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Feline obesity elicits a plethora of metabolic responses leading to comorbidities, with potential reversal during weight loss. The specific metabolic alterations and biomarkers of organ dysfunction are not entirely understood. Untargeted, high-throughput metabolomic technologies may allow the identification of biological components that change with weight status in cats, increasing our understanding of feline metabolism. The objective of this study was to utilize untargeted metabolomic techniques to identify biomarkers and gain mechanistic insight into the serum metabolite changes associated with reduced food intake and weight loss in overweight cats. During a four-wk baseline period, cats were fed to maintain body weight. For 18 wk following baseline, cats were fed to lose weight at a rate of ~1.5% body weight/wk. Blood serum metabolites were measured at wk 0, 1, 2, 4, 8, 12, and 16. A total of 535 named metabolites were identified, with up to 269 of them being altered (p- and q- values &lt, 0.05) at any time point. A principal component analysis showed a continual shift in metabolite profile as weight loss progressed, with early changes being distinct from those over the long term. The majority of lipid metabolites decreased with weight loss, however, ketone bodies and small lipid particles increased with weight loss. The majority of carbohydrate metabolites decreased with weight loss. Protein metabolites had a variable result, with some increasing, but others decreasing with weight loss. Metabolic mediators of inflammation, oxidative stress, xenobiotics, and insulin resistance decreased with weight loss. In conclusion, global metabolomics identified biomarkers of reduced food intake and weight loss in cats, including decreased markers of inflammation and/or altered macronutrient metabolism.

The objective of the present study was to evaluate the changes in blood metabolites, AA profile, and oxidative stress markers in American Foxhound dogs fed a nutrient-fortified endurance diet while undergoing unstructured endurance exercise over several months. Thirty-six adult American Foxhound dogs (mean age: 4.5, range 2 to 10 yr and mean BW: 34.7, range: 23.1 to 46.9 kg) were selected to participate in the study. Prior to the study, all dogs consumed a commercial diet for 16 wk. After collecting baseline blood samples, dogs were assigned to a standard commercial performance diet (control) or a nutrient-fortified dog food (test). Dogs were balanced by gender, age, body weight, and athletic performance between diets. During the study, dogs underwent 78 bouts of exercise, with approximately 22 km/bout. Blood samples were collected after 40, 75, 138, and 201 d on study (October 2012 to March 2013). All blood metabolites were similar at baseline and serum chemistry profile remained within normal ranges throughout the study. Over time, plasma taurine and vitamin E concentrations decreased (P < 0.05) in dogs fed the control diet but were maintained or increased (P < 0.05) in dogs fed the treatment diet. Also, plasma creatinine and triglycerides were lower (P < 0.05) and blood phosphorus and alkaline phosphatase were higher (P < 0.05) in dogs fed the treatment diet. Vitamin E and taurine status of dogs appear to be affected by extended endurance exercise. These data suggest dogs undergoing endurance exercise may benefit from supplementation of vitamin E and taurine to minimize oxidation and maintain taurine status.

OBJECTIVE To determine effects of restriction feeding of a moderate-protein, high-fiber diet on loss of body weight (BW), voluntary physical activity, body composition, and fecal microbiota of overweight cats. ANIMALS 8 neutered male adult cats. PROCEDURES After BW maintenance for 4 weeks (week 0 = last week of baseline period), cats were fed to lose approximately 1.5% of BW/wk for 18 weeks. Food intake (daily), BW (twice per week), body condition score (weekly), body composition (every 4 weeks), serum biochemical analysis (weeks 0, 1, 2, 4, 8, 12, and 16), physical activity (every 6 weeks), and fecal microbiota (weeks 0, 1, 2, 4, 8, 12, and 16) were assessed. RESULTS BW, body condition score, serum triglyceride concentration, and body fat mass and percentage decreased significantly over time. Lean mass decreased significantly at weeks 12 and 16. Energy required to maintain BW was 14% less than National Research Council estimates for overweight cats and 16% more than resting energy requirement estimates. Energy required for weight loss was 11% more, 6% less, and 16% less than American Animal Hospital Association recommendations for weight loss (80% of resting energy requirement) at weeks 1 through 4, 5 through 8, and 9 through 18, respectively. Relative abundance of Actinobacteria increased and Bacteroidetes decreased with weight loss. CONCLUSIONS AND CLINICAL RELEVANCE Restricted feeding of a moderate-protein, high-fiber diet appeared to be a safe and effective means for weight loss in cats. Energy requirements for neutered cats may be overestimated and should be reconsidered.

