A comparative evaluation of the microbiome effects on easy and hard keeper horses

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University of Delaware
Horses with different metabolic tendencies are anecdotally referred to as “easy” or “hard” keepers. Easy keepers (EK) tend to gain weight easily while hard keepers (HK) require extra feed to maintain body condition. Horses that do not struggle with maintaining a healthy weight are referred to as “medium keepers” (MK). The horse, as an obligate herbivore, relies on the gut microbiome to provide more than half of its energy requirements. Therefore, equine energetics and metabolism is greatly influenced by the gut microbiome. It is not yet known what causes a horse to be an EK, MK, or HK but, I hypothesize that the gut microbial structure and function play a vital role in equine metabolic tendencies. The dynamic interactions between the horse and its gut microbiome likely reflect individual capacities and genetics to harbor specific populations as well as host-specific abilities to utilize available nutrients. To test this central hypothesis regarding the microbial side of the conversation, these research projects focus on the bacterial and protozoal fractions. ☐ The first objective of this work was to develop a reliable and standardized tool for determining equine keeper status. The lack of a standardized method to identify equine keeper status requires reliance on the owner reported keeper statuses which is unreliable and irreproducible. The Equine Keeper Status Scale (EKSS) was developed and validated on data gathered from 240 horses. With EKSS assignments, incorrect keeper status assignments provided by owners was reduced by 60%. The EKSS was used in all further studies to identify EK, MK, and HK study cohorts. ☐ The second objective of the project was to compare bacterial composition (16S rRNA surveys) of EKSS statuses. 16S rRNA surveys of equine feces in an observational study (n=73) found differences in alpha and beta diversities and taxa abundances based on EKSS assignments. However, when a controlled cohort (n=12) was used, significances in alpha and beta diversities were lost, but unique bacterial cores and representative bacteria of each EKSS status were found. Determining the bacterial core of each EKSS status will aid during probiotic choice and probiotic development to target these key groups and improve EK and HK weight management strategies. ☐ The third objective was to compare protozoal composition (18S rRNA surveys) of EKSS statuses. As an extension to this objective, we sought to obtain reference sequences for uncharacterized protozoans to improve molecular methods to identify protozoans. Two previously unsequenced equine protozoan species (Blepharocorys valvata and Blepharoconus benbrooki) and two other equine protozoans (Tripalmaria dogieli, Cochliatoxum periachtum), were successfully single sorted and sequenced. After the addition of these sequences to the classifier, the protozoan (18S rRNA) profile of horses (n=39) in the Mid-Atlantic Region and EKSS statuses were evaluated. Thirty-five species level protozoans were identified in the Mid-Atlantic Region, and protozoal richness was lowest in HK horses compared to EK and MK animals (P = 0.05). Describing the commensal protozoal fraction is the first step towards ultimately understanding this population’s purpose during microbial metabolism and host health. ☐ The fourth objective was to determine if bacterial activities differed between EKSS statuses. Bacterial functionality between EKSS statuses was tested using PICRUSt to hypothesize fermentative differences between the gut microbiomes, and 48h in vitro challenge protocols. PICRUSt predictions were performed on the 16S rRNA surveys from the observational study (n=73) and found overall, 18 metabolism pathways were differentially abundant in EKSS statuses (P < 0.10); seven of which were significantly enriched in the EK representing both foregut metabolism (i.e. starch and lipid digestion), and hindgut metabolism (i.e. fiber and amino acid digestion). PICRUSt is inherently limited but these results indicate that the EK has an enhanced metabolic potential to breakdown feed and harvest energy compared to the MK and HK. ☐ In vitro experiments (n=12) demonstrated that the HK microbiome had the quickest and most drastic reaction to both carbohydrate and protein challenges. The slower metabolic response demonstrated by EK cultures may be a resiliency mechanism that these communities utilize to slow overall metabolism while increasing ATP production. These different bacterial responses suggest unique microbial strategies between keeper communities to maintain stability that may ultimately change metabolism patterns. ☐ In conclusion, the data collected from these studies supports our central hypothesis that the microbiome plays a pivotal role in equine keeper status. Our results suggest that bacterial activity and functionality are more influential towards equine keeper status than the bacterial composition. Results further suggest that the protozoan population is a significant contributor in equine keeper status and this population deserves further investigation.
Equine, In vitro, Metabolism, Microbiome, Next generation sequencing, Protozoan