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与透射电子显微镜，评估经实验污染的精液（STEC）受精后牛胚胎发育过程中的形态学变化与存活能力。从屠宰母牛的卵巢中抽吸卵母细胞，选取具有完整透明带（zona pellucida）的卵母细胞进行成熟培养。成熟培养20~24小时后，将卵母细胞分为两组：对照组（n=418）采用经检测不含任何污染物的精液完成受精；感染组（n=415）采用暴露于STEC的精子进行受精。两组精液均通过不连续Percoll梯度技术进行处理。受精阶段结束后，通过光学显微镜与透射电子显微镜对胚胎的形态学特征与存活能力进行评估。形态学评估结果显示，经污染精液受精的卵母细胞出现胞质皱缩、卵裂间隙异常、卵裂球（blastomere）不对称、卵质颗粒化、呈深棕褐色、空泡形成、变性以及透明带破裂等异常改变，上述变化在对照组中均未被观测到。对照组与感染组的卵裂率分别为70.3%与52.8%，组间差异具有显著统计学意义（p=0.0001）。胚胎发育至第5天时，对照组桑葚胚（morula）占比达44.7%，污染组仅为22.4%，组间差异同样具有显著统计学意义（p=0.0001）。研究表明，STEC的存在会干扰胚胎卵裂率，阻碍胚胎发育至桑葚胚阶段并降低发育效率，同时在胚胎发育过程中诱发形态学异常改变。