The objective of this experiment was to determine the effects of in vitro fermentation of coconut endosperm fiber (CEF), chicory pulp (CHP), and selective blends of these substrates on SCFA production and changes in microbiota using canine fecal inocula. A total of 6 individual substrates, including short-chain fructooligosaccharide (scFOS; a well-established prebiotic source), pectin (PEC; used as a positive control), pelletized cellulose (PC; used as a negative control), beet pulp (BP; considered the gold standard fiber source in pet foods), CEF, and CHP, and 3 CEF:CHP blends (75:25% CEF:CHP [B1], 50:50% CEF:CHP [B2], and 25:75% CEF:CHP [B3]) were tested. Triplicate samples of each substrate were fermented for 0, 8, and 16 h after inoculation. A significant substrate × time interaction (P < 0.05) was observed for pH change and acetate, propionate, butyrate, and total SCFA concentrations. After 8 and 16 h, pH change was greatest for scFOS (-2.0 and -3.0, respectively) and smallest for PC (0.0 and -0.1, respectively). After 16 h, CEF had a greater butyrate concentration than CHP and all the CEF:CHP blends and it was not different than PEC. The substrate × time interaction was significant for bifidobacteria (P < 0.05) and lactobacilli (P < 0.05). After 8 h, bifidobacteria was greatest for BP and lowest for PC (12.7 and 10.0 log10 cfu/tube, respectively). After 16 h, PC had the lowest and scFOS had the greatest bifidobacteria (6.7 and 13.3 log10 cfu/tube, respectively). In general, CEF, CHP, and their blends had similar bifidobacteria populations after 8 and 16 h of fermentation when compared with BP and scFOS. After 16 h, lactobacilli populations were greatest for B1, B2, B3, BP, and scFOS, intermediate for PEC, and lowest for PC (P < 0.05). Overall, our data suggest that CEF had a butyrogenic effect and that CEF, CHP, and their blends had similar bifidobacteria and lactobacilli populations as popular prebiotic and fiber substrates. Future research should investigate the effects of CEF, CHP, and their blends on gastrointestinal health and fecal quality in dogs.

Objective—To determine whether gonadectomy or age at gonadectomy was associated with the risk that dogs would subsequently become overweight. Design—Retrospective cohort study. Animals—1,930 dogs gonadectomized between 1998 and 2001 at ≤ 6 months of age (n = 782), > 6 months to ≤ 1 year of age (861), or > 1 to ≤ 5 years of age (287) and 1,669 sexually intact dogs. Procedures—Dogs were followed-up through medical records for ≥ 10 years or until a diagnosis of overweight (defined as overweight, obese, or having a body condition score ≥ 4/5) was recorded. Information extracted included age at study entry, sex, breed, breed-size category, hospital visit frequency, and diagnosis (yes or no) of overweight or diseases that might affect body condition. Relative risk of a diagnosis of overweight was assessed among age groups of gonadectomized dogs and between gonadectomized and sexually intact dogs. Results—No difference was detected among dogs grouped according to age at gonadectomy with respect to the risk of being overweight. This risk was significantly greater in gonadectomized dogs than in sexually intact dogs, but only during the first 2 years after gonadectomy. Sexually intact male dogs were approximately 40% less likely to have this diagnosis (hazard ratio, 0.61; 95% confidence interval, 0.52 to 0.72) than were sexually intact female dogs; no difference in risk between the sexes was evident for gonadectomized dogs. Conclusions and Clinical Relevance—Gonadectomized dogs had a greater risk of being overweight than did sexually intact dogs, but this risk was not influenced by age at gonadectomy. Opportunities exist for veterinarians to provide counseling during the first years after gonadectomy to help dogs maintain a healthy weight.

