Testing hypotheses involving binomial responses such as pregnant/not pregnant, requires huge numbers of animals per treatment to obtain statistical significance unless treatment differences are fairly large. One approach to amplifying treatment differences in fertility is competitive, or heterospermic, fertilization, mixing sperm of different treatments or males before insemination, and determining the proportion of embryos, fetuses or offspring derived from each male or treatment. Here we compare fertility after sexing sperm by flow cytometry/cell sorting for DNA content at two sorter pressures using a heterospermic approach with sex as the genetic marker.
Sperm from each of two bulls was sorted into X-chromosome and Y-chromosome populations at ~95% accuracy with the pressure of the sorter at either 30 or 50 psi. After concentrating sperm post-sorting by centrifugation, 106 X- sperm sorted at 30 psi were placed in 0.25-mL straws with 106 Y-sperm sorted at 50 psi within each bull, as well as the converse in other straws: 106 Y-sperm at 30 psi plus 106 X-sperm at 50 psi. These sperm, along with unsorted controls, were then frozen, thawed some months later, and inseminated into the body of the uterus of 85 Holstein heifers either 12 or 24 h after observed estrus with subgroups balanced across two inseminators. Two months post-insemination, 81% of the 43 heifers becoming pregnant had fetuses of the sex (determined by ultrasound) corresponding to the sex of sperm processed at 30 psi. This differed from the 50:50 sex ratio expected (P<0.01) if there was no difference in fertility of sperm sorted at the two pressures. A disadvantage of the lower pressure is that rates of sorting sperm are slightly lower. The pregnancy rate with sexed sperm at 2 x 106 sperm per dose was 51% (43/85); this was similar to the controls of 20 x 106 unsexed sperm per dose from the same ejaculates, 39% (9/23). This procedure using sex as the genetic marker also can be used to rank fertility of males and to rank fertility of sperm treatments not involving sperm sexing if the males or treatments do not interact with sperm sexing procedures. One disadvantage of this approach to testing fertility is that no estimates of the magnitude of the treatment or male to male differences are produced. Another disadvantage is 5% errors in assigning parentage via fetal sex when sperm are sorted at 95% accuracy. Nevertheless, this heterospermic approach was rapid, sensitive, and noninvasive, and required relatively few animals to detect a significant treatment difference.