Changes in morphology and viability on the development of bovine embryos in vitro fertilized with experimentally contaminated semen to Escherichia coli Shiga toxin producing stx2

ABSTRACT:The objective of this study was to evaluate by optical microscopy and transmission electron, changes in morphology and viability of the development of bovine embryos, fertilized with semen experimentally contaminated (STEC). Oocytes were aspirated from ovaries of slaughtered cows and the intact zona pellucida were selected and matured. After 20-24 hours of maturation, the oocytes were divided into 2 groups. The first, control group (n = 4l8),fertilized with semen tested and without any type of contaminant and the second, the infected group (n = 415), fertilized with sperm exposed to STEC. Both semen were treated by the technique of discontinuous Percoll gradient. After the period of fertilization, embryos were evaluated for their morphology and viability by optical and electron microscopy. In morphologic evaluation, the oocytes fertilized with contaminated semen showed cytoplasmic shrinkage, gaps in the division, asymmetry of blastomeres, ooplasm grainy, dark brown color, vacuoles formation, degeneration and zona pellucid disruption. These changes were not observed in the control group. The cleavage rate was 70.3 and 52.8%, respectively, for control and infected groups, significant differences (p = 0.0001). After the 5th day of embryonic development, where it was observed 44.7% of morula in the control group, and 22.4% in the contaminated group, showing a significant difference (p = 0.0001). The presence of STEC interferes with the cleavage rate of embryos and also prevents and causes a decline in embryonic development to the morula stage and cause morphological changes during this development.

摘要：本研究旨在通过光学显微镜（Optical Microscopy）与透射电子显微镜（Transmission Electron Microscopy, TEM），评估使用经实验污染产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli, STEC）的精液受精后，牛胚胎发育过程中的形态与活力变化。研究人员从屠宰母牛的卵巢中抽吸获取卵母细胞，筛选出完整透明带（zona pellucida）的卵母细胞进行体外成熟培养。成熟培养20~24小时后，将卵母细胞分为两组：对照组（n=418），采用经检测无任何污染物的精液完成受精；感染组（n=415），采用暴露于STEC的精子完成受精。两组精液均经不连续Percoll梯度离心技术处理。受精周期结束后，通过光学及电子显微镜对胚胎的形态与活力进行评估。形态学评估结果显示，经污染精液受精的卵母细胞可出现胞质皱缩、卵裂间隙异常、卵裂球不对称、胞质颗粒化、深褐色外观、空泡形成、变性及透明带破裂等一系列异常改变，上述异常在对照组中均未被观察到。对照组与感染组的卵裂率分别为70.3%与52.8%，组间差异极显著（p=0.0001）。胚胎发育至第5天时，对照组桑葚胚（morula）形成率为44.7%，污染组仅为22.4%，组间差异同样极显著（p=0.0001）。综上，STEC的存在会干扰胚胎卵裂率，阻碍并降低胚胎发育至桑葚胚阶段的比例，同时在胚胎发育过程中引发显著的形态学改变。