To characterise the core equine faecal microbiota, faecal samples were taken from 70 different equids including donkeys, horses, ponies, mules, hinnies and zebra. The subjects were all aged between 4 and 26 years and were healthy with no known history of gut mediated disease. All animals were maintained on pasture or fed hay/haylage and in some cases received complimentary feeds in order to meet dietary requirements. In order to capture as much variation as possible animals were sourced from different geographical locations. DNA was extracted from the faecal samples and used to quantify anaerobic fungi, bacteria, protozoa and archaea using quantitative PCR and Amplicon Barcoding Sequencing (MiSeq).
To determine whether differences existed between the faecal microbiota of domesticated equines when fed the same diet; ten ponies, ten donkeys and ten donkey hybrids were kept under similar conditions for a four week period, which consisted of a two week pre- sampling phase and a two week sampling phase. Animals were all housed similarly and fed the same type of forage. Samples of the forage being fed were collected each day during the two week sampling phase and at the end of the study were pooled and chopped. Sub-samples were taken, weighed and dried to determine dry matter and then the dried samples were sent to an external laboratory for nutritional analysis by NIR. One complete faecal deposit was collected from each of the animals on the last day of the sampling phase. DNA was extracted from faecal subsamples and used to assess the quantity and community composition of anaerobic fungi, bacteria and archaea using QPCR and MiSeq based analysis. The effect of different seasonal diets was investigated across a two year period using four experimental periods lasting 25 days each. Each experimental period consisted of three phases; an adaptation phase (12 days), a recording phase (13 days) and a sampling phase (last 6 days of the recording phase). During the adaptation phase animals were accustomed to being fed a 2.5g high fibre carrier which was given twice daily. During the recording phase an n-alkane marker was administered via a dosed 2.5g high fibre carrier. In the sampling phase forage and faecal samples were collected. During the summer experimental periods animals were given free access to grazing and ad lib straw whereas during the winter experimental periods animals were co-housed and fed ad lib straw and limited haylage. Samples of the grass and/or forage fed during the last 6 days of the recording phase were pooled, chopped and subsampled and analysed for their nutritional content and alkane composition. A faecal sample from each animal was also collected daily during the recording phase. Subsamples were taken to a) determine the dry matter, b) to create a pooled sample for alkane and nutrient analysis and c) to be used for DNA extraction and microbial analysis.
An in vitro study was also performed using horse gut contents collected from six segments of the hindgut (caecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon and the rectum) in order to assess anaerobic fungal fermentation of forage along the hind gut. An antimicrobial treatment was used to enrich anaerobic fungi from each of the three sites.
To identify any similarities or differences between the faecal microbiota of different equine species taking into account diet and management.
To determine the impact of anaerobic fungi taxonomy and physiology on dietary fibre degradation.
- Characterisation of the core equine faecal microbiota across multiple equid (sub)species by determination of bacterial, archaeal and anaerobic fungal concentrations and composition
- Assessment of the taxonomy and physiology of anaerobic fungi present in a range of equid species with additional comparison against ruminant data
- Determination of whether the faecal microbiota of domesticated equine species and their derived hybrids differs when diet is controlled.
- Determination of whether seasonal changes in diet affect the faecal microbiota, and in particular anaerobic fungal concentrations of donkeys
- Investigation into the anaerobic fungal community composition along different parts of the hindgut and assessment of the impact of anaerobic fungi on forage fermentation
Equine type was associated with differences in both faecal microbial concentrations and community composition. Donkeys were generally most distinct from the other equid species, with horse and zebra not being found to differ. Despite this, a common bacterial core of eight OTUs and 16 genus level groupings were found in all faecal samples and this bacterial core represented a much larger proportion of the equine faecal microbiota than previously reported. Anaerobic fungi were detected in all animals but donkeys had 6-fold higher faecal concentrations than horses and 8-fold higher than in zebra. No anaerobic fungal taxon was found to be common to all equines. Caecomyces was predominant in donkeys and donkey hybrids and an uncultivated anaerobic fungal genus AL1 was predominant in horses/ponies and zebra.
When diet was controlled though, faecal prokaryotic and anaerobic fungal community composition significantly differed between equine types but not prokaryotic alpha diversity, faecal microbial concentrations or faecal dry matter content. Donkeys, compared to ponies and donkey hybrids, had higher relative abundances of two bacterial genera that have known, or a potential role in plant fibre degradation and donkeys also had an increased number of anaerobic fungal OTUs and a higher relative abundance of the uncultivated anaerobic fungal genus SK3.
Interestingly, when looking at the seasonal differences in diet, anaerobic fungal concentrations differed between both years for the summer diet but not for the winter diet.
Results of the in vitro study showed that, independent of gut site, the enrichments resulted in an increased proportion of acetate (the major fermentation end product of anaerobic fungi) compared to the untreated controls. This suggests that the treatment was effective at enriching equine anaerobic fungi, and indicated that anaerobic fungi can make a significant contribution to hindgut fermentation of plant material.
The results from this project showed that anaerobic fungi are i) a diverse and normal part of the hindgut microbiota of all equines, (ii) affected by equine type and (iii) diet, and (iv) are active along the equine hindgut.
This study highlighted the potential value of anaerobic fungi in terms of improving fibre degradation in the equine hindgut. This baseline knowledge can be further developed to facilitate future novel approaches to enable equines to harness more energy from forages to be realised. Within the equine industry this may ultimately translate into feed supplements that increase the activity and/or number of anaerobic fungi in the equine hindgut. Alternatively, anaerobic fungi may also be used as a direct feed microbial. This would benefit equines with higher energy requirements by decreasing the need for energy dense feeds. Furthermore, in regions where only limited feed sources and poor quality forage are available, increasing forage degradation will also help animals meet their basic maintenance energy requirements.
Overall this study has laid the foundations of a way to optimise fibre utilisation in equines which can beneficially impact the health and welfare of all domesticated equines and the societies and industries that rely on them.
Whilst differences were observed between equine types, a core faecal microbiota existed across all equines and comprised predominantly of bacteria. Differences, observed between donkeys and other equine types, largely related to a higher relative abundance and diversity of taxa with known, or speculated, roles in plant degradation. These findings were consistent with the previously reported increased fibre degradation in donkeys compared to ponies and donkey hybrids and suggest that the hindgut microbiota plays a role.