111 From abattoir-derived oocytes to in vitro-produced embryos: pathogen surveillance and washing procedures

In 2022, nearly 80% of all transferrable cattle embryos produced globally were in vitro produced (IVP), yet biosafety concerns prohibit the importation of IVP embryos in many countries. As a first step in assessing potential disease risks associated with IVP embryos, follicular fluid from cattle was surveilled for bacterial and selected viral contamination. The efficacy of current International Embryo Transfer Society standards for sanitary handling of in vivo-derived (IVD) embryos to remove those contaminants from IVP embryos was also tested. It was hypothesised that contaminants present in follicular fluid of abattoir-derived ovaries would be eliminated by washing embryos with trypsin according to IETS recommendations for IVD embryos. Twice monthly for 12 months, 10 ovaries collected from local abattoirs processing either dairy or beef cattle were transported to the laboratory in saline and externally sterilized with ethanol followed by a saline rinse. Follicular contents were aspirated, and the pellet of cumulus–oocyte complexes (COC) removed. Remaining follicular fluid was centrifuged to remove cellular debris and stored at −80°C. Grade 1 and 2 oocytes were fertilized and cultured to Day 7 according to standard procedure. Half of the resulting embryos were washed in 0.2% trypsin (washed) before snap freezing, while the other half were rinsed with phosphate-buffered saline and directly snap-frozen (unwashed). Homogenised embryos and follicular fluid were plated on brain heart infusion agar and incubated for at least 5 days at 37°C. Resultant colonies were subjected to Sanger sequencing for identification of bacteria. Follicular fluid was also tested for bovine viral diarrhoea virus (BVDV) antigen. Almost all follicular fluid collected from abattoir-derived ovaries indicated the presence of bacterial cultures; 20 and 10 different genera of bacteria were cultured from follicular fluid of beef and dairy cattle, respectively. Both environmental contaminants and pathogenic bacteria were identified. Bacteria within the genera histophilus and truperellapyogenes were only identified in follicular fluid from dairy cattle, while Escherichia coli, klebsiella, and shigella were only identified in follicular fluid from beef cattle. Bacterial cultures remained present in 14.2% of unwashed beef embryos and 27.3% of unwashed dairy embryos. Embryos washed with trypsin did not grow bacteria. All samples in this study were BVDV negative. Although bacteria were present in nearly all follicular fluid, standard washing procedures associated with the IVP process eliminated greater than 70% of contamination, while washing with trypsin yielded no detectable bacterial contamination (100% effectiveness). These data suggest that pathogens in the follicular fluid of beef and dairy cattle can be effectively removed if handed appropriately.