310 USE OF COMPETITIVE FERTILIZATION TO EVALUATE A SIMPLER LASER FOR FLOW CYTOMETRIC SEXING OF BOVINE SPERM

Statistically significant correlations between laboratory assays of sperm quality and fertility require large sample populations. Experimental differences can be assessed accurately using limited observations with competitive fertilization and fetal sexing, as clearly demonstrated previously (Seidel GE Jr. et al. 2003 Theriogenology 59, 515 abst). Our objectives were to compare pregnancy rates in Holstein heifers inseminated with: (1) 2 × 106 hetero-sex-selected sperm interrogated with different light sources or, (2) 2 × 106 or 10 × 106 X-chromosome bearing sperm using a continuous wave (CW) laser, to those of unsexed inseminates containing 10 × 106 total sperm. Sperm were sexed by flow cytometry/cell sorting at 40 psi with the aid of light produced from either a continuous wave (CW) or a quasi-cw (PULSED; Vanguard 350-HDM, Spectra-Physics, Mountain View, CA, USA) laser operating at 150 mW during flow cytometric/cell sorting. Subsequent fertility was evaluated by competitive fertilization with fetal sex as the genetic marker. Sperm from Holstein bulls were sorted into X- and Y-chromosome populations at >90% accuracy using either the CW or the PULSED laser. After concentration of sperm post-sorting by centrifugation, an equal number of X-sorted sperm illuminated with the CW laser were pooled with Y-sorted sperm illuminated by the PULSED laser within each bull, as well as the converse. Total sorted sperm (2 × 106) were placed in 0.25 mL straws. In addition to these sperm, homospermic inseminates containing 2 or 10 × 106 total sperm, sorted using the CW laser, and unsorted controls (10 × 106 total sperm) were then frozen. Holstein heifers (n = 763) were synchronized for estrus in five groups (July–December) with a CIDR in place for 7 days followed by 25 mg PGF-2α i.m. Heifers (n = 626) were body inseminated either 12 or 24 h after observed estrus. Sexed and unsorted inseminates were balanced across sperm from three bulls and two inseminators. Two months post-insemination, pregnancy and fetal sex were determined using ultrasound. Data were subjected to ANOVA. Pregnancies marked by fetal sex achieved with competitive fertilization resulted in a 52 (PULSED):48 (CW) ratio, which is not different from the expected 50:50 ratio (P > 0.05) if neither laser was more or less damaging to the fertilizing potential of sperm. Actual pregnancy rates for the competitive 2 × 106, homospermic 2 × 106, and 10 × 106 sexed inseminates were 54, 56, and 62%, respectively, with n = 179, 179, and 180, and were similar to unsorted controls (61%, n = 88) (P > 0.05). This study demonstrated no deleterious effects, in terms of pregnancy rate, to sperm illuminated with a PULSED laser during sorting when compared to conventional instrumentation. Also, pregnancy rates with sexed sperm were similar to those of unsexed controls. The use of the PULSED laser for sperm sorting has economic advantages because it requires less energy, is air-cooled, and has a longer operating life than the water cooled CW laser.