tyrosine residues by IF), with progressive motility (CASA) and with the intact acrosome (PSA-FITC, IF) compared to the control was observed (p<0.05). Moreover, the decrease in motility through the time was less in these SPZ than in the cryopreserved SPZ incubated in the absence of OECs. This sperm population could be recovered for its use in different ART.Improvements in handling and selection of cryopreserved SPZ not only generate tools to solve the problem that IVF presents in equines, but would also improve efficiency in other ART such as ICSI, allowing the use of a population of higher quality gametes which it would positively impact the quality of the embryos obtained.