Potential Mechanical and Antiviral Methods to Ensure a Porcine Reproductive and Respiratory Syndrome Virus-Free Semen Supply

As artificial reproduction techniques become refined and more widespread in the pork industry, precautions must be taken to prevent infectious agents, such as porcine reproductive and respiratory syndrome virus (PRRSV), from being transmitted through semen. Hence, on farm methods that are not only easy but consistent in their results are required. A uni-layer density gradient was evaluated for removing PRRSV from semen. Novel inserts were utilized in 50 ml conical tubes to layer the reagents. No viral RNA was detected in 31 of 44 (71 %) sperm samples collected from PRRSV-inoculated boars after purification by the gradient. Semen pellet samples that were not fully cleared of PRRSV by the single layer gradient were inoculated intraperitoneally (IP) into PRRSV seronegative piglets and found to be infectious regardless of the real-time PCR cycle threshold (CT) value. Several antivirals (cysteine protease inhibitors) were also evaluated for PRRSV inhibition in vitro with the long-term goal of using the antiviral in semen to prevent seminal transmission of virus. The protease inhibitor, chymostatin, was shown to inhibit PRRSV replication in vitro by 90% at a 62.5μm concentration. The other antivirals did not demonstrate viral inhibition up to 2000 μm concentrations. Chymostatin was dissolved in Dimethyl sulfoxide and spiked into semen samples at specific doses; l x with infected semen, 1 Ox with infected semen, and l Ox with noninfected semen. Chymostatin was also given IP in DMSO without semen on day -1 and l to evaluate potential toxicity levels. Piglets averaging four weeks in age and seronegative for PRRSV antibodies were inoculated IP with the doses. All animals were seropositive for PRRSV antibodies by 7 days post inoculation by IDEXX HerdChek ® ELISA and were positive for PRRS viral RNA by PCR tests. Viral RN A loads were reduced in 71 % of the PCR positive semen samples purified by the gradient washing methods thus reducing the risk of transmission through semen. Chymostatin was added to PCR positive semen and inoculated into piglets. All challenged animals were positive for viral RNA and PRRSV antibodies in serum. Together these data indicate a potential to reduce PRRSV transmission. The methods utilized to purify semen and the additions of antivirals to PRRSV positive semen samples are excellent candidates for on-farm techniques to reduce PRRSV transmission in a breeding herd. They are easily performed by technicians, less time consuming than previous studies with 2 layer gradients, and with further study and development have the potential to consistently reduce PRRSV in semen samples. However, further study is needed to evaluate different doses of chymostatin, to determine if there is a toxicity level. More research is needed to refine the novel insert utilized in purifying semen samples as well.