245 RISK OF TRANSMISSION OF BOVINE LEUKOSIS VIRUS (BLV) USING SEROPOSITIVE BULLS FOR IN VITRO FERTILIZATION EMBRYO PRODUCTION

Abstract

Bovine leukosis virus (BLV) is a pathogen that affects the bovine immune system and leads to lymphosarcoma, leukemia, decreased milk production, and increased culling rates in cattle. BLV-infected cattle herds can be found worldwide; in the United States, specifically, 38% of beef herds, 84% of all dairy herds, and 100% of large-scale dairy operation herds are infected (Buehring et al. 2014 Emerg. Infect. Dis. 5, 772–782). The main transmission between cattle in herds is affected leukocytes in blood. Several farm practices, such as dehorning, rectal palpation, and vaccinating can lead to the pathogen transmission. Due to international trade laws and biosecurity concerns, semen from a BLV-positive bull is illegal to sell within certain countries. Prior studies have looked at use of seropositive bulls in AI with little risk in affecting the dam (Burger et al. 2000 AVJR 60, 819). Other studies used semen that was artificially infected with the virus then used for IVF (Bielanski et al. 2000 Vet. Rec. 146, 255–256). The aim of this research was to evaluate naturally infected BLV donor semen using abattoir-derived oocytes and the possible contamination of in vitro-produced (IVP) embryos. Semen was collected and frozen by a private company. Three seropositive bulls and 1 negative control bull were selected. All positive bulls were selected based on availability of seropositive BLV status. Prior to the experiment, all bulls used were evaluated for motility, concentration, and morphology. The negative control was used in prior IVF experiments that produced acceptable results for use in this experiment. Frozen sperm were thawed at 37°C for 40 s and pelleted by centrifugation (25 min at 300 × g) on a Percoll discontinuous gradient (45–80% in Tyrode’s modified medium without glucose and BSA). The matured oocytes were purchased from DeSoto Biosciences (Seymour, TN, USA) and were IVF according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Using 200 oocytes per replicate, the 3 positive bulls and 1 control bull were allocated 50 oocytes per bull in each replicate. After 20 to 22 h of gametes co-incubation, zygotes were denuded and cultured for 7 days in SOF, followed by the evaluation of embryos (from tight morula until hatching blastocyst). Positive bull #1 produced and tested 48 embryos. Positive bull #2 produced and tested 41 embryos. Positive bull #3 produced and tested 46 embryos. The negative control produced and tested 55 embryos. Embryonic DNA extraction was performed using standard procedures (Sattar et al. 2011 Reprod. Domest. Anim. 46, 1090–1097). Nested PCR followed the Fechner evaluations methods (Fechner et al. 1996 J. Vet. Med. B 43, 621–630). To detect BLV presence, electrophoresis was used with a 2% agarose gel containing 0.1% ethidium bromide. A total of 190 embryos were evaluated that were produced in 3 replicates. All samples analysed showed no evidence of BLV. In conclusion, use of BLV seropositive donor semen showed no transmission of the virus upon IVF of the oocytes.