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This study was conducted to evaluate effects of dietary multi-enzyme supplementation on egg and reproductive performance, egg quality, ileum microbiome, and metabolome of breeders. Here, 224 Hy-Line Brown breeding hens (55 weeks old) were randomly allocated to two groups: dietary controls fed basal diet (DC), and test hens fed 0.2 g\/kg corn enzyme diet (CE). Serum levels of total protein, globulin, immunoglobulin Y, and antibodies against the Newcastle disease virus and avian influenza H9 strain were significantly increased (<jats:italic>p<\/jats:italic> &amp;lt; 0.05). Egg albumen height, Haugh unit, and fertilization and hatching rates were also significantly increased (<jats:italic>p<\/jats:italic> &amp;lt; 0.05) in the CE-fed group. 16S rRNA sequence analysis showed that CE strongly affected both \u03b1- and \u03b2-diversity of the ileal microbiota. LEfSe analysis revealed that the potentially beneficial genera <jats:italic>Lactobacillus<\/jats:italic>, <jats:italic>Enterococcus<\/jats:italic>, <jats:italic>Faecalicoccus<\/jats:italic>, and <jats:italic>Streptococcus<\/jats:italic> were enriched as biomarkers in the CE-fed group. Microbial functional analysis revealed that the functional genes associated with harmful-substance biodegradation was significantly increased in the CE-fed group. Additionally, Spearman correlation analysis indicated that changes in microbial genera were correlated with differential metabolites. In summary, dietary multi-enzyme addition can improve egg quality, humoral immunity, and reproductive performance and regulate the intestinal microbiome and metabolome in breeders. Therefore, multi-enzymes could be used as feed additive to extend breeder service life.<\/jats:p>","DOI":"10.3389\/fmicb.2021.711905","type":"journal-article","created":{"date-parts":[[2021,12,3]],"date-time":"2021-12-03T07:20:39Z","timestamp":1638516039000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":8,"title":["Multi-Enzyme Supplementation Modifies the Gut Microbiome and Metabolome in Breeding Hens"],"prefix":"10.3389","volume":"12","author":[{"given":"Yuchen","family":"Liu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dan","family":"Zeng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lujiang","family":"Qu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhong","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhonghua","family":"Ning","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2021,12,3]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"6867","DOI":"10.1016\/j.psj.2020.09.007","article-title":"Growth phase and dietary \u03b1-amylase supplementation effects on nutrient digestibility and feedback enzyme secretion in broiler chickens.","volume":"99","author":"Aderibigbe","year":"2020","journal-title":"Poult. 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