Since the first publications 30 years ago showing that flow cytometry was a reliable method to separate X and Y chromosome bearing sperm, the process has been subject to continual refinement. Numerous experiments have been performed in the last few years with the objective of developing an improved sex-sorted product, branded SexedULTRA™ (Sexing Technologies, Navasota, TX, USA) that retains sperm integrity to improve post-thaw sperm quality, in vitro embryo production, and field fertility compared with the previous XY method. Laboratory evaluations were performed on semen from 12 bulls at the Sexing Technologies laboratory in Navasota (TX, USA). Each ejaculate was divided in 2 aliquots and then processed in 1 of 2 methods (XY method or SexedULTRA™). Post-thaw sperm motilities were classified into percent total and progressively motile after thawing (0 h) and after a 3-h incubation at 37°C using a computer-assisted sperm motility analyzer (Hamilton Thorne IVOS II system, Hamilton Thorne Biosciences, Beverly, MA, USA). Percent intact acrosomes was also estimated after a 3-h incubation. Results were analysed by a mixed model ANOVA with the fixed effect of treatment and random effect of bull. Percent total motile SexedULTRA™ sperm was greater (P < 0.001) than sperm processed following the XY method at 0 (78.8 v. 67.2%) and 3 h (51.0% v. 39.0%) post-thaw. Likewise, there was a higher percent of progressively motile sperm both at 0 (50.7 v. 44.9%) and 3 h (31.5 v. 4.4%) post-thaw in the SexedULTRA™ sperm. Percent intact acrosomes was also greater in SexedULTRA™ sperm compared with the sperm processed following previous method (78.0 v. 64.0%). In vitro fertilizations were performed as a measure of sperm competence using 8 ejaculates from 2 bulls in Sexing Technologies IVF laboratory in Laceyville (PA, USA). Five to 10 oocytes and 5,000 motile sperm/oocyte were placed per IVF drop for the analysis. A total of 3 straws and a minimum of 800 oocytes per treatment group (ejaculate × treatment) were included in the comparison for development to 8-cell stage (cleavage rate) and to Day 7 blastocyst stage, measured as total (grades 1 to 4) and freezable (grades 1 and 2) embryos. Results were analysed using a mixed model ANOVA with treatment as a fixed effect and bull, ejaculate within bull, and IVF cycle as random effects. Results from IVF trials are shown in Table 1. Total and freezable embryo numbers were significantly higher (P < 0.05) when using SexedULTRA™ compared with XY sperm. Maintaining a suitable environment for sperm to progress through the various steps of the sex-sorting process results in better semen quality post-thaw as well as improved in vitro fertility. The SexedULTRA™ method confers a significant benefit in maintaining sperm integrity that, if translated into field fertility, could reduce the conception rate gap between conventional and sex-sorted bovine sperm.
Table 1.
Results from IVF and embryo culture using frozen-thawed, sex-sorted semen processed using the XY or the SexedULTRA™ method
The swim-up technique separates bovine sperm by metabolic rates, motility and tail length