Insemination of mares with low numbers of either unsexed or sexed spermatozoa

Two experiments were conducted to determine pregnancy rates in mares inseminated 1) with 5, 25 and 500 × 10 6 progressively motile spermatozoa (pms), or 2) with 25 × 10 6 sex-sorted cells. In Experiment 1, mares were assigned to 1 of 3 treatments: Group 1 (n = 20) was inseminated into the uterine body with 500 × 10 6 pms. Group 2 (n = 21) and Group 3 (n = 20) were inseminated into the tip of the uterine horn ipsilateral to the preovulatory follicle with 25 and 5 × 10 6 pms, respectively. Mares in all 3 groups were inseminated either 40 (n = 32) or 34 h (n = 29) after GnRH administration. More mares became pregnant when inseminated with 500 × 10 (18/20 = 90%) than with 25 × 10 6 pms (12/21 = 57%; P < 0.05), but pregnancy rates were similar for mares inseminated with 25 × 10 vs 5 × 10 6 pms (7/20 = 35%) (P > 0.1). In Experiment 2, mares were assigned to 1 of 2 treatments: Group A (n = 11) was inseminated with 25 × 10 6 spermatozoa sorted into X and Y chromosome-bearing populations in a skimmilk extender. Group B (n = 10) mares were inseminated similarly except that spermatozoa were sorted into the skimmilk extender + 4% egg yolk. Inseminations were performed 34 h after GnRH administration. Freshly collected semen was incubated in 224 μM Hoechst 33342 at 400 × 10 6 sperm/mL in HBGM-3 for 1 hr at 35 °C and then diluted to 100 × 10 6 sperm/mL for sorting. Sperm were sorted by sex using flow cytometer/cell sorters. Spermatozoa were collected at approximately 900 cells/sec into either the extender alone (Group A) or extender + 4% egg yolk (Group B), centrifuged and suspended to 25 × 10 6 sperm/mL and immediately inseminated. Pregnancy rates were similar (P > 0.1) between the sperm treatments (extender alone = 3/10, 30% vs 4% EY + extender = 5/10, 50%). Based on ultrasonography, fetal sex at 60 to 70 d correlated perfectly with the sex of the sperm inseminated, demonstrating that foals of predetermined sex can be obtained following nonsurgical insemination with sexed spermatozoa.