Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability

The objective of this study was to develop an adequate sperm handling protocol in order to obtain a sex-sorted sperm population with an optimal fertilizing ability. For this purpose, different aspects of the sorting procedure were examined. The effects of the high dilution rates (experiment 1), type of collection medium used (experiment 2), and sheath fluid composition (experiment 3) on sorted boar sperm quality and function were evaluated. Sperm quality was assessed by motility and viability tests, whereas sperm function was evaluated by an in vitro fertilization assay which determined the penetration and polyspermy rates as well as the mean number of sperm penetrating each oocyte. In experiment 1, the results obtained indicated that the high dilution rates did not cause a decrease either in the sperm quality parameters evaluated or the in vitro fertilization ability of spermatozoa. In experiment 2, although sperm quality was not affected, fertilizing ability was compromised after sorting, regardless of the collection medium that was used. In the experiment 3, all groups displayed adequate sperm quality values, but higher in vitro fertility parameters were obtained for spermatozoa sorted in presence of EDTA in the sheath fluid and egg yolk (EY) in the collection media when compared with those sorted in absence of these protective agents. No differences in penetration rates between unsorted highly diluted (control) and sorted sperm in the presence of EDTA and EY were observed. In conclusion, fertilizing ability was compromised in sex-sorted sperm. The addition of EDTA to sheath fluid and EY to collection medium improved boar sperm fertilizing ability, and both agents should be included as essential media components in future studies.