Trachte E, Stringfellow D, Riddell K, Galik P, Riddell M, Wright J
Department of Large Animal Surgery and Medicine, College of Veterinary Medicine, Auburn University, AL 36849-5519, USA.
Theriogenology. 1998 Oct 1;50(5):717-26. doi: 10.1016/s0093-691x(98)00177-0.
Gametes, somatic cells and materials of animal origin in media are potential sources for introducing bovine viral diarrhea virus (BVDV) into systems for production of IVF bovine embryos. Further, the efficacy of washing and trypsin treatment for removal of BVDV from IVF embryos is questionable. Washing and trypsin treatments recommended by the International Embryo Transfer Society for in vivo-derived embryos were applied to in vitro-derived, virus-exposed, bovine embryos in this side-by-side comparison of treatments. Embryos for the study were produced in a virus-free system in which follicular oocytes were matured and fertilized in vitro and presumptive zygotes were co-cultured with bovine uterine tubal cells for 7 d. A total of 18 trials was performed, 9 using a noncytopathic BVDV and 9 using a cytopathic BVDV. In each trial, 4 equal groups of 10 or less, zona pellucida-intact embryos/ova were assembled, including 2 groups of morulae and blastocysts (M/B) and 2 groups of nonfertile or degenerated ova (NFD). Each group was prewashed and exposed to 10(4) to 10(6) TCID50/mL of either noncytopathic (SD-1) or cytopathic (NADL) BVDV for 2 h. Following in vitro viral exposure, one group of M/B and one group of NFD were washed. The other groups of M/B and NFD were trypsin-treated. Both treatments were consistent with IETS guidelines. After in vitro exposure to noncytopathic BVDV and washing, viral assays of 100% (9/9) and 78% (7/9) of the groups of M/B and NFD ova, respectively, were positive. After in vitro exposure to cytopathic BVDV and washing, viral assay of 33% (3/9) of the groups of both M/B and NFD ova were positive. After in vitro exposure to noncytopathic BVDV and trypsin treatment, viral assay of 44% (4/9) of groups of M/B and 67% (6/9) of groups of NFD ova were positive. Finally, after in vitro exposure to cytopathic BVDV and trypsin treatment, viral assay of 22% (2/9) of the groups of M/B and 44% (4/9) of the groups of NFD ova were positive. Contingency table analysis, in which data was stratified by embryo type and virus biotype, was used to compare results. While a difference existed between results of the 2 treatments of groups of M/B within the noncytopathic biotype (P = 0.01, Mantel Haenszel Chi-square), no difference was observed between comparison of treatment between all groups in both biotypes (P > 0.05).
培养基中动物来源的配子、体细胞和材料是将牛病毒性腹泻病毒（BVDV）引入体外受精牛胚胎生产系统的潜在来源。此外，通过洗涤和胰蛋白酶处理从体外受精胚胎中去除BVDV的效果存在疑问。在这项并排比较的处理中，将国际胚胎移植协会推荐的用于体内来源胚胎的洗涤和胰蛋白酶处理方法应用于体外来源的、暴露于病毒的牛胚胎。本研究中的胚胎在无病毒系统中产生，其中卵泡卵母细胞在体外成熟并受精，假定的合子与牛输卵管子宫细胞共培养7天。总共进行了18次试验，9次使用非细胞病变型BVDV，9次使用细胞病变型BVDV。在每次试验中，将4组数量相等且每组10个或更少、透明带完整的胚胎/卵子进行分组，包括2组桑椹胚和囊胚（M/B）以及2组未受精或退化的卵子（NFD）。每组预先洗涤后，暴露于10⁴至10⁶ TCID₅₀/mL的非细胞病变型（SD - 1）或细胞病变型（NADL）BVDV中2小时。体外病毒暴露后，一组M/B和一组NFD进行洗涤。另一组M/B和NFD进行胰蛋白酶处理。两种处理均符合国际胚胎移植协会的指南。在体外暴露于非细胞病变型BVDV并洗涤后，M/B组和NFD组卵子的病毒检测阳性率分别为100%（9/9）和78%（7/9）。在体外暴露于细胞病变型BVDV并洗涤后，M/B组和NFD组卵子的病毒检测阳性率均为33%（3/9）。在体外暴露于非细胞病变型BVDV并进行胰蛋白酶处理后，M/B组卵子的病毒检测阳性率为44%（4/9），NFD组卵子的病毒检测阳性率为67%（6/9）。最后，在体外暴露于细胞病变型BVDV并进行胰蛋白酶处理后，M/B组卵子的病毒检测阳性率为22%（2/9），NFD组卵子的病毒检测阳性率为44%（4/9）。使用列联表分析（按胚胎类型和病毒生物型对数据进行分层）来比较结果。虽然在非细胞病变生物型中M/B组的两种处理结果之间存在差异（P = 0.01，Mantel Haenszel卡方检验），但在两种生物型的所有组之间的处理比较中未观察到差异（P > 0.05）。
