Effectiveness of washing procedures for removing Brucella abortus from in vivo- and in vitro-derived wood bison embryos

Washing procedures for zona pellucida (ZP)–intact bovine embryos have been developed to enable international movement of pathogen-free embryos. We evaluated the effectiveness of a washing procedure to remove Brucella abortus from in vivo – and in vitro –produced bison embryos exposed to the pathogen in vitro . In Experiment 1, ZP-intact embryos from superovulated bison were incubated with B. abortus biovar 1 (1.9 × 10⁵ to 3.8 × 10⁸ CFU/mL) for 2 h at 37ºC. Embryos underwent a 10-step wash using medium (PBS + 0.4% BSA) with or without antibiotics (penicillin and streptomycin). Medium samples were cultured after washes 1, 3, 6, and 9, and embryos were individually cultured on blood agar after the final wash. B. abortus was not detected in samples after the third wash in either group. PCR detected B. abortus in 2/27 embryos washed without antibiotics, but in none of those washed with antibiotics (0/27). In Experiment 2, cumulus-oocyte complexes were collected from superstimulated bison 34 h after hCG treatment, fertilized in vitro immediately, and cultured for 7 days. ZP-intact embryos were exposed to B. abortus and washed as in Experiment 1. B. abortus was not detected in samples after wash 3, and all embryos were culture-negative (0/84). In summary, B. abortus was detected only in in vivo -derived embryos washed without antibiotics (2/27), whereas no positives were observed in embryos washed with antibiotics or in vitro -derived embryos. Washing effectively removed B. abortus from in vivo - and in vitro -derived wood bison embryos, but disease-free calf production remains unconfirmed.