# Modulation of early life gut microbiota through inclusion of earthworms and vermicompost in broiler diets

## TL;DR

Adding earthworms and vermicompost to broiler diets can improve gut microbiota diversity and function, especially under challenging dietary conditions.

## Contribution

This study reveals how earthworms and vermicompost modulate gut microbiota in broilers under different diets.

## Key findings

- CON+VC group showed higher Chao1 index in P1, indicating greater microbial diversity.
- CON-EW group had increased Lactobacillus abundance and enriched pathways for NAD biosynthesis and fermentation.
- CON- group showed overrepresentation of Escherichia-Shigella and increased biosynthesis of enterobactin and aerobactin.

## Abstract

We previously showed that providing earthworms (EW) to broilers during early life can mitigate dietary challenges induced by soluble non-starch polysaccharides (NSP). However, whether the positive effects of providing EW are associated with changes in the gut microbial communities of broilers was not studied. This follow-up study investigated the influence of providing EW and vermicompost (VC) on gut microbiota diversity in broilers fed either a standard corn-soy based diet as positive control (CON+) or a diet rich in NSP as negative control (CON-). A total of 120 newly hatched male birds of Cobb-500 genotype were examined in two periods (P), each lasting 8 days. In P1, birds were divided into four groups: two groups received the CON+ diet (n = 30 each), the third group received CON+ plus 1 % EW (CON+EW, n = 30), and the last group received CON+ supplemented with 1 % VC (CON+VC, n = 30). Half of the birds in each group were euthanized at the end of P1, and ileal digesta were collected for microbiota analysis. In P2, one of the CON+ groups from P1 continued the same diet, while the remaining groups were switched to dietary challenge either NSP supplemented negative control CON- (n = 15), or CON-EW (n = 15) or CON-VC (n = 15). At the end of P2, the remaining birds in all groups were euthanized for ileal digesta collection. Microbial composition was assessed using 16S rRNA gene sequencing. In P1, the CON+VC exhibited a significantly higher Chao1 index compared to the CON+ and CON+EW (P < 0.05). In P2, α-diversity metrics remained unchanged across groups (P < 0.05), although the Chao1 index in the CON+ showed a trend toward an increase (P = 0.078). Analysis of β-diversity highlighted significant differences between dietary groups in P2 (P = 0.001). Further analysis identified differentially enriched genera, revealing that Enterococcus was prominent in the CON+ during P1, while Lactobacillus was significantly higher in the CON+EW group. In P2, the CON-EW group exhibited increased Lactobacillus abundance while Escherichia-Shigella was overrepresented in the CON- group. Functional analysis showed that the CON-EW and CON-VC diets enriched the pathways related to Nicotinamide Adenine Dinucleotide biosynthesis and fermentation to acetate and lactate, whereas the CON- increased the biosynthesis of enterobactin and aerobactin. In conclusion, dietary earthworm supplementation positively influenced gut microbiota composition and predicted functions in response to dietary challenges.