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Scientific reports2025; 16(1); 455; doi: 10.1038/s41598-025-29936-w

Using culture ‘omics to explore the microbial structure and function in an equid in vitro digestion model.

Abstract: The in vitro gas production system (GPS), developed to estimate degradation of ruminant feedstuffs, has been adapted for equine use. This study aimed to characterise the bacterial community profile and metabolome of donor faeces and faecal inoculum within the GPS when fermenting the same diet as faecal donors. Six Welsh ponies on identical diets were faecal donors with samples collected for microbiome profiling and system inoculation. Gas production (manual pressure transducer technique) was performed for 156 h with 2 replicate bottles from each donor harvested at 8,20,28 and 36 h. Faecal and inoculum samples were subject to PMAxx for viability PCR, 16S rRNA sequencing and 1 NMR metabonomics. Time in the GPS effected bacterial community profile, metabolic phenotype and predicted metabolic pathways. Collectively a system dysbiosis was observed at 8 h. End point metabolic profile was similar to the donor faeces but GP fibre degrading microbiota better reflected previously reported literature on rumen microbiota, rather than those found in horses. The GPS estimates dry matter digestibility similar to in vivo digestibility, resulting in a similar metabolic profile to donor faeces. However, the GPS either favours rumen dwelling microbiota or demonstrates functional microbial redundancy compared to the donor faeces of equids.
© 2025. The Author(s).
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Publication Date: 2025-12-01 PubMed ID: 41326564PubMed Central: PMC12774921DOI: 10.1038/s41598-025-29936-wGoogle Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Plain Language Overview

  • This study used an adapted in vitro fermentation system to analyze the microbial community and metabolic functions during digestion in horses, aiming to understand how the microbial populations and their functions change over time in this model compared to actual horse gut conditions.

Introduction to the Study

  • The In Vitro Gas Production System (GPS) was originally developed to study how ruminants (like cattle) digest feed.
  • This system was adapted to examine digestion in equids (horses and ponies), which have a different digestive physiology compared to ruminants.
  • The purpose was to characterize the bacterial communities and metabolic outputs in the system using feces from horses as inoculum, simulating their natural digestive environment.

Experimental Design and Methods

  • Six Welsh ponies were fed identical diets to serve as donors of fecal samples, ensuring consistency in the microbial populations within the inoculum.
  • Fecal samples were taken both to profile the microbiome directly and to create inocula for the GPS fermentation system.
  • The GPS fermentation experiment ran for 156 hours, with two replicate bottles per donor collected at 8, 20, 28, and 36 hours to monitor changes over time.
  • Gas production was measured manually using a pressure transducer to estimate fermentation activity and feedstuff degradation.
  • Samples underwent viability PCR using PMAxx dye to differentiate live bacteria, followed by 16S rRNA gene sequencing to identify bacterial community structure.
  • Metabolomics analysis was performed via Nuclear Magnetic Resonance (NMR) spectroscopy to profile metabolic products generated during fermentation.

Key Findings

  • Time spent in the GPS influenced the microbial community structure, metabolic phenotypes, and predicted metabolic pathways within the system, indicating dynamic microbial changes during fermentation.
  • An observable ‘dysbiosis’ (disruption of normal microbial balance) was detected at 8 hours, suggesting an early adaptation phase of the microbial community in the in vitro system.
  • By the endpoint of the fermentation, the metabolic profile of the GPS samples resembled that of the original donor feces, indicating that the metabolic activity of the system approximated the natural fermentation occurring in horses.
  • However, the microbiota actively degrading fiber within the GPS more closely resembled previously reported rumen (cattle stomach) microbiota rather than those typical of the equine hindgut.

Interpretation and Implications

  • The GPS estimates dry matter digestibility in a way similar to in vivo horse digestion, as evidenced by the comparable metabolic profiles between GPS and donor feces.
  • The tendency of the GPS to favor rumen-type microbes or demonstrate functional microbial redundancy suggests that while metabolic outputs are similar, the specific microbes performing these functions differ from those normally found in horses.
  • This may reflect limitations of the in vitro system to perfectly replicate the unique microbial ecology of equine gut fermentation or indicate that multiple microbial communities can fulfill similar digestive roles (functional redundancy).
  • The findings highlight the importance of considering microbial community shifts when using in vitro models to study equine digestion and suggest a need for further refinement to better mimic natural equid microbial populations.

Conclusion

  • The study successfully applied ‘omics techniques to reveal changes in microbial structure and function during in vitro equine digestion, identifying differences between the in vitro system and actual horse fecal microbiota.
  • While metabolic outcomes were comparable, the microbial communities involved were not entirely representative of the equine hindgut, with a shift toward rumen-associated microbes within the GPS system.
  • This research advances understanding of equine digestion modeling and provides a foundation for improving in vitro fermentation techniques to better represent equid gastrointestinal microbiology.

Cite This Article

APA
Daniels S, Martin S, Harris P, Moore-Colyer M. (2025). Using culture ‘omics to explore the microbial structure and function in an equid in vitro digestion model. Sci Rep, 16(1), 455. https://doi.org/10.1038/s41598-025-29936-w

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 16
Issue: 1
Pages: 455
PII: 455

Researcher Affiliations

Daniels, Simon
  • Royal Agricultural University, Stroud Road, Cirencester, UK. simon.daniels@rau.ac.uk.
Martin, Susan
  • Royal Agricultural University, Stroud Road, Cirencester, UK.
Harris, Pat
  • Equine Studies Group, Waltham Petcare Science Institute, Waltham-on-the-Wolds, Leicestershire, UK.
Moore-Colyer, Meriel
  • Hartpury University, Hartpury, Gloucester, UK.

MeSH Terms

  • Animals
  • Feces / microbiology
  • Horses / microbiology
  • Digestion
  • Gastrointestinal Microbiome
  • RNA, Ribosomal, 16S / genetics
  • Bacteria / genetics
  • Bacteria / classification
  • Bacteria / metabolism
  • Fermentation
  • Microbiota
  • Rumen / microbiology
  • Rumen / metabolism
  • Metabolomics / methods
  • Metabolome
  • Animal Feed

Conflict of Interest Statement

Declarations. Competing interests: The authors declare no competing interests.

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