2 Evaluation of the microbiome effects of easy and hard keeper horses with the application of the Equine Keeper Status Scale

Abstract

Horses with 2 different metabolic tendencies are referred to as “easy keepers” (EK) or “hard keepers” (HK). These tendencies are anecdotal and often related to the Henneke Body Condition Scoring system. EK tends to be over-conditioned (BCS ≥6) and HK need extra feed to maintain a BCS of 5. “Medium keepers” (MK) can maintain a BCS of 5 easily. This lab group has developed the Equine Keeper Status Scale (EKSS) and the objective of this work is to compare the microbiome composition of the 3 keeper statuses between owner reported keeper statuses (ORKS) and EKSS assignments. Fresh fecal samples from 73 horses were collected for 16S rRNA profiling. ORKS were obtained by a verbal estimate of their horse's keeper status in regard to their metabolic tendencies with no influence of the research team (ORKS: EK, n = 20; MK, n = 26; HK, n = 27). Feed weights and BCS scores of each horse were collected and digestible energy intakes were estimated by FeedXL software (input variables of the EKSS) and EKSS assignments were determined at a later time (EK, n = 31; MK, n = 22; HK, n = 20). QIIME2 (Quantitative Insights Into Microbial Ecology, v. 2020.8) and R was used for data processing and analysis. α diversity Shannon-Weaver measures between ORKS groups were not found ( P = 0.34) but were found after EKSS evaluation ( P = 0.06) and Tukey's pairwise tests found the difference between EK and MK to be the most different ( P < 0.05). β diversity weighted Unifrac measures of ORKS found group centroids were different ( P = 0.07) but it wasn't due to ORKS ( P = 0.58); while EKSS evaluation found different centroids due to EKSS ( P = 0.02, P = 0.03, respectively). Spearman correlation testing identified bacteria correlated with every keeper status except ORKS MK which is likely due to the inconsistencies of ORKS. Differential abundance testing performed with ANCOM identified 4 phyla differences (Planctomycetes, Euryarchaeota, Spirochaetes, Proteobacteria) between EKSS groups but not between ORKS. Planctomycetes thrive in low nutrient environments and are most abundant in EK. HK appears to have optimized plant fermentation characteristics due to higher abundances of the methane producers (Euryarcheaota), and denitrifying (Spirochaetes), and N oxidizers (Proteobacteria). The EKSS removes the objectivity and reliance on ORKS and with the more clearly defined EKSS keeper groups we demonstrate we are able to identify structural and functional differences in the microbiomes of EK and HK horses and how it relates to metabolism.