186 A NOVEL METHOD FOR ELIMINATING PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS FROM BOAR SEMEN AND ITS EFFECTS ON EMBRYO DEVELOPMENT

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSv) is known to cause venereal transmission of the disease via natural or artificial breeding and this constitutes a significant risk to AI programs in modern swine production. The objectives of this study were to determine the effectiveness of a novel density gradient centrifugation method incorporating trypsin to eliminate PRRSv from infected semen and to evaluate its effects on sperm viability and embryo development. Exp. 1: To assess the efficacy of the procedure on eliminating PRRSv, semen was collected from 21 infected boars. Concentrated sperm (1 mL) was layered on three Percoll (Sigma, St. Louis, MO, USA) density gradient columns, top to bottom: 1 mL 30%, 2 mL 45% with or without 0.25% trypsin (trypsin-treated and control, respectively), and 2 mL 90% with or without 10 μg/mL soy-based trypsin inhibitor (Sigma), and centrifuged (700g for 30 min). Trypsin-treated and control sperm were submitted for RT-PCR analysis pre- and post-treatment. Exp. 2: To evaluate the effect on sperm quality, semen samples (n = 10) were collected from non-infected boars and processed as described in Exp. 1. Sperm motility, viability, and acrosomal integrity were evaluated at 0 and 2 h post-treatment. Exp. 3.1: To assess the in vitro fertilizing capability of trypsin-treated sperm, in vitro-matured porcine oocytes (n = 64) were inseminated, and cleavage (48 h post-insemination (PI)) and blastulation (144 h PI) rates were compared to those of oocytes (n = 63) inseminated with control sperm. Exp. 3.2: Trypsin-treated or control sperm (3 × 109/dose) were used to AI sows (n = 10). In vivo-generated embryos were surgically recovered 4–6 d post-AI, and embryo number, stage, and morphological quality were recorded. Data were analyzed using ANOVA and differences were considered significant at P < 0.05. Sperm quality parameters are expressed as means ± SEM. Results showed that the procedure (with and without trypsin) was effective for eliminating PRRSv from infected boar semen. There were no differences at 0 or 2 h post-treatment between the control and the trypsin-treated boar sperm in motility (76 ± 4.9 and 56 ± 8.7 vs. 75 ± 4.4 and 48 ± 8.3%, respectively), viability (87 ± 2.6 and 75 ± 6.2 vs. 81 ± 3.2 and 80 ± 3.7%, respectively), and acrosomal integrity (96 ± 2.7 and 98.8 ± 0.8 vs. 98 ± 1.3 and 99 ± 0.4%, respectively). There was no difference between the control and trypsin-treated sperm used for IVF on cleavage (82 vs. 89%, respectively) and blastulation (20 vs. 32%, respectively) rates. There were significantly more transferable-quality embryos recovered from sows inseminated with trypsin-treated as compared to control sperm: 54/63 (85.7%) vs. 35/72 (48.6%), respectively. In conclusion, the novel trypsin gradient treatment was effective in eliminating PRRSv without detrimentally affecting sperm quality and has the potential to increase the numbers of transferable-quality embryos produced.