Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

Abstract In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 × 10 9 spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 × 10 9 spermatozoa) or with 1 × 10 9 spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 × 10 9 progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 °C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 × 10 9 progressively motile spermatozoa that had been cooled to 5 °C and stored for 24 h. Group 3 mares were inseminated once with 2 × 10 9 progressively motile spermatozoa that had been cooled to 5 °C and stored for 24 h. All mares were given 2500 IU iv hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered im 10 mg of PGF 2 α and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 × 10 9 progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 × 10 6 motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 °C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 × 10 9 progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P 9 progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 × 10 9 progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 × 10 progressively motile spermatozoa given on two consecutive days. However, a shorter interval (≤24 h rather than >0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.