Development of a body condition index to estimate adiposity in ponies and horses from morphometric measurements.

BACKGROUND: There is a high prevalence of obesity in ponies and pleasure horses. This may be associated with equine metabolic syndrome and an increased risk of laminitis. Body condition scoring (BCS) systems are widely used but are subjective and not very sensitive. OBJECTIVES: To derive a body condition index (BCI), based on objective morphometric measurements, that correlates with % body fat. STUDY DESIGN: Retrospective cohort study. METHODS: Morphometric measurements were obtained from 21 ponies and horses in obese and moderate body condition. Percentage body fat was determined using the deuterium dilution method and the BCI was derived to give the optimal correlation with body fat, applying appropriate weightings. The index was then validated by assessing inter-observer variation and correlation with % body fat in a separate population of Welsh ponies; and finally, the correlation between BCI and BCS was evaluated in larger populations from studies undertaken in Australia, the United Kingdom and the United States. RESULTS: The BCI correlated well with adiposity in the ponies and horses, giving a Pearson r value of 0.74 (P < 0.001); however, it was found to slightly overestimate the % body fat in leaner animals and underestimate in more obese animals. In field studies, the correlation between BCI and BCS varied particularly in Shetlands and miniature ponies, presumably due to differences in body shape. MAIN LIMITATIONS: Further work may be required to adapt the BCI to a method that is more applicable for Shetlands and miniature ponies. CONCLUSIONS: This BCI was able to provide an index of adiposity which compared favourably with condition scoring in terms of accuracy of estimating adiposity; and was more consistent and repeatable when used by inexperienced assessors. Therefore, this may be a useful tool for assessing adiposity; and may be more sensitive than condition scoring for tracking weight gain or weight loss in individual animals.

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

The equine faecal microbiota is often assessed as a proxy of the microbial community in the distal colon, where the microbiome has been linked to states of health and disease in the horse. However, the microbial community structure may change over time if samples are not adequately preserved. This study stored equine faecal samples from n = 10 horses in four preservation treatments at room temperature for up to 150 h and assessed the resulting impact on microbial diversity and the differential abundance of taxa. Treatments included "COLD" (samples packaged with a cool pack), "CLX" (2% chlorhexidine digluconate solution), "NAP" (nucleic acid preservation buffer), and "FTA" (Whatman FTA™ cards). The samples were assessed using 16S rRNA gene sequencing after storage for 0, 24, 72, and 150 h at room temperature under the different treatments. The results showed effective preservation of diversity and community structure with NAP buffer but lower diversity (p = 0.001) and the under-representation of Fibrobacterota in the FTA card samples. The NAP treatment inhibited the overgrowth of bloom taxa that occurred by 72 h at room temperature. The COLD, CLX, and NAP treatments were effective in preserving the faecal microbiota for up to 24 h at room temperature, and the CLX and NAP treatments improved the yield of Patescibacteria and Fibrobacterota in some cases. The cold and CLX treatments were ineffective in preventing community shifts that occurred by 72 h at room temperature. These findings demonstrate the suitability of the COLD, NAP, and CLX treatments for the room temperature storage of equine faeces for up to 24 h and of NAP buffer for up to 150 h prior to processing.

Confidence does not mediate a relationship between owner experience and likelihood of using weight management approaches for native ponies.

Native ponies are at increased risk of obesity and metabolic perturbations, such as insulin dysregulation (ID), a key risk factor for endocrinopathic laminitis. Management and feeding practices can be adapted to maintain healthy body condition and support metabolic health, but owners may inadvertently provide their ponies with inappropriate management leading to obesity and exacerbating risk of metabolic disease. Adoption of preventative weight management approaches (WMAs), including regular monitoring of body condition, providing appropriate preserved forage, promoting seasonal weight loss, and using exercise accordingly, are key in supporting native ponies' metabolic health. The factors influencing the adoption of WMAs, such as owners' experience and confidence, require exploration. The aim of the current study was to understand factors influencing owners' likelihood to undertake certain WMAs, to develop our understanding of suitable intervention targets. A total of 571 responses to an online cross-sectional questionnaire were analysed. Mediation analysis revealed that whilst long term (≥20 years) experience caring for native ponies was associated with owners increased, self-reported confidence in identifying disease and managing their native ponies, this did not translate to an increased likelihood of implementing WMAs. Conversely, respondents who managed ponies with dietary requirements related to obesity, laminitis, or equine metabolic syndrome were more likely to use WMAs related to feeding, seasonal weight management and exercise. Owner confidence was assessed and rejected as a mediator of the relationship between experience and WMA use. These results highlight the need for further work that elucidates the pathways leading owners to undertake action against obesity without the need for ponies to develop overt disease, as well as suggesting a need for long term managers of native ponies to update management practices with preventative care as the focus.

