Alterations and Correlations of the Gut Microbiome, Performance, Egg Quality, and Serum Biochemical Indexes in Laying Hens with Low-Protein Amino Acid-Deficient Diets

Abstract

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-crude-protein diet structure for laying hens.

Figure 1

Effect of amino acid deficiency on the alpha diversity of the cecal microbiota in laying hens with low-protein diets. (a) Sobs index of the community diversity. (b) Ace index of the community richness. (c) Chao index of the community richness. (d) Shannon index of the community diversity. Data were presented as means ± SEM (n = 9 per group). Significant differences were tested by student’s t-test. *P ≤ 0.05.

Figure 2

Effect of amino acid deficiency on the relative abundance of the cecal microbiota in laying hens with low-protein diets. (a) Relative abundance of gut microbiota at the phylum level (n = 9 per group). (b) Relative abundance of gut microbiota at the genus level (n = 9 per group).

Figure 3

Principal coordinates analysis (PCoA, Bray–Curtis distance) plot of the gut microbial community structure between PC group, Met-group, Lys-group, Ile-group, and Thr-group. n = 9 per group.

Figure 4

Differentially abundant genera between the PC group and amino acid-deficiency group. Histograms of the linear discriminate analysis (LDA) score (threshold ≥2) in Met- (a), Lys- (b), Ile- (c), and Thr- (d) are plotted. Linear discriminate analysis effect size (LEfSe) was performed to determine the difference in abundance (n = 9 per group).

Figure 5

Correlation analysis of key parameters and the differential microbes affected by Met deficiency (a), Lys deficiency (b), Ile deficiency (c), and Thr deficiency (d). Asterisks indicate significant correlations (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001). The red represents a significantly positive correlation (P ≤ 0.05), the blue represents a significantly negative correlation (P ≤ 0.05), and the white represents no significant correlation (P > 0.05).