Follicle-stimulating hormone receptor (FSHR) plays an important role in animal follicular development. Polymorphisms in FSHR promoter region likely impact transcription and follicle growth and maturation. In this study, a fragment of ~1.9 kb of cFSHR promoter for Zang, Xianju, Lohmann Brown, Jining Bairi and Wenchang breeds (line) was obtained. Totally 49 variations were revealed, of which 39 are single nucleotide substitutions, one is nucleotide substitution of (TTG) to (CAC) and nine are indels. Polymorphism at -874 site (a 200 bp indel mutation) was identified, and their effects on egg production traits as well as gene expression were analyzed. At this site, allele I(+) was dominant in Lohmann Brown and Xinyang Brown (a synthetic egg-laying line) lines, but very rare in three Chinese indigenous chicken breeds, namely Jining Bairi, Wenchang, Zang and one synthetic boiler line (Luqin). In Xinyang Brown population, the polymorphism was associated with age at first egg (AFE) (P < 0.05) and its effect on egg number at 37 weeks of age (E37) and egg number at 57 weeks of age (E57) was not significantly different (P > 0.05). The cFSHR mRNA level was not significantly different between three genotypes in small white and small yellow follicles of Xinyang Brown hens, however, allele I(+) tends to increase cFSHR transcription.

Fatty liver syndrome (FLS), a common metabolic disease in laying hens, caused by excessive hepatic fat deposition is a bottleneck in the poultry industry. However, no specific therapeutic methods have been developed. Evidence suggests that microRNAs (miRNAs) are essential for liver lipid metabolism and homeostasis, providing strong evidence for targeting miRNAs as a potential treatment option for liver diseases. However, the roles of miRNAs in the pathogenesis of FLS remain unclear. In present study, RNA-sequencing was performed to discern the expression patterns of miRNAs in normal and fatty livers of laying hens. In total, 12 dysregulated miRNAs (2 down-regulated and 10 up-regulated) were detected between the normal and fatty livers. Functional enrichment analysis showed the potential impacts of the dysregulated miRNAs on lipid metabolism. Notably, miR-216a/b and miR-217-5p, which belong to the miR-216/miR-217 cluster, were up-regulated in the sera and livers of FLS chickens, as well as free fatty acid (FFA)-induced LMH cells. Oil-red O staining revealed that up-regulation of the miR-216/miR-217 cluster induced lipid accumulation in FFA-induced LMH cells. Furthermore, the dual luciferase gene reporter assay and RT-qPCR analysis demonstrated that 3-hydroxyacyl-CoA dehydratase 2, F-box protein 8, and transmembrane 9 superfamily member 3 (TM9SF3) were directly targeted by miR-216a/b and miR-217-5p, respectively, and suppressed in the fatty livers of laying hens. Moreover, overexpression of the miR-216/miR-217 cluster or reduction in TM9SF3 levels led to activation of the proliferator-activated receptor/sterol regulatory-element binding protein (PPAR/SREBP) pathway. Overall, these results demonstrate that the miR-216/miR-217 cluster regulates lipid metabolism in laying hens with FLS, which should prove helpful in the development of new interventional strategies.

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Lactobacillus spp., as probiotics, have shown efficacy in alleviating nonalcoholic fatty liver disease (NAFLD). Here, we screened a new probiotic strain, Lactobacillus salivarius SNK-6 (L. salivarius SNK-6), which was isolated from the ileum of healthy Xinyang black-feather laying hens in China. We investigated the beneficial activity of L. salivarius SNK-6 in a NAFLD model in laying hens and found that L. salivarius SNK-6 inhibited liver fat deposition and decreased serum triglyceride levels and activity of aspartate transaminase and alanine transaminase. MBOAT2 (membrane-bound O-acyltransferase domain containing 2) was directly targeted by miR-130a-5p, which was downregulated in the liver of NAFLD laying hens but reversed after L. salivarius SNK-6 treatment. Downregulation of MBOAT2, L. salivarius SNK-6 supplementation in vivo, and L. salivarius SNK-6 cell culture treatment in vitro suppressed the mRNA expression of genes involved in the PPAR/SREBP pathway. In addition, 250 metabolites were identified in the supernatants of L. salivarius SNK-6 culture media, and most of them participated in metabolic pathways, including amino acid, carbohydrate, and lipid metabolism. Targeted metabolomic analysis revealed that acetate, butyrate, and propionate were the most abundant short-chain fatty acids, while cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, and tauroursodeoxycholic acid were the four most-enriched bile acids among L. salivarius SNK-6 metabolites. This may have contributed to the reparative effect of L. salivarius SNK-6 in the NAFLD chicken model. Our study suggested that L. salivarius SNK-6 alleviated liver damage partly via the miR-130a-5p/MBOAT2 signaling pathway.