EQUIFAT: A novel scoring system for the semi-quantitative evaluation of regional adipose tissues in Equidae

Anatomically distinct adipose tissues represent variable risks to metabolic health in man and some other mammals. Quantitative-imaging of internal adipose depots is problematic in large animals and associations between regional adiposity and health are poorly understood. This study aimed to develop and test a semi-quantitative system (EQUIFAT) which could be applied to regional adipose tissues. Anatomically-defined, photographic images of adipose depots (omental, mesenteric, epicardial, rump) were collected from 38 animals immediately post-mortem. Images were ranked and depot-specific descriptors were developed (1 = no fat visible; 5 = excessive fat present). Nuchal-crest and ventro-abdominalretroperitoneal adipose depot depths (cm) were transformed to categorical 5 point scores. The repeatability and reliability of EQUIFAT was independently tested by 24 observers. When half scores were permitted, inter-observer agreement was substantial (average κw: mesenteric, 0.79; omental, 0.79; rump 0.61) or moderate (average κw; epicardial, 0.60). Intra-observer repeatability was tested by 8 observers on 2 occasions. Kappa analysis indicated perfect (omental and mesenteric) and substantial agreement (epicardial and rump) between attempts. A further 207 animals were evaluated ante-mortem (age, height, breedtype, gender, body condition score [BCS]) and again immediately post-mortem (EQUIFAT scores, carcass weight). Multivariable, random effect linear regression models were fitted (breed as random effect; BCS as outcome variable). Only height, carcass weight, omental and retroperitoneal EQUIFAT scores remained as explanatory variables in the final model. The EQUIFAT scores developed here demonstrate clear functional differences between regional adipose depots and future studies could be directed towards describing associations between adiposity and disease risk in surgical and post-mortem situations

Preliminary Investigation into a Potential Role for Myostatin and Its Receptor (ActRIIB) in Lean and Obese Horses and Ponies

Obesity is a widespread problem across the leisure population of horses and ponies in industrialised nations. Skeletal muscle is a major contributor to whole body resting energy requirements and communicates with other tissues through the secretion of myokines into the circulation. Myostatin, a myokine and negative regulator of skeletal muscle mass, has been implicated in obesity development in other species. This study evaluated gene and protein expression of myostatin and its receptor, ActRIIB in adipose tissues and skeletal muscles and serum myostatin concentrations in six lean and six obese animals to explore putative associations between these factors and obesity in horses and ponies. Myostatin mRNA expression was increased while ActRIIB mRNA was decreased in skeletal muscles of obese animals but these differences were absent at the protein level. Myostatin mRNA was increased in crest fat of obese animals but neither myostatin nor ActRIIB proteins were detected in this tissue. Mean circulating myostatin concentrations were significantly higher in obese than in lean groups; 4.98 ng/ml (±2.71) and 9.00 ng/ml (±2.04) for the lean and obese groups, respectively. In addition, there was a significant positive association between these levels and myostatin gene expression in skeletal muscles (average R(2) = 0.58; p<0.05). Together, these results provide further basis for the speculation that myostatin and its receptor may play a role in obesity in horses and ponies

