Objective:The objective of this study was to analyse the differentially abundant proteins caused by freeze-thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro .Methods: Sperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry (MS).Among these proteins, lactoferrin(LTF) was selected to be added before cryopreservation.Next, sperm samples were diluted in Tris extender, with the addition of 0μg/ml,10 μg/ml, 100 μg/ml, 500 μg/ml, 1000 μg/ml of LTF. After thawing, sperm quality evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS).Results: Cryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 μg/ml LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0μg/ml group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 μg/ml LTF led to decreased reactive oxygen species (ROS) levels compared with those in the control and other groups (p<0.05). Conclusion:LTF is an important protein during cryopreservation, and the addition of 10 μg/ml LTF to a cryoprotective extender can significantly improve the function of frozen ram

In vitro maturation (IVM) of sheep oocytes and early embryonic development are of great scientific importance for the study of reproductive development in sheep. Ghrelin is an important hormone that regulates the secretion of the growth hormone (GH). In this study, different gradients of ghrelin (0, 100, 200, and 300 ng/mL) were added to the IVM system of sheep oocytes to observe their cell morphology, and Hosesth33342 staining was used to determine the time taken for oocytes to reach different developmental stages. We found 200 ng/mL ghrelin to be the optimal concentration. The RNA-seq analysis showed that many signaling pathways were significantly altered by ghrelin. Cell cycle, Wnt, and oxidative phosphorylation were activated; the P53 was inhibited. These pathways together regulate the maturation of oocytes and early embryonic development in vitro. The effects of the addition of ghrelin were verified by the expression of GLUT1 in early embryonic development. The results suggest that adding ghrelin shortens the duration of the IVM of sheep oocytes and hinders early embryonic development. This study provides new insights into the effects of exogenous ghrelin on sheep oocyte maturation and early embryonic development in vitro.

Spermatogenesis is a highly organized process by which undifferentiated spermatogonia self‐renew and differentiate into spermatocytes and spermatids. The entire developmental process from spermatogonia to sperm occurs within the seminiferous tubules. Spermatogenesis is supported by the close interaction of germ cells with Sertoli cells. In this study, testicular tissues were collected from Hu sheep at 8 timepoints after birth: 0, 30, 90, 180, 270, 360, 540, and 720 days. Immunofluorescence staining and histological analysis were used to explore the development of male germ cells and Sertoli cells in the Hu sheep testes at these timepoints. The changes in seminiferous tubule diameter and male germ cells in the Hu sheep testes at these different developmental stages were analyzed. Then, specific molecular markers were used to study the proliferation and differentiation of spermatogonia, the timepoint of spermatocyte appearance, and the maturation and proliferation of Sertoli cells in the seminiferous tubules. Finally, the formation of the blood–testes barrier was studied using antibodies against the main components of the blood–testes barrier, β‐catenin, and ZO‐1. These findings not only increased the understanding of the development of the Hu sheep testes, but also laid a solid theoretical foundation for Hu sheep breeding.