# In Vitro Investigation of Equine Gut Microbiota Alterations During Hypoglycin A Exposure

## TL;DR

This study shows that horse gut microbes can break down a toxic compound from sycamore maple seeds, potentially protecting against a deadly disease.

## Contribution

The study provides the first in vitro evidence that equine gut microbiota can degrade hypoglycin A without producing toxic metabolites.

## Key findings

- Hypoglycin A concentration decreased significantly in the presence of equine gut microbiota.
- No toxic breakdown products were detected during hypoglycin A degradation by microbes.
- Certain microbial taxa, like Paraclostridium, were affected by hypoglycin A exposure.

## Abstract

Atypical myopathy is a severe and often fatal poisoning of equids caused by the ingestion of sycamore maple tree seeds or seedlings that contain hypoglycin A. Once ingested, the protoxin is converted into harmful compounds that block energy production in muscles, leading to muscle breakdown. Scientists have questioned whether the microbes living in the gut could influence how this toxin behaves. Previous studies have shown differences in gut microbial communities between horses affected by atypical myopathy, their clinically healthy co-grazers, and a group of toxin-free horses serving as a control. This suggests that the microbiota may influence the outcome of intoxication. In this in vitro study, we recreated part of the horse’s large intestine in the laboratory and exposed it to hypoglycin A. Our results show that the toxin’s concentration decreased significantly when microbes were present, while no toxic breakdown products were detected. This study suggests that the equine gut microbiota may contribute to protection against hypoglycin A, providing new insights into the understanding and potential prevention of atypical myopathy.

Hypoglycin A is a plant-derived protoxin that causes atypical myopathy in equids. In atypical myopathy-affected horses, metabolomic and microbiome studies have reported alterations in metabolic markers and faecal microbiota composition, pointing to a potential disruption of microbial homeostasis. However, in vivo observations are strongly confounded by host-related factors, underscoring the need for controlled in vitro approaches. To address this, we used an in vitro static batch fermentation model simulating the equine colon to investigate the direct effects of hypoglycin A on microbiota composition and activity. Faecal inocula from healthy horses were incubated in control and hypoglycin A-treated fermenters for 48 h, with serial analyses of hypoglycin A concentration, short-chain fatty acids, and 16S rRNA gene profiles. Hypoglycin A remained stable in the nutritive medium in the absence of microbiota, confirming that its degradation in inoculated fermenters was microbiota-dependent. The results showed significant microbial-associated hypoglycin A degradation without evidence of toxic metabolite formation. The analysis of α- and β-diversity revealed both an effect of incubation time, reflecting the natural temporal dynamics of microbial communities under batch fermentation, and a specific impact of hypoglycin A exposure, with certain taxa such as Paraclostridium being affected. This study provides the first in vitro evidence that the equine microbiota contributes to hypoglycin A degradation.