The objective of this study was to determine if seasonal and/or pulsatile variations occur in plasma concentrations of thyrotropin (TSH) and leptin in mares while maintaining a constant energy balance. Blood samples were collected every 20 min during a 24h period in winter and again in summer from six Quarter Horse type mares. Plasma concentrations of TSH, leptin, and T(4) were determined by radioimmunoassay. No differences were observed in body weight between winter (388.1+/-12.5 kg) and summer (406.2+/-12.5 kg; P=0.11). Plasma concentrations of TSH were greater in the summer (2.80+/-0.07 ng/ml) when compared to winter (0.97+/-0.07 ng/ml; P0.001). Pulse frequency of TSH was not different between winter (6.17+/-0.78 pulses/24h) and summer (5.33+/-0.78 pulses/24h; P=0.49). Mean TSH pulse amplitude, pulse area, and area under the curve were all greater in summer compared to winter (3.11+/-0.10 ng/ml versus 1.20+/-0.10 ng/ml, 24.86+/-0.10 ng/ml min versus 13.46+/-1.90 ng/ml min, 3936+/-72.93 ng/ml versus 1284+/-72.93 ng/ml, respectively; P0.01). Mean concentrations of leptin were greater in summer (2.48+/-0.17 ng/ml) compared to winter (0.65+/-0.17 ng/ml; P0.001). Pulsatile secretion patterns of leptin were not observed in any horses during experimentation. Mean concentrations of T(4) were greater in winter (20.3+/-0.4 ng/ml) compared to summer (18.2+/-0.4 ng/ml; P0.001). These seasonal differences between winter and summer provide evidence of possible seasonal regulation of TSH and leptin.

Endocrine characteristics of Quarter Horse-type mares were determined during a 68 h feed deprivation and again in the same mares following surgical thyroidectomy (THX). A crossover experimental design was implemented, in which mares received brome hay available ad libitum (FED) or were food deprived (RES) for 68 h. Blood samples were collected every 20 min for 48 h, beginning 20 h after the onset of food deprivation. Concentrations of triiodothyronine and thyroxine were undetectable post-THX. Plasma concentrations of thyrotropin were greater post-THX versus pre-THX (PP< 0.01). The existence of leptin pulse secretion was found only in post-THX compared with the same horses pre-THX (P = 0.02). We theorize that non-pulsatile secretion of leptin may have contributed to the survival of this species, as it evolved in the regions of seasonal availability of food. Lack of pulsatile secretion of leptin may contribute to the accumulation of energy stores by modulating leptin sensitivity.

Objective—To quantify changes in endothelium-derived factors and relate those changes to various aspects of digital hemodynamics during the prodromal stages of carbohydrate overload (CHO)-induced laminitis in horses. Animals—20 adult horses without abnormalities of the digit. Procedures—Digital and jugular venous blood samples were collected at 1-hour intervals (for assessment of endothelin-1 [ET-1] immunoreactivity and measurement of glucose, insulin, and nitric oxide [NO] concentrations) or 4-hour intervals (CBC and platelet-neutrophil aggregate assessment) for 8 hours or 16 hours after induction of CHO-associated laminitis in horses treated with an ET-1 antagonist. Effects of treatment, collection site, and time and the random effects of horse on each variable were analyzed by use of a repeated-measures model. Where treatment and collection site had no significant effect, data were combined. Results—Compared with baseline values, CHO resulted in changes in several variables, including a significant increase from baseline in digital blood ET-like immunoreactivity at 11 hours; digital blood ET-like immunoreactivity was significantly greater than that in jugular venous blood at 8, 9, 11, and 12 hours. Digital and jugular venous blood concentrations of glucose increased from baseline significantly at 3, 4, and 5 hours; insulin concentration increased significantly at 5 hours; and the number of platelet-neutrophil aggregates increased significantly at 12 hours. Conclusions and Clinical Relevance—In horses, concurrent increases in venous blood ET-1 immunoreactivity, insulin and glucose concentrations, and platelet-neutrophil aggregates support a role of endothelial dysfunction in the pathogenesis of CHO-induced laminitis.

