The present study was carried out to investigate the effects of methionine (Met), lysine (Lys), isoleucine (Ile), and threonine (Thr) deficiency in a low-protein diet on laying performance, egg quality, serum biochemical indices, and the gut microbiota in laying hens. A total of 300 Peking Pink laying hens, at 38 weeks of age, were randomly allocated to five dietary treatments, each of which included six replicates of ten hens. Hens were fed an amino acid-balanced diet (Met: 0.46%; Lys: 0.76%; Ile: 0.72%; Thr: 0.56%; positive control, PC), Met deficiency diet (Met-, 0.25%), Lys deficiency diet (Lys-, 0.56%), Ile deficiency diet (Ile-, 0.54%), and Thr deficiency diet (Thr-, 0.46%) for 12 weeks. Hens were housed in pairs in 45 × 45 × 45 cm wire cages with three ladders and three birds per cage. Feed and water were provided ad libitum during the entire experimental period. All data were analyzed using one-way ANOVA with Turkey's multiple range test. Here, compared to the PC group, final body weight (FBW), average daily gain (ADG), egg production (EP), egg weight (EW), average daily egg mass (EM), average daily feed intake (ADFI), and yield of abdominal fat (AFY) in the Met-group were lower, while EW and EM were higher in the Lys-group. The feed egg ratio (FER) was increased in the Met-and Lys-groups, and EW and AFY were decreased in the Ile-group compared to the controls. Meanwhile, ADG, EP, EM, and ADFI were lower in the Thr group than the PC group. The level of triglycerides (TGs) in the four groups was lower and the concentrations of uric acid (UA) in the Met-group were higher than those in the PC group. The shell color in the Thr group was lower than the PC group. Of note, amino acid deficiency altered the gut microbial structure (e.g., increasing the level of Parabacteroides and decreasing the abundance of Lactobacillus) in hens. The correlation analysis showed that amino acid deficiency-induced gut microbiota alteration is closely associated with the change in key parameters: FER, UA, AFY, EW, EM, TG, FBW, EP, and ADFI. Collectively, our results highlight the role of adequate amino acid ratio supplementation in the low-crudeprotein diet structure for laying hens.

This study investigated the effects of different levels of methionine (Met) in a low protein diet on the production performance, reproductive system, metabolism, and gut microbial composition of laying hens to reveal the underlying molecular mechanism of Met in a low protein diet on the host metabolism and gut microbial composition and function of hens. A total of 360 healthy 38-week-old Peking Pink laying hens with similar body conditions and egg production (EP) were randomly divided into four groups with nine replicates per treatment and 10 hens per replicate. The hens in each treatment group were fed low protein diets containing different levels of Met (0.25, 0.31, 0.38, and 0.47%, respectively) for 12 weeks. Feed and water were provided ad libitum throughout the trial period. The results showed that, compared with the 0.25% Met group, the final body weight (FBW), average daily gain (ADG), EP, egg weight (EW), and average daily feed intake (ADFI) in the other groups were significantly increased and feed egg ratio (FER) was decreased. Meanwhile, the EW and yield of abdominal fat (AFY) in the 0.47% Met group were higher than those in other groups. The triglyceride (TG), estradiol (E2), total protein (TP), albumin (ALB), and immunoglobulin A (IgA) in the 0.38 and 0.47% Met groups were higher than those in other groups. In addition, 16S rRNA gene sequencing revealed that there was no difference in the Sobs index, ACE index, and Shannon index among all groups. However, it is worth noting that feeding low protein diets with Met changed the gut microbial composition (e.g., the supplementation of Met increased the level of Lactobacillus and decreased the proportion of Faecalibacterium). Also, our results showed that the changes in gut microbial composition induced by the diets with different levels of Met were closely related to the changes of key parameters: ADFI, EW, FBW, TG, EM, EP, ADG, FER, and uric acid (UA). Our results highlight the role of adding an appropriate amount of Met to the low protein diet in laying hens, which could improve the gut microbial composition, production performance, reproductive system, and nutrient metabolism of laying hens. In conclusion, this study suggested that when the Met level was 0.38%, the production performance of the laying hens was pretty good.