157 EFFECT OF DENSITY GRADIENT SELECTION ON EMBRYO RECOVERY RATES OF FRESH AND COOLED STALLION SEMEN

Abstract

The aim of this study was to improve the spermatic kinetic parameters from stallions with poor quality of fresh and chilled semen by the use of density gradient Equipure™ (Nidacon, Mölndal, Sweden). Semen from 5 Quarter Horse stallions aged 8 and 16 years with history of low embryo recovery rates were used. The kinetics sperm evaluation was performed by computerized semen analysis (CASA) and plasma membrane integrity with fluorescent probes. The average motility parameters for fresh semen before selection were total motility (MT) 60%, progressive motility (PM) 30%, and plasma membrane integrity (IMP) 60% and for cooled semen (24 h at 5°C) were: MT 50%, MP 18%, and 50% IMP. For the group of fresh and cooled semen with no density gradient selection (NS), mares were inseminated with 1 billion viable sperm diluted in skim milk extender in the uterine body, 24 h after ovulation induction with 1 mg of deslorelin. For Equipure^TM selection group (SE) semen was concentrated through Spermfilter membrane™ and resuspended in 5 mL of BotuSemen^TM (Botupharma^TM, Brazil). In a 15 mL conic tube 5 mL of Equipure^TM was added and another part containing 5 mL of the resuspended sperm was slowly added over the Equipure^TM column. The 15 mL conic tube was centrifuged at 400g for 20 min. After centrifugation, the sperm pellet was carefully aspirated and to the pellet was resuspended in 4 mL of Botucrio^TM. The sperm recovery rate with Equipure^TM was 40%. Deep uterine AI was performed 24 h after ovulation induction with 1 mg of deslorelin. Data were analyzed by ANOVA followed by Tukey’s test (P < 0.05). The analysis of semen after Equipure^TM selection resulted in average: 75, 35, 65, and 40%, respectively, for MT, MP, and IMP index and sperm recovery. The embryo recovery rate from the 5 stallions showed the following results: Stallion 1 (fresh semen): 12 mares (NS)/4 embryos (33%) and for group SE 22 mares/16 embryos, (72%; P ≤ 0.05). Stallion 2 (fresh semen): 9 mares (NS)/4 embryos, (44%) and for group SE, 12 mares/8 embryos (66%; P > 0.1). Stallion 3 (fresh semen): 4 mares (NS)/1 embryo (25%) and 7 mares for SE/4 embryos (57%; P > 0.1). Stallion 4 (chilled semen): 4 mares (NS)/0 embryo 0% and for SE group 8 mares/6 embryos 75%; P ≤ 0.001). Stallion 5 (chilled semen): 10 mares (NS)/2 embryos (20%) and for SE 6 mares/3 embryos (50%; P > 0.1). For the overall results, 39 inseminations were performed on the no selected group and 11 embryos recovered (28%) for the selected group 55 inseminations and 37 embryos recovered (67%; P < 0.01). The results clearly showed that selection by sperm density gradient Equipure^TM was a very effective technique that allowed an improvement on semen quality and fertility.

The authors acknowledge support from FAPESP and Botupharma.