Leptin is a protein hormone produced by adipose tissue that influences hypothalamic mechanisms regulating appetite and energy balance. In species tested thus far, including horses, concentrations of leptin increase as animal fat mass increases. The variables and mechanisms that influence the secretion of leptin are not well known, nor is it known in equine species how the secretion of leptin is influenced by acute alterations in energy balance, circadian patterns, and/or reproductive competence. Our objectives were to determine in horses: 1) whether plasma concentrations of leptin are secreted in a circadian and/or a pulsatile pattern; 2) whether a 48-h period of feed restriction would alter plasma concentrations of leptin, growth hormone, or insulin; and 3) whether ovariectomy and/or a melatonin implant would affect leptin. In Exp. 1, mares exposed to ambient photoperiod of visible light (11 h, 33 min to 11 h, 38 min), received treatments consisting of a 48-h feed restriction (RES) or 48 h of alfalfa hay fed ad libitum (FED). Mares were maintained in a dry lot before sampling and were tethered to a rail during sampling. Analyses revealed that leptin was not secreted in a pulsatile manner, and that mean leptin concentrations were greater (P < 0.001) in FED vs. RES mares (17.20 +/- 0.41 vs. 7.29 +/- 0.41 ng/mL). Plasma growth hormone was pulsatile, and mean concentrations were greater in RES than FED mares (2.15 +/- 0.31 vs. 1.08 +/- 0.31 ng/mL; P = 0.05). Circadian patterns of leptin secretion were observed, but only in FED mares (15.39 +/- 0.58 ng/mL for morning vs. 19.00 +/- 0.58 ng/mL for evening; P < 0.001). In Exp. 2, mares that were ovariectomized or intact received either a s.c. melatonin implant or a sham implant. Thereafter, blood was sampled at weekly intervals at 1000 and 1700. Concentrations of leptin in samples collected at 1700 were greater (P < 0.001) than in those collected at 1000 (28.24 +/- 1.7 vs. 22.07 +/- 1.7 ng/mL). Neither ovariectomy nor chronic treatment with melatonin affected plasma concentrations of leptin or the circadian pattern of secretion. These data provide evidence that plasma leptin concentrations in the equine are sensitive to acute changes in nutritional status and vary in a circadian pattern that is sensitive to fasting but not to melatonin treatment or ovariectomy.

Protracted laminitis and the resultant stress often results in prolonged, elevated cortisol secretion and this may contribute to the persistence and refractoriness of laminitis. Cushing’s disease is a condition in which increased secretion of pituitary pars intermedia-derived pro-opiomelanocortin (POMC) peptides leads to perpetually enhanced adrenal secretion of cortisol (hyperadrenocorticism), the physiological glucocorticoid (GC) in the equine species. The vasoconstrictive responses of equine digital arteries to catecholamines is potentiated by GCs leading to interference with hoof lamellar perfusion. The level of the steroid-transforming enzyme, 11β-hydroxysteroid dehydrogenase type 1 is sometimes elevated in hoof lamellar tissues during laminitis as a result of locally high cortisol concentrations within hoof lamellae. Obesity has been shown to be a significant risk factor for the development of laminitis. Obesity-associated laminitis may be similar to the metabolic syndrome (MS) in obese humans. Development of MS is a risk factor in humans for stroke and atherosclerosis and there may be a similar risk for laminitis in horses. GCs cause insulin resistance (IR) by inhibiting the action of insulin thus promoting the availability of glucose for cells in the CNS and other cells that do not depend on insulin for glucose uptake. GCs may impair lamellar perfusion by direct action on vascular smooth muscle and indirectly by causing IR. Equine hoof lamellar keratinocytes appear to have an exceptionally high glucose requirement and maintenance of the lamellar may rely on glucose delivery to and uptake by these keratinocytes. Chronic IR, characterized by hyperglycemia and hyperinsulinemia, subjects cells that are not dependent on insulin for glucose uptake to relatively high glucose levels over time. Glucotoxic endotheliopathy is characterized by increased production of endothelin-1 and reduced release of NO by endothelial cells and these constricting factors may impair lamellar perfusion and risk of laminitis. Horse owners and veterinarians should recognize that obesity is associated with multiple endocrinological changes that might predispose to laminitis. Feeding high glycemic index rations during long periods of physical inactivity promotes the development of obesity. A diagnosis of metabolic syndrome (MS) should be considered in horses with abnormal body fat distribution and laminitis. Identification of IR represents the most useful clinical approach to diagnosing MS in horses and rations for horses with MS should have a low glycemic index. Diagnosis of pituitary pars intermedia dysfunction (PPID) in teenage (and younger) horses before the development of the “classic” signs of hirsutism and weight loss is being reported more commonly using the e-ACTH criterion. Therefore, veterinary diagnosticians must be careful to differentiate whether obese teenage horses might be affected with either or both IR and PPID before the appearance of the more “classic” Cushing’s phenotype is evident. The extent to which these two endocrinological disturbances are related deserves further investigation.