Effect of age and the individual on the gastrointestinal bacteriome of ponies fed a high-starch diet.

Bacteria residing in the gastrointestinal tract of mammals are crucial for the digestion of dietary nutrients. Bacterial community composition is modified by age and diet in other species. Although horses are adapted to consuming fibre-based diets, high-energy, often high-starch containing feeds are increasingly used. The current study assessed the impact of age on the faecal bacteriome of ponies transitioning from a hay-based diet to a high-starch diet. Over two years, 23 Welsh Section A pony mares were evaluated (Controls, 5-15 years, n = 6/year, 12 in total; Aged, ≥19 years, n = 6 Year 1; n = 5 Year 2, 11 in total). Across the same 30-week (May to November) period in each year, animals were randomly assigned to a 5-week period of study and were individually fed the same hay to maintenance (2% body mass as daily dry matter intake) for 4-weeks. During the final week, 2g starch per kg body mass (micronized steam-flaked barley) was incorporated into the diet (3-day transition and 5 days at maximum). Faecal samples were collected for 11 days (final 3 days hay and 8 days hay + barley feeding). Bacterial communities were determined using Ion Torrent Sequencing of amplified V1-V2 hypervariable regions of 16S rRNA. Age had a minimal effect on the bacteriome response to diet. The dietary transition increased Candidatus Saccharibacteria and Firmicutes phyla abundance and reduced Fibrobactres abundance. At the genera level, Streptococcus abundance was increased but not consistently across individual animals. Bacterial diversity was reduced during dietary transition in Streptococcus 'responders'. Faecal pH and VFA concentrations were modified by diet but considerable inter-individual variation was present. The current study describes compositional changes in the faecal bacteriome associated with the transition from a fibre-based to a high-starch diet in ponies and emphasises the individual nature of dietary responses, which may reflect functional differences in the bacterial populations present in the hindgut.

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.

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-abdominal-retroperitoneal 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, breed-type, 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.

Nutritional Management of the Older Horse.

Leisure animals now comprise the majority of working horses in industrialized nations; a shift that has decreased workloads yet improved veterinary care and lifetime health. Although many horses now progress well into their 20s without any requirement for dietary modification, age-related changes are insidious, and older animals benefit from regular veterinary monitoring to identify, address, and ameliorate the inevitable onset of age-related "disease." Basal metabolic rate decreases with age; older animals expend less energy on controlled exercise, and there can be an increased propensity toward the development of obesity, which needs to be recognized and managed.

Considerations for the use of restricted, soaked grass hay diets to promote weight loss in the management of equine metabolic syndrome and obesity.

The addition of hay soaking to current nutritional advice for weight loss management for equine obesity lacks clinical evidence. Twelve overweight/obese horses and ponies were used to test the hypothesis that feeding soaked hay at 1.25% of body mass (BM) daily as dry matter (DM) before soaking would elicit weight losses within the target 0.5-1.0% of BM weekly. Six animals were used to evaluate the impact of nutrient-leaching on the digestibility and daily intakes of dietary energy and nutrients. Soaked hay DM was corrected in accordance with the 'insoluble' ADF content of fresh and soaked hays. The ADF-based method was validated using a test-soaking protocol. Animals fed soaked hay for 6 weeks lost 0.98 ± 0.10% of BM weekly. The most weight loss sensitive animal lost ~2% of BM weekly. Soaking hay did not alter DM gross energy concentrations, incurred losses of water soluble carbohydrates (WSC) and ash and increased acid detergent fibre (ADF) concentrations. Digestibilities of GE, DM, ash and WSC were unaltered but soaking increased uncorrected values for crude protein (+12%) and ADF (+13.5%) digestibility. Corrected DM provision was only 1% of BM daily, providing 64% of maintenance DE requirements, a 23.5% increase in the intended magnitude of energy restriction. Hay soaking leached nutrients, reduced DM and DE provision and was associated with accelerated weight losses over those expected had fresh-hay been fed to the same level. The ADF-based method will allow the predictive evaluation of individual hays to direct feeding management and prevent inadvertently severe DM and energy restriction.

