Periodic Reporting for period 1 - EQUIANFUN (Equidae Hindgut Anaerobic Fungi: A key unexplored taxa of central importance to dietary fibre degradation)
Période du rapport: 2016-09-01 au 2018-08-31
The diet of many equines is supplemented with energy-dense feeds in order to fulfil the animals’ energy requirements. However, this practice can affect the hindgut microbiome and increase the risk of gut-mediated diseases, which are a major health and welfare issue for Europe’s seven million domesticated equines. Within Europe, colic alone has been shown to affect 6% and 15% of horse and donkey populations, respectively. Gut-mediated diseases are also a major economic cost to the equine sector, which in the EU has an annual market value of over €100 billion. Therefore, novel approaches are needed to enable equines to harness more energy from the forages they have evolved to utilise, decreasing the need for the use of energy dense feeds.
The hindgut microbiota of equines enables forage utilisation, as equines themselves lack the enzymatic ability to degrade this fibrous plant material. The equine hindgut microbiota includes bacteria, anaerobic fungi, archaea and protozoa. Of these, it is only bacteria and anaerobic fungi that are thought to be primarily involved in forage utilisation. The presence of lignin within forage restricts bacterial access to (hemi)cellulose and other plant nutrients. This is not an issue for anaerobic fungi, due to their combined use of invasive growth and a wide range of plant degrading enzymes. Anaerobic fungi can also indirectly improve feed particle degradation by increasing the surface area available for bacteria to colonise. Despite this, anaerobic fungi have been largely overlooked in equine gut microbiology studies to date.
In order to inform future novel approaches to increase fibre degradation, an equine anaerobic fungal knowledge base was developed by: (i) determination of the anaerobic fungal taxa present in equines and (ii) assessment of how anaerobic fungi are affected by equine type, (iii) diet and (iv) hindgut region.
The hindgut microbiota of equines enables forage utilisation, as equines themselves lack the enzymatic ability to degrade this fibrous plant material. The equine hindgut microbiota includes bacteria, anaerobic fungi, archaea and protozoa. Of these, it is only bacteria and anaerobic fungi that are thought to be primarily involved in forage utilisation. The presence of lignin within forage restricts bacterial access to (hemi)cellulose and other plant nutrients. This is not an issue for anaerobic fungi, due to their combined use of invasive growth and a wide range of plant degrading enzymes. Anaerobic fungi can also indirectly improve feed particle degradation by increasing the surface area available for bacteria to colonise. Despite this, anaerobic fungi have been largely overlooked in equine gut microbiology studies to date.
In order to inform future novel approaches to increase fibre degradation, an equine anaerobic fungal knowledge base was developed by: (i) determination of the anaerobic fungal taxa present in equines and (ii) assessment of how anaerobic fungi are affected by equine type, (iii) diet and (iv) hindgut region.
An anaerobic fungal survey of equines was performed (i), with faecal samples collected from over 70 equines and analysed using methodology that was developed and validated within the project. Animals sampled included domesticated equines (horses, ponies, donkeys, mules and hinnies) as well as zebras kept under managed conditions. Anaerobic fungi were detected in all animals, but their faecal concentrations were approx. six-fold higher in donkeys compared to horses/ponies and zebras, with mules/hinnies being intermediate of the parent equine types (i.e. donkeys and horses/ponies). No anaerobic fungal taxon was found that was common to all equines, with Caecomyces being predominant in donkeys and mules/hinnies and an uncultivated anaerobic fungal genus AL1 that was found to be predominant in horses/ponies and zebras.
As the diet of the animals was not controlled in the survey, a study was performed where faecal microbiota was collected from ponies, donkeys and mules/hinnies after four weeks of being fed the same diet (ii). In contrast to the previous findings, anaerobic fungal concentrations were not affected by equine type, and Caecomyces was predominant in all the animals. However, the uncultivated genus SK3 was present only in donkeys, and anaerobic fungal diversity was also highest in donkeys. This is consistent with previous reports that dietary fibre degradation in donkeys was more complete compared to that of horses/ponies.
