233 NON-CROSS-CONTAMINATION OF BOVINE EMBRYOS WITH MICROBES USING THE OPS VITRIFICATION SYSTEM

Abstract

Here we report on the outcome of the application of vitrification open pulled straw (OPS) technology on the sanitary status of embryos exposed to pathogenic agents and cryopreserved using the commercial kit Vit-SeT^® (Minitube Canada, Ingersoll, Ontario, Canada). The Vit-SeT consists of 3 stainless steel chambers: the first for cooling the 0.5-mL AI straws, the second for vitrification of OPS straws, and the third for loading OPS into the AI straws. The CBC 0.5-mL straws (Cryo Bio System, Paris, France), OPS straws (Minitube Canada), and the heat sealer were used. The investigation involved: (1) pressure seal testing the CBC straws; (2) testing LN for contamination after vitrification; (3) testing the surface of the CBC straws; and (4) testing the contents of OPS straws and vitrified embryos for cross-contamination. In the first Exp., CBC straws were heat sealed at one end and the other end was connected to a compressor with a manometer. The straws were then tested for air leaks in water. In Exp. 2, OPS straws were loaded by capillary aspiration of clean or contaminated cultures (3 × 10⁸ E. coli, 13 × 10⁸ P. aeruginosa, or 10⁵ BVDV TCID₅₀ mL^-1, NY strain), then vitrified in LN (chamber 2) in random order, inserted into the CBC straws (chamber 3), and heat sealed at a reading of 225 on the sealer dial. In Exp. 3, IVF embryos were either exposed or not to the microbe cultures for 1 h, loaded into OPS (5 embryos per straw), and then vitrified and processed as described in Exp. 2. After vitrification, OPS straws were retrieved from CBC straws and thawed according to the original methodology (Vajta et al. 1997 Cryo-Lett. 18, 191–195). For bacterial isolation, standard methodology was used, and BVDV was detected by virus isolation followed by immunoperoxydase staining. In Exp. 1, no air leaks were detected up to 20 mm Hg pressure at the heat-sealed end of CBC straws (n = 15). In Exp. 2, two samples of LN tested positive for E. coli (after immersion of 10 OPS, chamber 2). All remaining samples of LN after vitrification of OPS tested negative for pathogenic agents. Surfaces of the 0.5-mL straws all tested negative (chamber 3). The pathogenic agents were retrieved from all of the positive control OPS straws (n = 15). In Exp. 3, all tested samples of LN and surfaces of 0.5-mL straws were negative. The pathogenic agents retrieved from samples of control embryos previously exposed to pathogens were as follows: E. coli, 5/15; P. aeroginosa, 8/15; and BVDV, 2/15. All samples containing embryos not exposed to pathogens, surfaces of 0.5-mL straws, and LN all tested negative. On the basis of limited experimental work, it is concluded that the potential for cross-contamination of samples by application of the Vit-SeT for vitrification of embryos using OPS is negligible if: (1) LN in the chambers is frequently replaced and the chambers are disinfected between embryo donors, and (2) the protective straws (0.5-mL, AI) are applied over OPS and are properly sealed. These straws should be used without a cotton plug. However, the obtained results should be not extrapolated to other means of so-called 'sterile' methods of vitrification without specific testing.