161 PROCESSING OF SEMEN WITH PYOSPERMIA ALLOWS ITS USE IN EQUINE EMBRYO TRANSFER PROGRAMS

Abstract

Previous studies in humans and current studies in horses show that the presence of leukocytes in the semen promotes increased reactive oxygen species and proinflammatory cytokines production. These substances cause damage to membrane integrity, mitochondrial function, DNA integrity, and lead to increased expression of apoptosis markers in the spermatic cell. As a result of these effects, decreased quality and longevity of the semen that lead to fertility reduction could be highlighted. A 6-year-old stallion was admitted in the Department of Animal Reproduction and Veterinary Radiology of Veterinary Medicine and Animal Science School, UNESP, Botucatu-SP, Brazil, with a history of pyospermia due to seminal vesiculitis. Therefore, because of the high demand of the stallion semen and attempting to allow its use in equine embryo transfer programs, a process to reduce leukocyte numbers was performed. For the evaluation of the percentage of leukocytes in the sample, a 10-µL aliquot of semen was smeared and stained with Quick Panotic^TM. Semen was diluted in skim milk-based extender BotuSemen^TM (1 : 1; Nidacon International AB, Mölndal, Sweden) and then analyzed for sperm kinetic parameters by computer assisted semen analysis (CASA). Sequentially, seminal plasma was removed by filtration with SpermFilter^TM (Nidacon International AB). The sample was resuspended with BotuSemen^TM to a concentration of 300 × 10⁶ sperm mL^–1 and subjected to a single layer gradient centrifugation (EquiPure^TM; Nidacon International AB) at 400g for 20 min. A proportion of 5 mL of diluted semen in 5 mL of EquiPure^TM (1 : 1) was used for the centrifugation. The pellet was resuspended with BotuSemen^TM at a concentration of 50 × 10⁶ sperm mL^–1. Before and after processing, sperm kinetic means were, respectively: Total motility (TM) = 46% and 89%, progressive motility (PM) = 20% and 55%, and rapid sperm cells (RAP) = 32% and 81%. The mean percentages of leukocytes in semen smears before and after processing was 80% and 5%, respectively. The processed semen was used for artificial insemination of 5 mares and none of them showed signs of post-breeding endometritis. In addition, in all cases, embryos were recovered when uterine flushes were done on Day 8 post-ovulation. In conclusion, the method used for the processing of semen with pyospermia was effective in reducing leukocyte loads and promoted an improvement of seminal kinetics parameters which allowed its use in embryo transfer programs.

The authors acknowledge FAPESP (2011/23139-9) for financial support and Botupharma for donation of BotuSêmen^TM extender and SpermFilter^TM.