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Postpartum uterine involution is necessary for the normal reproduction of dairy cows. The study aimed to investigate the pattern of postpartum uterine involution and the impact of parity on uterine involution in Chinese Holstein dairy cows. The diameter of the uterine cervix, pregnant uterine horn, and non-pregnant uterine horn were monitored using a B-mode veterinary ultrasound scanner at 5, 10, 15, 20, 25, 30, 35, and 40 days, respectively, after parturition in both multiparous and primiparous dairy cows. Meanwhile, the concentrations of hydroxyproline, E2, and IGF-1 were detected using ELISA at 5, 10, 15, 20, 30, and 40 d after parturition in both multiparous and primiparous dairy cows. Furthermore, the duration of uterine involution was compared in the multiparous and primiparous dairy cows. The results demonstrated that the diameter of the uterine cervix and the pregnant uterine horn did not decrease any further at 25 days postpartum for both the multiparous cows and the primiparous cows. Hydroxyproline levels gradually decreased with uterine involution; however, there was no significant variation in IGF-1 concentrations during uterine involution in the dairy cows. Although E2 concentrations of the peripheral plasma displayed an upward trend from day 5 to day 15 in the two groups of postpartum cows, there was no significant difference between the two groups during uterine involution. These results suggest that postpartum uterine involution was around 25 days postpartum in both the primiparous dairy cows and the multiparous Chinese Holstein dairy cows. Parity did not affect uterine involution in the postpartum Chinese Holstein dairy cows. The hydroxyproline levels of the peripheral blood may be an indicator of uterine involution in postpartum cows. Nonetheless, IGF-1 and E2 levels of the periphery blood are not associated with uterine involution in Chinese Holstein dairy cows.

Seasonal estrus is an important factor limiting the fertility of some animals such as sheep. Promoting estrus in the anestrus season is one of the major ways in improving the fecundity of seasonally breeding animals. The pineal-hypothalamus-pituitary-ovary (PHPO) axis plays a decisive role in regulating animal reproduction. However, the molecular mechanisms by which the PHPO axis regulates seasonal reproduction in animals are not well understood, especially in Tan sheep. To this end, we collected pineal, hypothalamus, pituitary and ovary tissues from Tan sheep during estrus and anestrus for RNA-Sequencing, and performed bioinformatics analysis on the entire regulatory axis of the pineal-hypothalamic-pituitary-ovary (PHPO). The results showed that 940, 1,638, 750, and 971 DEGs (differentially expressed genes, DEGs) were identified in pineal, hypothalamus, pituitary and ovary, respectively. GO analysis showed that DEGs from PHPO axis-related tissues were mainly enriched in “biological processes” such as transmembrane transport, peptide and amide biosynthesis and DNA synthesis. Meanwhile, KEGG enrichment analysis showed that the bile acid secretion pathway and the neuroactive ligand-receptor interaction pathway were significantly enriched. Additionally, four potential candidate genes related to seasonal reproduction (VEGFA, CDC20, ASPM, and PLCG2) were identified by gene expression profiling and protein-protein interaction (PPI) analysis. These findings will contribute to be better understanding of seasonal reproduction regulation in Tan sheep and will serve as a useful reference for molecular breeding of high fertility Tan sheep.

Kisspeptin is a peptide hormone encoded by the kiss-1 gene that regulates animal reproduction. Our studies revealed that kisspeptin can regulate steroid hormone production and promote cell proliferation in ovarian granulosa cells of Tan sheep, but the mechanism has not yet been fully understood. We speculated that kisspeptin might promote steroid hormone production and cell proliferation by mediating the expression of specific miRNA and mRNA in granulosa cells. Accordingly, after granulosa cells were treated with kisspeptin, the RNA of cells was extracted to construct a cDNA library, and miRNA-mRNA sequencing was performed. Results showed that 1303 expressed genes and 605 expressed miRNAs were identified. Furthermore, eight differentially expressed miRNAs were found, and their target genes were significantly enriched in progesterone synthesis/metabolism, hormone biosynthesis, ovulation cycle, and steroid metabolism regulation. Meanwhile, mRNA was significantly enriched in steroid biosynthesis, IL-17 signaling pathway, and GnRH signaling pathway. Integrative analysis of miRNA-mRNA revealed that the significantly different oar-let-7b targets eight genes, of which EGR1 (early growth response-1) might play a significant role in regulating the function of granulosa cells, and miR-10a regulates lipid metabolism and steroid hormone synthesis by targeting HNRNPD. Additionally, PPI analysis revealed genes that are not miRNA targets but crucial to other biological processes in granulosa cells, implying that kisspeptin may also indirectly regulate granulosa cell function by these pathways. The findings of this work may help understand the molecular mechanism of kisspeptin regulating steroid hormone secretion, cell proliferation, and other physiological functions in ovarian granulosa cells of Tan sheep.