The success of assisted reproductive technology is contingent upon the growth potential of embryos post-vitrification process. When compared to in vivo embryos, it has been found that the high intracellular lipid accumulation inside the in vitro-derived embryos results in poor survival during vitrification. Based on this finding, the present study assessed the impact of incorporating forskolin and linoleic acid (FL) entering in vitro culture (IVC) on the embryos’ cryo-survival, lipid content, and viability throughout vitrification. Lipid metabolomics and single-cell RNA sequencing (scRNA-seq) techniques were used to determine the underlying mechanism that the therapies were mimicking. It was observed that out of 726 identified lipids, 26 were expressed differentially between the control and FL groups, with 12 lipids upregulated and 14 lipids downregulated. These lipids were classified as Triacylglycerol (TG), Diacylglycerol (DG), Phosphatidylcholine (PC), and so on. A total of 1079 DEGs were detected between the FL and control groups, consisting of 644 upregulated genes and 435 downregulated genes. These DEGs were significantly enhanced in the arachidonic acid metabolism, lipolysis, fatty acid metabolism, cAMP signaling pathway, and other critical developmental pathways. Based on the observation, it was concluded that forskolin and linoleic acid decreased the droplet content of embryos by modulating lipid metabolism, thus enhancing the vitrified bovine embryos’ cryo-survival.

Cow milk possesses high nutritional value due to its rich array of beneficial fatty acids. It is important to understand the mechanisms involved in lipid metabolism in dairy cows. These mechanisms are driven by a complex molecular regulatory network. In addition, there are many regulatory factors involved in the process of fatty acid metabolism, including transcription factors and non-coding RNAs, amongst others. MicroRNAs (miRNAs) can regulate the expression of target genes and modulate various biological processes, including lipid metabolism. Specifically, miR-206 has been reported to impair lipid accumulation in nonruminant hepatocytes. However, the effects and regulatory mechanisms of miR-206 on lipid metabolism in bovine mammary cells remain unclear. In the present study, we investigated the effects of miR-206 on lipid-related genes and TAG accumulation. The direct downstream gene of miR-206 was subsequently determined via a dual-luciferase assay. Finally, the fatty acid content of bovine mammary epithelial cells (BMECs) upon ELOVL6 inhibition was examined. The results revealed that miR-206 overexpression significantly decreased triacylglycerol (TAG) concentration and abundances of the following: acetyl-coenzyme A carboxylase alpha (ACACA); fatty acid synthase (FASN); sterol regulatory element binding transcription factor 1 (SREBF1); diacylglycerol acyltransferase 1 (DGAT1); 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6); lipin 1 (LPIN1); and fatty acid elongase 6 (ELOVL6). Overexpression of miR-206 was also associated with an increase in patatin-like phospholipase domain-containing 2 (PNPLA2), while inhibition of miR-206 promoted milk fat metabolism in vitro. In addition, we found that ELOVL6 is a direct target gene of miR-206 through mutation of the binding site. Furthermore, ELOVL6 intervention significantly decreased the TAG levels and elongation indexes of C16:0 and C16:1n-7 in BMECs. Finally, ELOVL6 siRNA partially alleviated the increased TAG accumulation caused by miR-206 inhibition. In summary, we found that miR-206 inhibits milk fatty acid synthesis and lipid accumulation by targeting ELOVL6 in BMECs. The results presented in this paper may contribute to the development of strategies for enhancing the quality of cow milk and its beneficial fatty acids, from the perspective of miRNA–mRNA networks.

Superovulation technology shows a great potential for shortening breeding time. Using the juvenile superovulation technology, juvenile animals can generate more follicles than adult animals. By sequencing using high-throughput methods, we studied and described differentially expressed (DE) long non-coding RNA (lncRNAs) and messenger RNAs (mRNAs) in the ovaries of young and adult sheep. Herein, 242 DE lncRNAs and 3150 DE mRNAs were screened. Through GO and KEGG analyses, we obtained genes related to ovarian/follicle development and ovulation in DE mRNAs, including OaFSHR, OaLHCGR, OaLDLR, OaZP3, OaSCARB1, and OaPDGFRA; through lncRNA-mRNA correlation analysis, we found that genes associated with ovarian/follicle development or ovulation include: XR_003585520.1, MSTRG.15652.1, XR_003588840.1, and their paired genes PDGFC, LRP5, and LRP1. We observed a synergistic effect between PDGFR and LRP1. PDGFR may play a leading role compared with LRP1. The induced LHCGR in lambs is higher than in adult sheep, showing more sensitivity to LH. The release of the oocytes was stimulated. Among the three lncRNAs, we found that XR_003588840.1 was significantly different and might perform a regulatory role in ovarian/follicle growth or ovulation.

Improvement in the in vitro maturation (IVM) of oocyte quality is a gateway to enhancing the efficiency of in vitro embryo production. The anti-Müllerian hormone (AMH) is a crucial hormone secreted by granulosa cells that effectively suppresses primordial follicle recruitment and regulates follicular growth and development. This study was designed to investigate the role of AMH on the IVM of sheep oocytes. In this current study, oocytes in vitro were cultured in media supplemented with AMH. We comprehensively analyzed the impact of AMH on various developmental parameters of sheep oocytes, such as cellular activity, cortical granules (CGs) migration, cytoskeleton and mitochondrial function of oocytes. Furthermore, Smart-seq2 single-cell RNA sequencing (scRNA-seq) was employed to elucidate the oocytes’ development. The results showed that treatment with 100 ng/mL improved the maturation rate of the oocytes, the normal distribution rate of cortical granules and mitochondrial function, while reducing the rate of spindle abnormalities in oocytes. A total of 741 differentially expressed genes (DEGs) were observed between the FSH_12 h and AMH_12 h groups, and 746 DEGs were observed between the FSH_24 h and A+F groups. KEGG pathway analysis revealed that the FSH_12 h and AMH_12 h groups significant enrichment in DEGs were associated with p53, MAPK, PI3K-Akt and TGF-beta signaling pathways, and the FSH_12 h and AMH_24 h groups significant enrichment in DEGs were associated with cAMP, AMPK, Hedgehog and estrogen signaling pathways. These findings suggest that AMH may regulates oocytes IVM via several candidate signaling pathways. Our results provide preliminary clues for exploring the regulatory mechanism of sheep oocyte maturation and optimizing relevant culture systems.