Edited by: Asta Tvarijonaviciute, University of Murcia, Spain
Reviewed by: F. Capela e Silva, Universidade de Évora, Portugal; Maria Grazia Cappai, University of Sassari, Italy
This article was submitted to Animal Nutrition and Metabolism, a section of the journal Frontiers in Veterinary Science
†These authors have contributed equally to this work
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
This study aimed to investigate the effects of a weight loss program (WLP) on biochemical and immunological profile, and cardiovascular parameters in a cohort of dogs with naturally occurring obesity. Eleven obese dogs [body condition scoring (BCS), ≥7/9] were enrolled into the study and underwent clinical and cardiovascular examination, and blood testing before (T0) and after 6 months (T1) of WLP. Eleven normal weight (BCS, 4/5) healthy dogs were used as a control (CTR) group. Compared to the CTR group, at T0 obese dogs expressed higher serum leptin concentrations (
A common nutritional disease among animals and humans is obesity (
Quality of life is more inferior in human and dogs that are overweight (
Often, the biochemical profile of obese dogs is characterized by dyslipidemia, with an increased risk of onset of pancreatitis or gastrointestinal disorders over time (
Regarding cardiovascular disorders, recent studies reported increased systolic blood pressure and left ventricular concentric remodeling in obese dogs (
Leptin is a hormone secreted by white adipose tissue that mainly acts at hypothalamic level reducing feed intake and increasing energy metabolism (
In human beings, the development of pathological conditions with high morbidity and mortality (for example, atherosclerosis) are closely related to the persistent slow-grade inflammation and immune dysregulation that occurs in obesity. Altered levels of leptin, adiponectin, cytokines, and acute-phase proteins are well-known in obese humans (
This study investigated the effects of WLP on biochemical and immunological profile, blood leptin level, and cardiovascular parameters in a cohort of dogs with naturally occurring obesity, in order to address the effects due to the imbalance of these aspects on the clinical presentation and management of canine obesity.
Eleven obese, but otherwise healthy dogs, 10 females (8 spayed) and 1 male (neutered), as well as 11 healthy dogs at ideal body condition, 7 females (3 spayed) and 4 males (1 neutered), were enrolled into this study. The animals were from a client-owned referral population of the Veterinary Teaching Hospital, Department of Veterinary Medicine and Animal Productions (University of Naples Federico II), from November 2018 to November 2019. Written informed consent was obtained from the owners before the beginning of the trial, including a commitment to maintain the required management conditions during the study.
Each recruited dog was classified according to a body condition scoring (BCS) assessed by the same operator (LC), utilizing a 9-point scale system (
Both groups were homogeneous by age (dogs younger than 2 years or older than 10 years were ruled out). Mean age and weight at the enrollment were respectively 5 ± 2.4 years and 28.3 ± 15.8 kg for the obese group, while they were respectively 5 ± 1.8 years and 22.3 ± 16.2 kg for the CTR group. Regarding dog breeds, the obese group included six mixed breed, three Labrador Retriever, one Jack Russell, and one Epagneul Breton, while the CTR group included four mixed breed, two Golden Retriever, one Jack Russell, one Corso dog, one Labrador Retriever, one Bullmastiff, and one Maltese. All the recruited dogs were considered clinically healthy, based on the history and physical examination, including measurement of arterial blood pressure and complete cardiovascular evaluation. Exclusion criteria were endocrine, liver, renal, heart, and infectious diseases or inflammatory conditions. Animals that were in some physiological conditions, such as pregnancy or nursing, and those under pharmacological treatment and following a regular exercise program were ruled out.
Body condition score, bodyweight evaluation, morphometric measurements (
Obese dogs underwent 6 months WLP using a prescription dry dog feed diet for weight management. Diet was high in protein but low in fat and carbohydrates; therefore, it encouraged steady and effective fat loss while maintaining a patient's muscle mass, promoting satiety, and stimulating resting metabolic rate. Besides, the diet was high in complex carbohydrates and fibers to help even out blood sugar levels throughout the day. The composition of this diet is shown in
Chemical composition of the experimental diet administered to obese group.