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 R2 = 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.

Post-mortem stability of RNA in skeletal muscle and adipose tissue and the tissue-specific expression of myostatin, perilipin and associated factors in the horse.

Obesity, a major concern for equine welfare, is highly prevalent in the leisure horse population. Skeletal-muscle and adipose tissues are important determinants of maintenance energy requirements. The myostatin and perilipin pathways play key roles in the regulation of muscle mass and lipolysis respectively and have both been associated with obesity predisposition in other mammalian species. High quality samples, suitable for molecular biology, are an essential prerequisite for detailed investigations of gene and protein expression. Hence, this study has evaluated a) the post-mortem stability of RNA extracted from skeletal-muscle and adipose-tissues collected under commercial conditions and b) the tissue-specific presence of myostatin, the moystatin receptor (activin receptor IIB, ActRIIB), follistatin and perilipin, genes and proteins across a range of equine tissues. Objectives were addressed using tissues from 7 Thoroughbred horses presented for slaughter at a commercial abattoir; a) samples were collected at 7 time-points from Masseter muscle and perirenal adipose from 5 minutes to 6 hours post-mortem. Extracted RN was appraised by Optical Density analysis and agarose-gel electrophoresis. b) Quantitative real time PCR and Western Blotting were used to evaluate gene and protein expression in anatomically-defined samples collected from 17 tissues (6 organs, 4 skeletal muscles and 7 discrete adipose depots). The results indicate that, under the present collection conditions, intact, good quality RNA could be extracted from skeletal-muscle for up to 2 hours post-mortem. However, RNA from adipose tissue may be more susceptible to degradation/contamination and samples should be collected no later than 30 minutes post-mortem. The data also show that myostatin and ActRIIB genes and proteins were almost exclusively expressed in skeletal muscle. The follistatin gene showed a more diverse gene expression profile, with expression evident in several organs, adipose tissue depots and skeletal muscles. Perilipin gene and protein were almost exclusively expressed by adipose tissue.

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.

Weight loss resistance: a further consideration for the nutritional management of obese Equidae.

Evidence-based, weight loss management advice is required to address equine obesity. Changes in body mass (BM), body condition score (BCS), heart (HG) and belly circumference (BG), direct (ultrasonographic) and indirect (D(2)O dilution, bioelectrical impedance analysis [BIA]) measures of body fat as well as indices of insulin resistance (IR) were monitored in 12 overweight (BCS ≥ 7/9) horses and ponies of mixed breed and gender for 16 weeks. Animals were randomly assigned to two groups (Group 1, n=6, BCS 7.6/9 ± 0.6, 489 ± 184.6 kg; Group 2, n=6, BCS 8.1/9 ± 0.6, 479 ± 191.5 kg). Daily dry matter intake (DMI) was restricted to 1.25% BM as one of two, near-isocaloric (DE ∼0.115 MJ/kg BM/day), forage-based diets (Group 1, 0.8% BM chaff-based feed: 0.45% BM hay; Group 2, 1.15% BM hay: 0.1% BM nutrient-balancer). Statistical modelling revealed considerable between-animal heterogeneity in proportional weight losses (0.16-0.55% of Week 1 BM weekly). The magnitude of weight loss resistance (WLR) or sensitivity to dietary restriction was independent of diet or any measured outset variable and was largely (65%) attributed to animal identity. Predicted rates of weight loss decreased over time. BCS and BIA were poor estimates of D(2)O-derived body fat%. Reciprocal changes in depths of retroperitoneal and subcutaneous adipose tissues were evident. Changes in BG were associated with losses in retroperitoneal fat and BM (r(2), 0.67 and 0.79). Indices of IR improved for 9/12 animals by Week 16. For obese animals, weight loss should be initiated by restricting forage DMI to 1.25% BM. Subsequent restriction to 1% BM may be warranted for WLR animals.

Body condition scoring as a predictor of body fat in horses and ponies.

