240 RECOMBINANT BOVINE TRYPSIN MADE IN MAIZE INACTIVATES BOVINE HERPES VIRUS-1 ADSORBED TO THE BOVINE ZONA PELLUCIDA

Abstract

For embryo culture, it is often desirable to use media that are devoid of animal products. One problem is that the trypsin required for treatment of embryos for export is an animal product. Therefore, we studied the efficacy of recombinant trypsin produced from the bovine gene in maize (Trypzean^TM, Sigma-Aldrich Corp., St Louis, MO, USA) for inactivating bovine herpes virus-1 (BHV-1), initially without ova present. We found that 525 U mL⁻¹ Trypzean is equivalent in efficacy to the standard 0.25% bovine trypsin treatment. Active virus was titrated on MDBK cells using a standard plaque assay. Next, ova recovered from superovulated cows were incubated for 45 min with BHV-1-infected cells for virus adsorption to the zona pellucida. Both unfertilized ova (n = 26) and embryos (n = 22) were allotted to 4 treatments: (1) control, not exposed to virus; (2) exposed to virus and then washed 10×; (3) treatment 2 plus trypsin treatment as recommended in the IETS Manual; and (4) treatment 2 plus 525 U Trypzean mL⁻¹ and 1 mM EDTA substituted for the 4th and 5th washes of 1 min each; the 6th wash of 10 washes was with soybean trypsin inhibitor at 80 µg mL⁻¹ (Sigma). The chemically defined medium used for handling ova was Syngro^® (AB Technology, Pullman, WA, USA), except that trypsin and inhibitor were made up in Hank's balanced salt solution with 0.2% polyvinyl alcohol, but without Ca²⁺ or Mg²⁺. Ova were stored in 0.5 mL at −80°C until sonication and inoculation onto MDBK cells in duplicate undiluted and at 0.1× dilution. The virus was allowed to adsorb for 45 min, and then overlaid with MEM containing 0.5% agarose, 5% FBS, and antibiotics. Two days later an identical overlay was added, but containing 0.005% neutral red; plaques were counted the following day. Unfertilized ova and embryos led to similar results, which are pooled. No virus was detected in the 5 control ova not exposed to virus. As has been shown by others, BHV-1 remained adhered to zonae even after 10 washes (Table 1). In contrast, treatment with Trypzean or the IETS trypsin protocols reduced this to near zero. We thus confirm that trypsin treatment is effective in inactivating BHV-1 adhered to bovine zonae pellucidae, whether trypsin is derived from animals or genetically engineered plants. We have found that Trypzean is more efficacious than some ‘trypsin-like’ enzymes for this purpose.

Table 1.  Mean plaque-forming units (pfu) per ovum incubated with virus (2 replicates per ovum pooled)