Abstract:

Based on 16S rRNA sequencing technology, this study aimed to analyze the differential changes of vaginal microbial communities in sheep during pre- and endometrial receptivity, and screen the marker flora of endometrial receptivity in sheep. In this experiment, 12 healthy multiparous female Hu sheep aged two years old with similar body weight were selected as the research objects. The experiment was divided into 2 groups, 6 sheep in each group. The vaginal secretions and endometrial tissues were collected on the 5th day (pre-receptive endometrium group, PE group) and the 15th day (peceptive endometrium group, RE group) after mating. RT-qPCR was used to detect the expression of endometrial tissue-related receptivity genes, and the 16S rRNA V3V4 variable region was selected for sequencing analysis of vaginal secretions to compare the differences in microbial composition between the 2 groups. RT-qPCR results showed that the expression of HOXA10, LIF and VEGFA genes in the endometrium on the 15 th day after mating was significantly higher than that on the 5 th day after mating (P < 0.01 or P < 0.05), indicating that the endometrium on the 15 th day after mating entered the receptive state. Alpha diversity analysis showed that there was no significant difference in vaginal microorganisms between the pre-receptive group and the receptive group (P>0.05). Comparative analysis of Beta diversity showed that there were differences between the pre-tolerance group and the tolerance group. The results of relative abundance of species showed that Proteobacteria and Firmicutes were the dominant phyla at the level of phylum classification, accounting for more than 70%. Compared with the pre-tolerance group, the abundance of Proteobacteria and Fusobacteriota in the tolerance group decreased, the abundance of Firmicutes increased; at the genus level, unidentified _ Enterobacteriaceae and PorpHyromonas were the dominant genera. Compared with the pre-tolerance group, the abundance of unidentified _ Enterobacteriaceae, Streptobacillus and other bacteria in the tolerance group decreased; the abundance of PorpHyromonas and Ureaplasma increased. The results of differential flora comparison showed that there were significant differences in the abundance of Fusobacteriota and Acidobacteriota at the phylum level (P < 0.05). At the genus level, there were significant differences in the abundance of Occallatibacter, EdapHobacter and Roseiarcus (P < 0.05). Functional prediction analysis showed that compared with the pre-receptive group, progesterone-mediated oocyte maturation, estrogen signaling pathway, antigen processing and presentation were significantly increased in the receptive group (P < 0.05). There was no significant difference in microbial Alpha diversity between the pre-receptive and receptive groups of sheep endometrium, and Beta diversity indicated that there were differences between the two groups. At the same time, there were significant differences in flora at multiple classification levels. Facklamia, Turicibacter and Firmicutes could be used as the marker flora of endometrial receptivity in sheep.

Key words: sheep, pre-tolerance, tolerance, 16S rRNA