Abstract
In artificial insemination the use of sex-sorted bovine sperm results in reduced conception, the causes of which are only partly understood. Therefore, we set out to investigate the effects of sexing on bovine sperm function and early embryonic development. Computer-assisted semen analysis (CASA) of sperm of the same bulls (n = 5), before and after sexing, demonstrated significantly reduced fast (A) and slow (B) progressively motile sperm (p < 0.05) after sexing. Sexed-sperm also revealed significantly less hyperactivated sperm (p < 0.05). As shown by time-lapse videomicroscopy of in vitro produced embryos (n = 360), embryos derived from sexed-sperm displayed significantly increased incidences of arrest at the 4-cell stage (p < 0.05). The relative risk for shrinkage/fusion of blastomeres with subsequent lysis was 1.71 times higher in the embryos derived from sexed-sperm as compared to conventional embryos (p < 0.05) resulting in significantly reduced blastocyst rates (p < 0.001). The relative risk for cleavage was 2.36 times lower in the embryos derived from sex-sorted sperm (p < 0.001). Additionally, sexed-sperm-derived embryos showed reduced survival times (hazard ratio HR = 1.54, p < 0.001) which were bull dependent (p < 0.001). However, the percentage of apoptotic cells was similar to conventional embryos. Furthermore, embryos derived from sexed-sperm were found to reach developmental stages at similar timings as conventional embryos. Our results suggest that reduced conception rates after sexing are due to altered sperm morphokinetics, decreasing the chance of sperm to reach and fertilise the oocyte, and aberrant early embryonic development.
Highlights
Flow cytometric sorting of spermatozoa is based on differences in the amount of DNA between X and Y bearing sperm, which is detected using the DNA-binding stain Hoechst 333421–3
Computer-assisted semen analysis (CASA) analysis of the sperm before and after sexing (n = 5 bulls) demonstrated significantly decreased percentages of sperm with fast (A) and slow (B) progressive motility compared to conventional sperm (25.6% and 4.3% compared to 60.8% and 13.3%, respectively) (Fig. 1A, paired t-tests, p < 0.05)
With regards to progressive sperm, linearity of movement (LIN) and wobble (WOB) were significantly decreased in sexed sperm compared to conventional sperm of the same bull (Fig. 1B, paired t-test, p < 0.05)
Summary
Flow cytometric sorting of spermatozoa is based on differences in the amount of DNA between X and Y bearing sperm, which is detected using the DNA-binding stain Hoechst 333421–3. In 2016, more than 4.5 million straws of sexed semen were processed in the US, over 90% being of dairy sire origin[9] This increased demand in the dairy industry is because male calves are of low economic value and are associated with a higher risk of dystocia compared to heifer calves[8,10]. Pregnancy rates are 60 to 80% of the conventional sperm range or higher[17,18], especially with the use of the recently developed SexedULTRATM technology which included increasing the number of sperm per insemination from 2 to 4 million[19,20]. Calves born of in vitro produced embryos from reverse X-sorted semen reveal higher cumulative mortality from 90 to 180 d compared to AI offspring. The introduction of SexedULTRATM, a new semen extender designed to provide increased physiological conditions for sperm during sorting and freezing, but not available for the research presented has resulted in significantly more acrosome intact sperm after 3 h at 37 C as well as in significantly increased sperm motility after 24 h compared to conventional sperm[42]
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