Obesity has been a major concern in the horse industry for many years, and the recent discovery of leptin and leptin receptors in numerous nonequine species has provided a basis for new approaches to study this problem in equine. The objectives were to: 1) clone a partial sequence of the equine leptin and leptin receptor genes so as to enable the design of primers for RT-PCR determination of leptin and leptin receptor gene presence and distribution in tissues, 2) develop a radioimmunoassay to quantify peripheral concentrations of leptin in equine, 3) determine if peripheral concentrations of leptin correlate with body condition scores in equine, and 4) determine if changing body condition scores would influence peripheral concentrations of leptin in equine. In Experiment 1, equine leptin (GenBank accession number AF179275) and the long-form of the equine leptin receptor (GenBank accession number AF139663) genes were partially sequenced. Equine leptin receptor mRNA was detected in liver, lung, testis, ovary, choroid plexus, hypothalamus, and subcutaneous adipose tissues using RT-PCR. In Experiment 2, 71 horses were categorized by gender, age, and body condition score and blood samples were collected. Sera were assayed for leptin using a heterologous leptin radioimmunoassay developed for equine sera. Serum concentrations of leptin increased in horses with body condition score (1 = thin to 9 = fat; r = 0.64; P = 0.0001). Furthermore, serum concentrations of leptin were greater in geldings and stallions than in mares (P = 0.0002), and tended to increase with age of the animal (P = 0.08). In Experiment 3, blood samples, body weights, and body condition scores were collected every 14 d from 18 pony mares assigned to gain or lose weight over a 14-wk interval based on initial body condition score. Although statistical changes (P = 0.001) in body condition scores were achieved, congruent statistical changes in peripheral concentrations of leptin were not observed, likely due to the small range of change that occurred. Nonetheless, serum concentrations of leptin tended to be greater in fat-restricted mares than in thin-supplemented mares (P = 0.09). We conclude that leptin and leptin receptors are present in equine tissues and that peripheral concentrations of leptin reflect a significant influence of fat mass in equine.

Leptin, a protein hormone secreted by adipocytes, plays an important role in energy homeostasis and regulation of body composition. We previously observed that acute feed restriction resulted in a rapid decline in concentrations of leptin in obese pony mares. This acute response prompted us to characterize the temporal changes in concentrations of leptin, GH, and insulin in obese pony mares during the transition between fed and feed-restricted conditions. Nine obese pony mares of mixed breed, previously maintained on fescue pasture, were randomly allotted to 2 groups. Treatments consisted of a 48-h feed restriction, a 48-h refeeding, and a 24-h feed restriction (RFR; n = 4), or 48 h of alfalfa hay ad libitum, a 48-h feed restriction, and a 24-h refeeding (FRF; n = 5). Blood samples were taken every 15 min during restriction and feeding transitions (0600 to 1400 on d 2 and 4), and every 30 min thereafter until 0830 of the following days (d 3 and 5). In the FRF treatment, plasma concentrations of leptin declined precipitously 6 h after the removal of feed (sample by treatment interaction; P < 0.01), and remained low and unresponsive to refeeding. Similarly, in the RFR group, plasma concentrations of leptin were initially low, and did not respond to feeding during the second (refeeding) sampling period. After feed restriction in each of the 2 treatment sequences, plasma insulin decreased and GH mean concentration, pulse frequency, pulse amplitude, and area under the curve increased (P < 0.05). Refeeding reversed these effects on insulin and GH. These data provide evidence that peripheral concentrations of insulin and GH are dynamically responsive to feed removal (decrease in insulin; increase in GH) and replacement (increase in insulin; decrease in GH), whereas leptin decreases in response to feed restriction but is slow to recover from a transient nutritional insult.