The Equine Gastrointestinal Microbiome:Impacts of Age and Obesity

Gastrointestinal microbial communities are increasingly being implicated in host susceptibilities to nutritional/metabolic diseases; such conditions are more prevalent in obese and/or older horses. This controlled study evaluated associations between host-phenotype and the fecal microbiome / metabolome. Thirty-five, Welsh Mountain pony mares were studied across 2 years (Controls, n = 6/year, 5–15 years, Body Condition Score (BCS) 4.5–6/9; Obese, n = 6/year, 5–15 years, BCS > 7/9; Aged, n = 6 Year 1; n = 5 Year 2, ≥19 years old). Animals were individually fed the same hay to maintenance (2% body mass as daily dry matter intake) for 2 (aged / obese) or 4 (control), 4-week periods in a randomized study. Outset phenotype was determined (body fat%, markers of insulin sensitivity). Feces were sampled on the final 3 days of hay feeding-periods and communities determined using Next Generation Sequencing of amplified V1–V2 hypervariable regions of bacterial 16S rRNA. Copy numbers for fecal bacteria, protozoa and fungi were similar across groups, whilst bacterial diversity was increased in the obese group. Dominant bacterial phyla in all groups were Bacteroidetes > Firmicutes > Fibrobacter. Significant differences in the bacterial communities of feces were detected between host-phenotype groups. Relative to controls, abundances of Proteobacteria were increased for aged animals and Bacteroidetes, Firmicutes, and Actinobacteria were increased for obese animals. Over 500 bacterial operational taxonomic units (OTUs) differed significantly between host-phenotype groups. No consistent pattern of changes in discriminant OTUs between groups were maintained across groups and between years. The core bacterial populations contained 21 OTUs, 6.7% of recovered sequences. Distance-based Redundancy Analyses separated fecal bacterial communities with respect to markers of obesity and insulin dysregulation, as opposed to age. Host-phenotype had no impact on the apparent digestibility of dietary GE or DM, fecal volatile fatty acid concentrations or the fecal metabolome (FT-IR). The current study demonstrates that host-phenotype has major effects on equine fecal microbial population structure. Changes were predominantly associated with the obese state, confirming an obesity-associated impact in the absence of nutritional differences. Clear biomarkers of animal-phenotype were not identified within either the fecal microbiome or metabolome, suggesting functional redundancy within the gut microbiome and/or metabolome.</p

Homemade Nucleic Acid Preservation Buffer Proves Effective in Preserving the Equine Faecal Microbiota over Time at Ambient Temperatures

Funding This research was funded by Mars Petcare UK and the Scottish Funding Council Research Excellence Grant (REG). Authors WR and MN receive salary support from the Rural and Environmental Sciences and Analytical Services Division (RESAS).Peer reviewedPublisher PD

Strong Stability and Host Specific Bacterial Community in Faeces of Ponies

The horse, as a hindgut fermenter, is reliant on its intestinal bacterial population for efficient diet utilisation. However, sudden disturbance of this population can result in severe colic or laminitis, both of which may require euthanasia. This study therefore aimed to determine the temporal stability of the bacterial population of faecal samples from six ponies maintained on a formulated high fibre diet. Bacterial 16S rRNA terminal restriction fragment length polymorphism (TRFLP) analyses of 10 faecal samples collected from 6 ponies at regular intervals over 72 hour trial periods identified a significant pony-specific profile (P<0.001) with strong stability. Within each pony, a significantly different population was found after 11 weeks on the same diet (P<0.001) and with greater intra-individual similarity. Total short chain fatty acid (SCFA) concentration increased in all ponies, but other changes (such as bacterial population diversity measures, individual major SCFA concentration) were significant and dependent on the individual. This study is the first to report the extent of stability of microbes resident in the intestinal tract as represented with such depth and frequency of faecal sampling. In doing so, this provides a baseline from which future trials can be planned and the extent to which results may be interpreted

Distribution of physical attributes and EQUIFAT scores in the population of animals used to describe associations between EQUIFAT scores and BCS (<i>n</i> = 207).

<p>Histograms were constructed with normal distribution overlaid for age (A), BCS (B), withers height (C), omental fat score (D), mesenteric fat score (E), epicardial fat score (F), retroperitoneal fat score (G), rump fat score (H), and crest fat score (I).</p

Example EQUIFAT scoring system for mesenteric adipose depot.

<p>The EQUIFAT scoring system was developed from the ranking of anatomically-defined depot-specific photographic images in order of increasing visually apparent adiposity. Detailed descriptors for each score (1–5) were generated and representative images are provided to aid in the use of the scoring system.</p

Associations between EQUIFAT scores and BCS.

<p>Associations between EQUIFAT scores and BCS.</p

Marginal mean plots illustrating predicted changes in BCS with retroperitoneal and omental EQUIFAT scores.

<p>Marginal mean plots were created from the final multivariable model 1 to demonstrate predicted changes in BCS with increasing retroperitoneal and omental EQUIFAT scores. Error bars signify 95% confidence intervals.</p

Inter-observer agreement of the EQUIFAT scores.

<p>Inter-observer agreement of the EQUIFAT scores.</p