134 Effectiveness of the timing of trypsin washing to remove pathogens from in vitro-produced bovine embryos at different stages of development
E. Xiao-Kim A , J. Kincade A , A. Bosco-Lauth A and J. P. Barfield AA
Although the number of in vitro-produced (IVP) embryos has surpassed the number of in vivo-derived (IVD) embryos produced annually, regulations still prohibit importation of IVP embryos into many countries due to biosecurity concerns. The IETS guidelines for sanitary handling of embryos were developed using IVD embryos; thus, regulators have questioned the safety of IVP embryos. Unlike IVD embryos, IVP embryos are accessible throughout development, and thus trypsin treatment and washing can occur at any stage. Our objective was to determine if there is an influence on embryo production or the effectiveness of washing procedures when washing is performed at different stages of the IVP process. Thirty abattoir-sourced ovaries were collected 5 times from a local slaughterhouse and transported to the laboratory. Follicular contents were aspirated into a 50-mL conical tube and inoculated with 105 colony-forming units (CFU)/mL of Brucella abortus strain RB51 for 1 h at 38.5°C. Following the 1 h exposure, follicular fluid was plated on brain-heart infusion (BHI) agar and cultured to confirm the presence of live B. abortus. Grade 1 and 2 oocytes were isolated from the remaining follicular fluid and divided into 6 groups relative to timing of embryo washing: (1) at the cumulus-oocyte-complex (COC) stage before being moved to IVM medium (n = 250), (2) at the presumed zygote stage after removal of cumulus cells and before being put into the first culture medium (n = 250), (3) at the cleavage stage 56 h after fertilization before being put into the second culture medium (n = 200), (4) at the blastocyst stage on Day 7 (n = 250), (5) an unwashed, inoculated control group (n = 250), and (6) a non-inoculated, unwashed control group (n = 250). Groups 1–4 were further divided into two groups where they were washed through 1 drop of PBS or 10 drops of PBS with a trypsin wash in between the 5th and 6th wash drop. All groups were cultured to Day 7. Embryos were homogenised before plating on brain heart infusion agar and incubated at 37°C for 3–4 days and evaluated for the presence of B. abortus. Embryo production rates of treatments were compared using a pairwise test of equal proportions. Results indicated that while live B. abortus was present in the follicular fluid of inoculated groups, no B. abortus was found in any treatment groups on Day 7. Embryo production rates were similar in all groups (25 ± 3% to 32 ± 2%) except when COCs were washed in trypsin (11 ± 2%; P < 0.05 for all comparisons). Based on these results, embryos can be washed at any stage after fertilization without affecting embryo production rates. Evidence also suggests that the IVP process itself may eliminate pathogens without the need for washing, with or without trypsin. This study provides valuable insight into the safety of IVP embryos that could be used to revise current import restrictions.