Body condition scoring systems were originally developed to quantify flesh cover in food animals and are commonly used to evaluate body fat in Equidae. The relationship between concurrent estimates of body fat content (eTBF%, deuterium oxide dilution; range, 2.7-35.6%) and subjective appraisals of body 'fatness' (body condition score, BCS; range, 1.25-9/9), was investigated in 77 mature horses and ponies. Univariate (UVM, r(2)=0.79) and multivariable (MVM, r(2)=0.86) linear regression models described the association, where BCS and eTBF% were explanatory and outcome variables, respectively. Other measures (age, sex, breed, body mass, ultrasound-generated subcutaneous and abdominal retroperitoneal fat depths, withers height, heart and belly circumferences) were considered as potential confounders but only height, belly circumference and retroperitoneal fat depth remained in the final MVM. The association between BCS and eTBF% was logarithmic. Appraisal of the transformed regression (UVM), actual eTBF% values and 95%CIs of the model forecast, suggested that the power of log-transformed BCS as a predictor of eTBF% decreased as BCS increased. The receiver operating characteristic curve for the prediction of horses with an eTBF% of >20%, suggested that the UVM correctly classified 76% of horses using a 'cut-off' of BCS 6.83/9 (sensitivity, 82.5%; specificity, 70.8%). Negative values for eTBF% were obtained for two thin ponies which were excluded from analyses, and caution is advised in the application of deuterium dilution methodologies where perturbed tissue hydration could be predicted. The data suggest that BCS descriptors may warrant further consideration/refinement to establish more clinically-useful, sub-classifications for overweight/obese animals.

Voluntary ingestion of wood shavings by obese horses under dietary restriction.

Dietary restriction for the weight-loss management of obese horses limits the natural trickle-feeding behaviour. During feed restriction, wood shavings are often advised as bedding to prevent dietary supplementation from non-feed sources. Data from twelve overweight/obese horses and ponies of mixed breed and sex, bedded on wood shavings during 16 weeks of feed restriction, were retrospectively evaluated. DM intake (DMI) was restricted to 1.25 % of body mass (BM) daily. Animals were randomly assigned to one of two diets (hay/chaff, n 6; hay/balancer meal, n 6). BM was recorded weekly. Feeding behaviour was recorded by continual observation over 24 h during week 15. The apparent digestibility (gross energy (GE), acid-detergent fibre (ADF) and DM) of feed was determined for all animals by total faecal collection (72 h, week 16). Rates of weight loss were independent of diet type, DM (R(2) 0.15), GE (R(2) 0.20) and ADF digestibilities (R(2) 0.18). Despite similar DMI, faecal DM ranged between 0.52 and 1.16 % of BM daily and was associated with wide ranges in apparent digestibility (GE--11.34 to 53.08 %; ADF--50.37 to 42.83 % and DM 2.14 to 57.32 %), which were improbably low for some animals. Apparent digestibilities were associated with DM output (GE R2 0.96; ADF R(2) 0.99 and DM R(2) 0.99) and time spent feeding (GE R(2) 0.62; DM R(2) 0.61 and ADF R(2) 0.59), indicating that feed intake was supplemented with wood shavings in at least five of the twelve animals. Quantities of wood shavings ingested (negligible to >3.0 kg/d) were back-calculated from predicted feed digestibilities. All animals remained healthy. Implications of 'feed-bulking/energy dilution' for feed-restricted animals need further consideration.

Effects of season and body condition on appetite, body mass and body composition in ad libitum fed pony mares.

Changes in appetite, body mass (BM), body condition score (BCS), direct (ultrasonographic) and indirect (deuterium oxide dilution technique) measures of body fat were monitored in Welsh Mountain pony mares (n=11, 5-19 years of age) offered ad libitum access to a complete diet (gross energy 16.9±0.07 MJ/kg dry matter) for 12 weeks during summer (n=6; 246±20 kg) and winter (n=5; 219±21 kg). At the outset, each group comprised two thin (BCS 1-3/9), moderate (BCS 4-6/9) and obese (BCS 7-9/9) animals. For ponies that were non-obese at the outset, BM was gained more rapidly (P=0.001) in summer (0.8±0.1 kg/day) than winter (0.6±0.0 kg/day). This was associated with a seasonal increase in dry matter intake (DMI) which became maximal (summer, 4.6±0.3% BM as DMI/day; winter, 3.5±0.1% BM as DMI/day) during the second month. The appetite of the obese ponies was half that reported for non-obese animals in the summer and BM remained constant irrespective of season. Body 'fatness' increased progressively for non-obese but not obese ponies. Body fat content was exponentially associated with increasing BCS but BCSs >6 were not useful indicators of actual body fat. Endogenous circannual mechanisms to suppress winter weight gain were insufficient to prevent the development of obesity in ad libitum fed ponies.