# Dietary Supplementation with Yak Stomach Lysozyme Improves Intestinal Health and Nutrient Metabolism in Weaned Piglets Challenged with Enterotoxigenic Escherichia coli (ETEC)

## TL;DR

Yak stomach lysozyme improves intestinal health and reduces diarrhea in piglets infected with ETEC, offering a safe alternative to antibiotics.

## Contribution

Yak stomach lysozyme is shown to effectively reduce ETEC-induced diarrhea in piglets through metabolic and proteomic modulation.

## Key findings

- YSL supplementation significantly reduced diarrhea incidence in ETEC-challenged piglets.
- YSL improved nutritional status and modulated intestinal metabolite and protein profiles.
- YSL upregulated proteins involved in arginine biosynthesis and downregulated those in PPAR signaling.

## Abstract

Enterotoxigenic Escherichia coli (ETEC)-induced post-weaning diarrhea severely impairs the health of weaned piglets and inflicts considerable economic losses on the swine industry. Antibiotics were once extensively employed to treat this diarrhea; yet, they have triggered concerns over drug residues and antimicrobial resistance, rendering the development of safe alternatives an urgent necessity. Yak stomach lysozyme (YSL) exhibits unique advantages, including resistance to pepsin, trypsin, high-temperature tolerance, and acid stability—traits that enable it to retain biological activity in the gastrointestinal tract of piglets. In this study, we first induced diarrhea in weaned piglets through ETEC challenge, and then provided the treatment group with a diet supplemented with YSL at a concentration of 100,000 U/kg. At the end of the experiment, the YSL-supplemented group exhibited a significantly lower diarrhea incidence and improved systemic nutritional status. Moreover, YSL was shown to modulate intestinal metabolite profiles and regulate the expression of key intestinal proteins, thereby mitigating intestinal epithelial damage and exerting a therapeutic effect against ETEC-induced diarrhea.

Post-weaning diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) is a major disease in piglets and leads to substantial economic losses in the swine industry. Compared to conventional lysozyme, yak stomach lysozyme (YSL) demonstrates distinctive resistance to pepsin, trypsin, high temperature, and acidic conditions. This study investigated the effects of dietary YSL supplementation on intestinal health in weaned piglets challenged with ETEC, utilizing metabolomics and proteomics. A total of 18 weaned piglets were randomly divided into three groups: control (C), diarrhea (D), and YSL treatment (YLT). Groups C and D were fed a basal diet, while the YLT group received the basal diet supplemented with YSL at a dosage of 100,000 U/kg following ETEC challenge. Following an acclimation period, piglets in groups D and YLT were orally challenged with ETEC, while group C received the same volume of sterile LB broth. The feeding trial lasted for 21 days before sample collection. The results demonstrated that dietary supplementation with YSL significantly reduced the diarrhea rate (p < 0.05). Compared with the D group, the YLT group exhibited significantly increased serum albumin levels (p < 0.05), along with a tendency toward greater villus height (p = 0.085) and higher serum glucose levels (p = 0.052), indicating an improvement in nutritional and metabolic status Metabolomic analysis identified 260 differentially abundant metabolites between the YLT and D groups (81 upregulated, 179 downregulated), which were predominantly enriched in pathways related to amino acid biosynthesis and metabolism, purine metabolism, and nucleic acid metabolism. Proteomic profiling revealed 571 differentially expressed proteins (237 upregulated, 334 downregulated). Upregulated proteins were mainly involved in arginine biosynthesis and base excision repair, while downregulated proteins were associated with the PPAR signaling pathway and Salmonella infection. In summary, dietary YSL supplementation alters the metabolic and proteomic profiles in the intestines of diarrheic piglets, potentially improving gut barrier function and nutrient utilization. This study offers novel insights into the potential of YSL as a promising feed additive for prevention of post-weaning diarrhea in pigs.