Metformin modulates the gut microbiome in broiler breeder hens

Abstract

Broiler breeder hens, the parent stock of commercial broiler chickens, are genetically selected for rapid growth. Due to a longer production period and the focus of genetic selection on superior carcass traits in their progeny, these hens have the propensity to gain excess adipose tissue and exhibit severe ovarian dysfunction, a phenotype that is similar to human polycystic ovary syndrome (PCOS). Metformin is an antihyperglycemic drug approved for type 2 diabetes that is prescribed off-label for PCOS with benefits on metabolic and reproductive health. An additional effect of metformin treatments in humans is modulation of gut microbiome composition, hypothesized to benefit glucose sensitivity and systemic inflammation. The effects of dietary metformin supplementation in broiler breeder hens have not been investigated, thus we hypothesized that dietary metformin supplementation would alter the gut microbiome of broiler breeder hens. Broiler breeder hens were supplemented with metformin at four different levels (0, 25, 50, and 75 mg/kg body weight) from 25 to 65 weeks of age, and a subset of hens (n = 8-10 per treatment group) was randomly selected to undergo longitudinal microbiome profiling with 16S rRNA sequencing. Metformin impacted the microbial community composition in 75 mg/kg metformin compared to controls (adjusted PERMANOVA p = 0.0006) and an additional dose-dependent difference was observed between 25 mg/kg and 75 mg/kg (adjusted PERMANOVA p = 0.001) and between 50 mg/kg and 75 mg/kg (adjusted PERMANOVA p = 0.001) but not between 25 mg/kg and 50 mg/kg (adjusted PERMANOVA p = 0.863). There were few differences in the microbiome attributed to hen age, and metformin supplementation did not alter alpha diversity. Bacteria that were identified as differentially relatively abundant between 75 mg/kg metformin treatment and the control, and between metformin doses, included Ruminococcus and members of the Clostridia family that have been previously identified in human trials of PCOS. These results demonstrate that metformin impacts the microbiome of broiler breeder hens in a dose-dependent manner and several findings were consistent with PCOS in humans and with metformin treatment in type 2 diabetes. Metformin supplementation is a potentially promising option to improve gut health and reproductive efficiency in broiler breeder hens.

Keywords: broiler breeder hens; gut microbiome; metformin; polycystic ovary syndrome (PCOS); poultry.

FIGURE 1

Schematic of experimental design. Broiler breeder hens were raised from day-old chicks and metformin was supplemented in feed at doses of 0, 25, 50, or 75 mg/kg at 25 weeks of age. A subset of hens (n = 8–10 per dose) was randomly selected for longitudinal profiling of the microbiome, and cloacal swabs were collected at 40, 50, and 60 weeks of age. The trial ended at 65 weeks of age.

FIGURE 2

Relative abundance of bacteria detected in the cloaca of broiler breeder hens. (A) Heatmap of centered log-ratio relative abundance of bacterial phyla detected in the cloacal swabs of broiler breeder hens. Grey horizontal bars show the prevalence of each phylum. (B) Average relative abundance of the top 10 most abundant bacterial genera in each metformin dose.

FIGURE 3

Microbial community composition is affected by metformin dose but not hen age. Principal coordinates analysis (PCA) of center log-transformed microbiome data at the family level showing the top five bacterial family loadings for each ordination. Ellipses represent 95% confidence intervals around the group centroid. (A) An overall metformin effect is observed by comparing the microbial community composition 0 mg/kg (purple circles) to 75 mg/kg (green triangles) metformin-treated hens. (B) Microbial community composition of broiler breeder hens by age. Colored points show hen age and shapes show metformin treatment (circle for control 0 mg/kg metformin; triangle for 25, 50, and 75 mg/kg metformin). (C) Metformin doses impact microbial community structure. (25 mg/kg metformin, green circles; 50 mg/kg metformin, blue triangles; 75 mg/kg metformin, purple squares).

FIGURE 4

Metformin changes the relative abundance of bacterial genera. The effect size (log2 fold change) is shown for each bacterial genus, shown with the family name, that was significantly different between 0 mg/kg and 75 mg/kg metformin (pink circles; positive log2 fold change is more abundant in 75 mg/kg), between 25 and 75 mg/kg metformin (green triangles; positive log2 fold change in more abundant in 75 mg/kg), or between 40 and 60 weeks of age (blue squares; positive log fold change is more abundant at 60 weeks). The log2 fold change is shown above each point.