Due to the different findings of the two studies regarding anaerobic fungal concentrations, a third study was conducted where the effect of seasonal differences in diet on faecal anaerobic fungal concentrations was assessed (iii). Faecal samples were collected from a group of donkeys that were sampled during summer (pasture/straw) and winter (haylage/straw) periods over two consecutive years. Interestingly, anaerobic fungal concentrations differed between both years for the summer diet, but not for the winter diet, warranting further analysis as well as more extensive and longer surveys in the future. Determination of the forage consumed by the animals, using an alkane marker technique, is currently in progress, which will provide additional insight into the findings.
Faecal samples have been widely used in the project, however, how representative they are of anaerobic fungal community composition along the hindgut is not known. A collaborative study found large differences in both anaerobic fungal community composition and concentrations along the hindgut of a horse (iv). Following up on this, an in vitro study was performed using horse gut content collected from three different hindgut sites in order to assess anaerobic fungal fermentation of forage along the equine hindgut. An antimicrobial treatment was used to enrich anaerobic fungi from each of the three sites. 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.
Three manuscripts have been submitted for peer-review (i & ii), with two other papers planned after data analyses have been finalized (iii & iv). As a result of collaborative activities, two papers have also been published and a further two are planned. Study findings and a congress paper on equine anaerobic fungi have been presented at three international equine congresses, where delegates included equine professionals as well as researchers. Outreach activities have also brought anaerobic fungal information to the general public via a microbial exhibit at the Artis Micropia museum in Amsterdam (NL) and three anaerobic fungal videos and a project video that were shared on YouTube and other social media. Information about the project activities and outputs have also been regularly disseminated via the project website. No exploitation activities were conducted within the duration of the project.
As the diet of the animals was not controlled in the survey, a study was performed where faecal microbiota was collected from ponies, donkeys and mules/hinnies after four weeks of being fed the same diet (ii). In contrast to the previous findings, anaerobic fungal concentrations were not affected by equine type, and Caecomyces was predominant in all the animals. However, the uncultivated genus SK3 was present only in donkeys, and anaerobic fungal diversity was also highest in donkeys. This is consistent with previous reports that dietary fibre degradation in donkeys was more complete compared to that of horses/ponies.
Due to the different findings of the two studies regarding anaerobic fungal concentrations, a third study was conducted where the effect of seasonal differences in diet on faecal anaerobic fungal concentrations was assessed (iii). Faecal samples were collected from a group of donkeys that were sampled during summer (pasture/straw) and winter (haylage/straw) periods over two consecutive years. Interestingly, anaerobic fungal concentrations differed between both years for the summer diet, but not for the winter diet, warranting further analysis as well as more extensive and longer surveys in the future. Determination of the forage consumed by the animals, using an alkane marker technique, is currently in progress, which will provide additional insight into the findings.
Faecal samples have been widely used in the project, however, how representative they are of anaerobic fungal community composition along the hindgut is not known. A collaborative study found large differences in both anaerobic fungal community composition and concentrations along the hindgut of a horse (iv). Following up on this, an in vitro study was performed using horse gut content collected from three different hindgut sites in order to assess anaerobic fungal fermentation of forage along the equine hindgut. An antimicrobial treatment was used to enrich anaerobic fungi from each of the three sites. 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.
Three manuscripts have been submitted for peer-review (i & ii), with two other papers planned after data analyses have been finalized (iii & iv). As a result of collaborative activities, two papers have also been published and a further two are planned. Study findings and a congress paper on equine anaerobic fungi have been presented at three international equine congresses, where delegates included equine professionals as well as researchers. Outreach activities have also brought anaerobic fungal information to the general public via a microbial exhibit at the Artis Micropia museum in Amsterdam (NL) and three anaerobic fungal videos and a project video that were shared on YouTube and other social media. Information about the project activities and outputs have also been regularly disseminated via the project website. No exploitation activities were conducted within the duration of the project.
The scientific results of 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 information highlights the potential value of anaerobic fungi in terms of improving fibre degradation in the equine hindgut. Following these first steps, 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 fed microbial. This will 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. In conclusion, the EQUIANFUN project has laid the foundations of a way to optimise fibre utilization in equines which can beneficially impact the health and welfare of all domesticated equines, and the societies and industries that rely on them.