Moisture | % | 7.5 | – |
Protein | % | 29 | 9.8 |
Fat | % | 6.05 | 2.0 |
Linoleic acid | % | 1.4 | 0.5 |
ω-3 | % | 0.09 | 0.03 |
DHA | % | 0.01 | 0.0 |
Carbohydrates | % | 41.0 | 13.8 |
Starch | % | 23.1 | 7.8 |
Total sugars | % | 1.7 | 0.6 |
Crude fiber | % | 10.0 | 3.4 |
Soluble fiber | % | 1.6 | 0.5 |
Insoluble fiber | % | 18.7 | 6.3 |
Vitamin A | IU/kg | 21,936 | 741 |
Vitamin D3 | IU/kg | 962 | 32.5 |
Vitamin E | IU/kg | 300 IU/kg | 10.1 |
kcal/g | 2.96 kcal/g | – |
Bodyweight evaluation and morphometric measurements (
Morphometric measurements were obtained to apply the equations developed to predict lean body mass (LBM) (kg), fat mass (FM) (kg), and body fat percentage (BFP) according to Witzel et al. (
Complete blood count, serum biochemical panel, cardiovascular biomarkers (ET-1 and cTnI), insulin, leptin, cytokines (TNF-α and IL-6), and blood immunophenotype (CD3+CD4+, CD3+CD8+ T cells, CD4/CD8 ratio, CD21+ B cells, Treg cells) evaluations were acquired in the obese group before (T0) and after 6 months of the WLP (T1). In the CTR group, the above determinations, except cytokines, were performed at the enrollment (T0).
Blood sample collection was cruelty-free, according to the national legislation. Ethical committee approval was required (see ethics statement). Ten milliliters of blood was collected by jugular venepuncture after 12 h of fasting at 9:00 a.m. for both obese and CTR groups. Blood sample obtained was divided immediately into three aliquots. The first aliquot was sited in tubes containing potassium ethylenediaminetetraacetic acid (EDTA) for CBC, performed within 30 min from the collection. The second aliquot of blood was collected similarly in anticoagulated tubes containing EDTA and stored at room temperature up to 5–6 h before immunological assays. The third aliquot was retained in tubes without anticoagulant, allowed to clot, and centrifuged at 908 g for 15 min at 4°C to obtain blood serum. Serum samples were stored at −80°C and defrosted immediately before biochemical profile, cardiovascular biomarkers, insulin, leptin, and cytokines evaluation.
The CBC was performed using a semiautomatic cell counter (Genius S, SEAC Radom Group). A semiautomatic chemical chemistry analyzer (OLOT, Spinreact) was utilized to measure blood urea, creatinine, glucose, triglycerides (TG), total cholesterol (T-Chol), alanine aminotransferase (ALT), alkaline phosphate (ALP), total bilirubin (T-Bil), sodium, potassium, albumins, and total proteins (TP), using reagents from Spinreact (Girona, Spain). Serum protein electrophoresis was quantified using a densitometer analyzer (Selvet 24, SELEO S.r.l., Orta di Atella, CE, Italy) with reagents from Spinreact (Girona, Spain).
Urinary samples were collected by cystocentesis for the obese group at T0 and T1 and for the CTR group only at the enrollment time (T0). Urinary protein/creatinine ratio (UP/C) was calculated starting from their spectrophotometric determination (OLOT, Spinreact) within 30 min.
Serum insulin analysis was performed for each sample using an ELISA kit (canine insulin ELISA, Mercodia AB). The insulin/glucose ratio (I/G) was calculated as the serum insulin (μU/ml) × 100/serum glucose (mg/dl), according to Bailhache et al. (
Serum leptin was estimated by an ELISA kit (Canine Leptin ELISA Cat. EZCL-31K, Millipore, Billerica, MA, USA). The detection limit was 0.2 ng/L, and intra- and interassay coefficients of variation (CV) were <5%. Absorbance was determined by a spectrophotometer with a wavelength of 450 nm (Epoch, BioTek Instruments Inc., Winooski, VT, USA).
Serum TNF-α and IL-6 were measured by canine cytokine ELISA kit (Genorise, Glen Mills, PA, USA). TNF-α detection range assay was 1–2,200 pg/ml with intra- and interassay CV <7 and <9%, respectively. IL-6 detection range assay was 50–3,200 pg/ml with intra- and interassay CV <6 and <9%, respectively. Absorbance was determined using a microplate reader (GDV programmable MPT Reader DV 990BV4, Agilent Technologies, Santa Clara, CA, USA) at 450 nm.
Serum ET-1 was assessed using a specie-specific commercial sandwich ELISA kit (Claud-Clone Corp, Katy, TX, USA). The detection range was 6.17–500 pg/ml, and intra- and interassay CV were <10 and <12%, respectively. Absorbance was determined using a microplate reader (GDV programmable MPT Reader DV 990BV4, Agilent Technologies, Santa Clara, CA, USA) at 450 nm.
