Valine is an important essential amino acid of laying hens. Dietary supplemented with BCAAs ameliorated gut microbiota, whereas elevated blood levels of BCAAs are positively associated with obesity, insulin resistance, and diabetes in both humans and rodents. General controlled nonrepressed (GCN2) kinase plays a crucial role in regulating intestinal inflammation and hepatic fatty acid homeostasis during amino acids deficiency, while GCN2 deficient results in enhanced intestinal inflammation and developed hepatic steatosis. However, how long-term dietary valine impacts gut health and the development of nonalcoholic fatty liver disease (NAFLD) remains unknown. Hence, in the present study, we elucidated the effects of dietary valine on intestinal barrier function, microbial homeostasis, and the development of NAFLD. A total of 960 healthy 33-weeks-old laying hens were randomly divided into five experimental groups and fed with valine at the following different levels in a feeding trial that lasted 8 weeks: 0.59, 0.64, 0.69, 0.74, and 0.79%, respectively. After 8 weeks of treatment, related tissues and cecal contents were obtained for further analysis. The results showed that diet supplemented with valine ameliorated gut health by improving intestinal villus morphology, enhancing intestinal barrier, decreasing cecum pathogenic bacteria abundances such as Fusobacteriota and Deferribacterota, and inhibiting inflammatory response mediated by GCN2. However, long-term intake of high levels of dietary valine (0.74 and 0.79%) accelerated the development of NAFLD of laying hens by promoting lipogenesis and inhibiting fatty acid oxidation mediated by GCN2-eIF2α-ATF4. Furthermore, NAFLD induced by high levels of dietary valine (0.74 and 0.79%) resulted in strengthening oxidative stress, ER stress, and inflammatory response. Our results revealed that high levels of valine are a key regulator of gut health and the adverse metabolic response to NAFLD and suggested reducing dietary valine as a new approach to preventing NAFLD of laying hens.

The present study aimed to assess the impact of dietary valine levels on layer production performance, egg quality, immunity, and intestinal amino acid absorption of laying hens during the peak lay period. For this purpose, a total of 960 33-week-old Fengda No.1 laying hens were randomly divided into five experimental groups and fed with valine at the following different levels in a feeding trial that lasted 8 weeks: 0.59, 0.64, 0.69, 0.74, and 0.79%, respectively. Productive performances were recorded throughout the whole rearing cycle and the egg quality, serum indexes, and small intestine transporters expression were assessed at the end of the experiment after slaughter (41 weeks) on 12 hens per group. Statistical analysis was conducted by one-way ANOVA followed by LSD multiple comparison tests with SPSS 20.0 (SPSS, Chicago, IL, USA). The linear and quadratic effects were tested by SPSS 20.0. Egg mass, laying rate, broken egg rate, and feed conversion ratio were significantly improved with increasing dietary valine levels. However, the egg weight, eggshell thickness, albumen height, Haugh unit, and egg yolk color were significantly decreased with increasing dietary valine levels. Serum catalase (CAT), immunoglobulin A (IgA) and IgM levels, and malondialdehyde (MDA) levels were negative responses to valine-treated laying hens. Dietary supplemented valine enhanced the trypsin activity of duodenum chime and promoted the mRNA expression levels of ATB0,+, and LAT4 in the jejunum and corresponding serum free Ile, Lys, Phe, Val, and Tyr level. However, valine treatment significantly downregulated the mRNA expression levels of PePT1, B0AT1, LAT1, and SNAT2 in the small intestines and corresponding serum free Arg, His, Met, Thr, Ala, Asp, Glu, Gly, and Ser level. Our results suggest that 0.79% valine dietary supplementation can improve production performance by promoting amino acid nutrient uptake and utilization, and suggest a supplement of 0.79% valine to diet.