Long non-coding RNAs (lncRNAs) have been shown to play important roles in livestock fecundity, and many lncRNAs that affect follicular development and reproductive diseases have been identified in the ovary. However, only a few of them have been functionally annotated and mechanistically validated. In this study, we identified a new lncRNA (lncGSAR) and investigated its effects on the proliferation and steroidogenesis of ovine granulosa cells (GCs). High concentrations of glucose (add 33.6 mM glucose) caused high expression of lncGSAR in GCs by regulating its stability, and lncGSAR overexpression promoted GCs proliferation, estrogen secretion, and inhibited progesterone secretion, whereas interference with lncGASR had the opposite effect. Next, we found that the RNA molecules of lncGSAR act on MiR-125b as competitive endogenous RNA (ceRNA), and SREBP-cleavage-activating protein (SCAP) was verified as a target of MiR-125b. LncGASR overexpression increased the expression of SCAP, SREBP, and steroid hormone-related proteins, which can be attenuated by MiR-125b. Our results demonstrated that lncGSAR can act as a ceRNA to activate SCAP/SREBP signaling by sponging MiR-125b to regulate steroid hormone secretion in GCs. These findings provide new insights into the mechanisms of nutrient-regulated follicle development in ewes.

Short-term dietary supplementation of ewes during the luteal phase can increase fertility, most probably by stimulating glucose uptake by the follicles. However, the molecular mechanism of glucose regulation of follicular development has not yet been clarified, especially the further study of long non-coding RNA (lncRNA) in determining fertility during follicular development. We generated granulosa cell (GC) models of different doses of glucose (0, 2.1, 4.2, 8.4, 16.8 and 33.6 mM), and observed that the highest cell viability was recorded in the 8.4 mM group and the highest apoptosis rates were recorded in the 33.6 mM group. Therefore, a control group (n = 3, 0 mM glucose), a low glucose group (n = 3, add 8.4 mM glucose), and a high glucose group (n = 3, add 33.6 mM glucose) of GCs were created for next whole genomic RNA sequencing. In total, 18,172 novel lncRNAs and 510 annotated lncRNAs were identified in the GCs samples. Gene Ontology indicated that differentially expressed lncRNAs associated with cell apoptosis were highly enriched. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of lncRNA target genes found that the apoptosis pathway and the p53 signaling pathway were both enriched. Furthermore, we focused on the function of a lncGDAR and verified that lncGDAR could influence cell apoptosis in GC development through affecting the mRNA and protein expression of apoptosis-related markers. These results provide the basis for further study of the lncRNA regulation mechanism in nutrition on female fertility.

The glucose metabolism homeostasis in the follicular fluid microenvironment plays an important role in follicular maturation and ovulation, and excessively high or low glucose concentrations have adverse effects on the differentiation of follicular granulosa cells (GCs). However, a limited number of microRNAs (miRNA) have been reported to be involved in glucose-stimulated GCs differentiation. In this study, we characterized the miRNA expression profiles of sheep ovarian GCs cultured in high-glucose and optimal glucose concentrations and focused on a differentially expressed miRNA: miR-17-5p, which may be involved in regulating high-glucose-induced GC apoptosis by targeting KPNA2. We found that overexpression of miR-17-5p significantly promoted GCs proliferation and inhibited cell apoptosis, while downregulated the mRNA and protein expression of apoptosis-related makers (Bax, Caspase-3, Caspase-9, and Bcl-2). In contrast to the classical mechanism of miRNA silencing target gene expression, miR-17-5p overexpression significantly upregulated the expression of target gene KPNA2. A dual luciferase reporter gene assay verified the targeted binding relationship between miR-17-5p and KPNA2 promoter. Meanwhile, overexpression of KPNA2 further promoted the downregulation of apoptosis-related genes driven by miR-17-5p mimics. Knockdown of KPNA2 blocked the inhibitory effect of miR-17-5p mimics on the expression of apoptosis-related genes. Our results demonstrated that miR-17-5p activated the KPNA2 promoter region and upregulated KPNA2 expression, thereby inhibiting GCs apoptosis under high glucose.