Neuropeptide Y (NPY) provides an important hypothalamic link between nutritional status and neuroendocrine mechanisms regulating growth and reproduction. The objective of the following series of experiments was to determine the effects of single or continuous administration of NPY on secretion of luteinizing hormone (LH) and (or) growth hormone (GH). In experiment 1, four ovariectomized (OVX) ewes and four OVX + estrogen-treated ewes each received, in a 4 x 4 Latin Square arrangement of treatments, a single injection of 0, 0.5, 5, or 50 microg NPY via an intracerebroventricular (i.c.v.) cannulae to determine the effects on secretion of GH. NPY significantly elevated serum GH at the 50 microg dose regardless of estrogen exposure (P = 0.003). In experiment 2, eight OVX ewes were infused i.c.v. with NPY or saline (n = 4/trmt) continuously for 20 h in a linearly increasing dose, ending at 50 microg/h NPY. Blood samples were collected via jugular cannulae every 10 min during hour -4-0 (interval 1, pre-treatment), hour 6-10 (interval 2) and hour 16-20 (interval 3) relative to the initiation of infusion (0 h). Mean LH and LH pulse frequency were lower in NPY- versus saline-infused ewes during intervals 2 and 3 (P0.01), but NPY had no discernable effect on serum GH (P0.10). In experiment 3, four OVX ewes were continuously infused with NPY as in experiment 2, except that the maximum 50 microg/h dose was achieved after only 10 h of infusion. Blood samples were collected every 10 min, beginning 4 h before and continuing until 4h after the NPY infusion. Mean serum LH changed significantly over time (P = 0.0001), decreasing below pre-treatment levels by hour 3 of NPY infusion (P0.01), and returning to pre-treatment concentrations following the end of infusion (P0.15). Serum GH also changed significantly over time (P0.001). Mean GH levels tended to be greater than pre-treatment levels by hour 2 of infusion (P0.08), but thereafter returned to basal levels. Serum GH also increased following the end of NPY infusion (P0.03). From these data we conclude that NPY exerts a persistent inhibitory effect on secretion of LH, and may stimulate the secretion of GH during the initiation and cessation of infusion of NPY. These observations support a role for NPY in mediating the effects of undernutrition on both LH and GH, and also provide evidence for potential mechanisms by which leptin, acting through NPY, may stimulate the secretion of GH.

Leptin is an adipocyte-derived hormone that suppresses feed intake and increases energy expenditure. Leptin is also involved in regulating body temperature. Thus, the presence of leptin in milk, which can be absorbed through the gut of neonates immediately after birth, may aid in the survival of neonates born in cold weather. Our objectives were to determine the temporal relationship between concentrations of leptin in postpartum ewe blood serum and ewe milk serum, and to determine whether ewe blood and milk serum leptin concentrations were correlated with concentrations of leptin in lamb blood serum in their off-spring. Approximately 1 wk before the expected date of lambing, blood samples, weights, and body condition scores (BCS; 0 to 5 scale) were collected from 27 mixed-parity ewes. Following parturition, ewe blood and milk samples were collected within 2 h of parturition (d 0), 12 h (d 0.5) and 24 h (d 1) after parturition, again on d 5, and weekly thereafter until d 47. Lambs were blood-sampled and weighed within 2 h of parturition (d 0), bled daily until d 5, and bled and weighed weekly thereafter to d 47. Prior to lambing, ewe blood serum leptin was positively correlated with congruent BCS (r2 = 0, 10, P = 0.06), but not weight (P = 0.14). Following parturition, ewe blood serum leptin was positively correlated with BCS, weight, and milk serum leptin (r2 = 0.14, P < 0.0001, r2 = 0.12, P < 0.0001, and r2 = 0.028, P = 0.04). Leptin in milk serum was correlated with ewe weight (r2=0.05, P = 0.007) but not ewe BCS (P = 0.7); however, concentrations of leptin in both ewe blood and milk serum varied with day of lactation (P = 0.0001), being maximal within 24 h of parturition and declining to nadir concentrations by d 5. Leptin in lamb serum was correlated with milk serum leptin, (r2 = -0.05; P = 0.001), but not ewe blood serum leptin (P = 0.5). Concentrations of leptin in lamb serum increased from birth to d 5 and declined thereafter to nadir concentrations by d 19. Elevated concentrations of leptin in milk during the early stages of lactation may provide a mechanism for thermoregulation, satiation, and homeostatic endocrine control in the neonate.