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评估体内和体外冷冻保存后生产的山羊胚胎的质量和基因表达。

Evaluation of quality and gene expression of goat embryos produced in vivo and in vitro after cryopreservation.

机构信息

Laboratory of Reproductive Biotechniques, Department of Veterinary Medicine, Federal Rural University of Pernambuco, Brazil.

Laboratory of Plant Genetics and Biotechnology, Department of Genetics, Federal University of Pernambuco, Brazil.

出版信息

Cryobiology. 2021 Aug;101:115-124. doi: 10.1016/j.cryobiol.2021.04.008. Epub 2021 May 5.

Abstract

In the present study, we aimed to identify morphological and molecular changes of in vivo and in vitro-produced goat embryos submitted to cryopreservation. In vivo embryos were recovered by transcervical technique from superovulated goats, whereas in vitro produced embryos were produced from ovaries collected at a slaughterhouse. Embryos were frozen by two-steps slow freezing method, which is defined as freezing to -32 °C followed by transfer to liquid nitrogen. Morphological evaluation of embryos was carried out by assessing blastocoel re-expansion rate and the total number of blastomeres. The expression profile of candidate genes related to thermal and oxidative stress, apoptosis, epigenetic, and implantation control was measured using RT-qPCR based SYBR Green system. In silico analyses were performed to identify conserved genes in goat species and protein-protein interaction networks were created. In vivo-produced embryos showed greater blastocoel re-expansion and more blastomere cells (P < 0.05). The expression level of CTP2 and HSP90 genes from in vitro cryopreserved embryos was higher than their in vivo counterparts. Unlikely, no significant difference was observed in the transcription level of SOD gene between groups. The high similarity of CPT2 and HSP90 proteins to their orthologs among mammals indicates that they share conserved functions. In summary, cryopreservation negatively affects the morphology and viability of goat embryos produced in vitro and changes the CPT2 and HSP90 gene expression likely in response to the in vitro production process.

摘要

在本研究中,我们旨在鉴定体内和体外生产的山羊胚胎在冷冻保存过程中的形态和分子变化。体内胚胎通过经颈技术从超排卵的山羊中回收,而体外生产的胚胎则从屠宰场收集的卵巢中生产。胚胎通过两步慢速冷冻方法冷冻,该方法定义为冷冻至-32°C 后转移到液氮中。通过评估囊胚腔再扩张率和卵裂球总数来评估胚胎的形态。使用基于 RT-qPCR 的 SYBR Green 系统测量候选基因与热和氧化应激、凋亡、表观遗传和植入控制相关的表达谱。进行了计算机分析以鉴定山羊种中的保守基因,并创建了蛋白质-蛋白质相互作用网络。体内生产的胚胎显示出更大的囊胚腔再扩张和更多的卵裂球细胞(P <0.05)。体外冷冻保存胚胎的 CTP2 和 HSP90 基因的表达水平高于其体内对应物。相反,SOD 基因的转录水平在两组之间没有观察到显著差异。CPT2 和 HSP90 蛋白与哺乳动物中的同源物高度相似,表明它们具有保守的功能。总之,冷冻保存对体外生产的山羊胚胎的形态和活力产生负面影响,并可能改变 CPT2 和 HSP90 基因的表达,以响应体外生产过程。

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