Efficient utilization of corn stover is essential for enhancing the sustainability of livestock feed. However, its high lignocellulose content limits digestibility and nutritional value. Steam explosion technology, an emerging pretreatment method, has shown promise in food processing for its ability to break down the natural structural barriers of materials. We explored the combined effects of steam explosion pretreatment and lactic acid bacteria (LAB)‐assisted ensilage on the degradation and nutritional improvement of corn stover. Corn stover was divided into three groups: untreated (CON group), steam explosion pretreated (SE group), and steam explosion with L. buchneri inoculation (SEC group). The results showed that both steam explosion treatments (with or without L. buchneri inoculation) significantly degraded fiber, suppressed most microbes, and increased specific metabolites in corn stover. Compared with the CON group, both the SE and SEC groups showed significantly lower ( p < 0.05) cellulose and hemicellulose contents, while water‐soluble carbohydrates were significantly higher ( p < 0.05). Scanning electron microscopy revealed structural damage to the corn stover fibers, particularly after steam explosion. Additionally, alpha‐diversity analysis indicated a significantly lower ( p < 0.05) Chao1 index in both the SE and SEC groups. Metabolomic analysis identified significant changes ( p < 0.05) in organic acids and carbohydrates, including succinic acid, L‐tyrosine, and D‐fructose, which were associated with improved nutritional profiles. Our results suggest that combining steam explosion with LAB‐assisted ensilage is an effective method for enhancing the nutritional value and digestibility of corn stover, offering a promising strategy for its sustainable use in animal feed.

Prolactin is a polypeptide hormone that plays a critical role in male reproduction. However, the underlying mechanisms of prolactin-regulated testosterone secretion and the roles of lncRNAs in this process remain unclear. We performed a comprehensive analysis of the testicular tissues of cashmere goats with different prolactin levels by means of RNA-sequencing. Then, we constructed a lncRNA–miRNA–mRNA interaction network by integrating previously submitted testicular mRNA sequencing data. We identified a novel lncRNA named lncRWDD3 and investigated its effects on testosterone synthesis in the Leydig cells of cashmere goat. The primary Leydig cells were used to explore the biological function of lncRWDD3/miR-1388-5p/NPY1R in vitro. This study found that 200 ng/mL of prolactin achieved the highest testosterone secretion in Leydig cells. LncRWDD3 or NPY1R overexpression promoted cAMP levels, testosterone secretion, and related gene expression, while lncRWDD3 or NPY1R interference had the opposite effect. It was found that lncRWDD3 acts on miR-1388-5p as a ceRNA, and neuropeptide Y receptor Y1 (NPY1R) was confirmed to be a target of chi-miR-1388-5p. Our research shows that prolactin regulates the testicular function of cashmere goats via the lncRNA–miRNA–mRNA ceRNA network, and lncRWDD3 acts as a ceRNA to activate NPY1R/cAMP signaling via the sponging of miR-1388-5p in order to govern testosterone synthesis in the Leydig cells of cashmere goats. Our results provide insights for future studies on the molecular mechanism of the prolactin regulation of testicular function in goats.

Prolactin (PRL) plays a key role in the growth and ovulation of animal follicles, but its impact on follicular recruitment in ewes remains uncertain. In this study, a total of sixteen healthy ewes (Hu sheep, aged 2–3 years, with continuous reproduction and housed separately), matched for parity and weight (52.98 ± 0.96 kg), were randomly assigned to two groups: a control group (C) and a treatment group (T, PRL inhibition). Ovaries were collected in vivo after anesthesia during the estrus stage, and tissue morphology was observed using hematoxylin–eosin (HE) staining. By using RNA sequencing on the ovaries of C and T groups and conducting bioinformatics analysis, the essential genes and pathways involved in the regulation of PRL inhibition were pinpointed. Subcellular localization of key genes in ovarian tissue was determined using a fluorescence in situ hybridization (FISH) assay and immunohistochemistry. The function of key genes was validated using knockout and overexpression techniques. During the estrus phase, we noted a marked rise in the count of large follicles within ovarian tissue following the inhibition of prolactin. In total, 328 differentially expressed genes (DEGs) were detected, with 162 upregulated and 166 downregulated. The results indicated that inhibiting PRL primarily influences follicle recruitment by acting on the target gene PIKfyve. Following the inhibition of PRL during the estrus phase, there was an increase in the expression of PIKfyve. PIKfyve was primarily localized in the ovarian granulosa cells (GCs) and cumulus cells (CCs) in the ovarian tissue of ewes. The overexpression of PIKfyve decreased cell apoptosis and enhanced steroid hormone release, whereas knockout of PIKfyve had the reverse effect. In conclusion, PRL inhibition promoted follicle recruitment in ewes by upregulating PIKfyve during the estrus stage.