The serum troponin I levels were assessed using a human commercial kit [ADVIA Centaur High Sensitivity Troponin I (TNIH)], previously validated in canine species (
The arterial blood pressure (ABP) was recorded for the obese group at T0 and T1 and for the CTR group only at the enrollment time (T0).
Blood pressure was measured by an automated oscillometric device (HDO, S + B MedVet). A cuff of appropriate size was placed on a limb or the tail, taking into account the dog body size. The first measurements were discarded, and a total of five to seven consecutive measurements were taken by the same operator, when the patient was calm after a short period of acclimatization in a quiet room, in ventral or lateral recumbency. The highest and lowest values of systolic, mean, and diastolic arterial blood pressure were ruled out, and an average value was obtained on the three remaining measurements. Dogs with systolic arterial blood pressure (SABP) >160 mmHg were considered to be affected by hypertension (
Electrocardiographic (ECG) and echocardiographic evaluations were performed for the obese group at T0 and T1 and for the CTR group only at the enrollment time (T0).
A standard six-lead electrocardiogram (ECG model 08SD, BTL Italia) was obtained with animals in right lateral recumbency, recording at least a 2-min strip (paper speed, 50 mm/s; calibration at 1 mV = 1 cm).
A conventional echocardiographic examination (two-dimensional, M-mode, spectral, and color flow Doppler) was performed with conscious dogs in lateral recumbency, using an ultrasound machine (Mylab50, Esaote) equipped with multifrequency (1–11 MHz) phased-array transducers. The echocardiographic study was performed in accordance with standardized methodologies (
M-mode measurements included left ventricle free wall thickness at end diastole (LVFWd) and end systole (LVFWs), interventricular septal thickness at end diastole (IVSd) and end systole (IVSs), and LV internal diameter at end diastole (LVIDd) and at end systole (LVIDs). LVFWd and IVSd were indexed by allometric scaling based on body weight (
Two-dimensional measurements included left atrium/aorta diameter ratio (LA/AO) and ejection fraction (EF), calculated starting from the ventricular volumes obtained using the long-axis area-length (A-L) method.
Regarding spectral Doppler parameters, aortic and pulmonary peak velocities were measured from a left apical five-chamber view and a right short-axis view, respectively. Transmitral flow velocities were obtained using a left apical four-chamber view. Regarding the transmitral flow profile, the ratio of E wave peak velocity to A wave peak velocity (E/A) was obtained as a parameter of diastolic function; values were not used for analysis if the E and A waves were combined. Deceleration time (DT) was obtained from the E wave flow profile, considering the slope of the line starting from the maximal velocity E wave point to the baseline (
In the obese group, indexed two-dimensional and mono-dimensional parameters, FS, FE (A-L), and spectral flow Doppler measurements were compared with those of the CTR group, and with the reference ranges, considering the actual weight of the dogs (
Peripheral blood CD21+ B cells, CD3+, CD3+CD4+, CD3+CD8+ T cells, CD4/CD8 ratio, and CD4+CD25high Foxp3+Treg cells were evaluated by cytometry analysis (ATTUNE NxT FACS and Attune NxT Software, Life Technologies, Italy). Dead cells were ruled out as described (
Fluorescein isothiocyanate (FITC), phycoerythrin (PE), and PE-cyanine7 anticanine CD3 (CA17.2A12 and CD3-12), CD4 (YKIX302.9), CD8 (YCATE55.9), and CD21 (CA2.1D6) monoclonal antibodies (mAbs) and isotype-matched mAb controls were used (Serotec Ltd London, UK). Foxp3 detection was measured using murine Foxp3 antibody (Clone FJK-16 s, eBioscience, San Diego, CA, USA), and the Foxp3detection Kit (Staining Set, eBioscience). Treg detection was performed as described (
Statistical evaluation was performed by Mann–Whitney test or Wilcoxon's matched pairs signed-rank test, as indicated. InStat version 3.0 software (GraphPad Software Inc., San Diego, CA, USA) was used. Results were considered significant at
All enrolled dogs (obese and CTR group) were considered healthy based on clinical examination performed at the beginning of the weight loss period (T0). A second clinical examination at the end of the weight loss follow-up (T1) was made to determine the health state of obese dogs and to know the owner's impressions of the effects related to the weight loss of his pet. All obese dogs showed clinical improvement after the WLP. The mean of BCS recorded in the obese group at T0 was 8.18 (range, 7–9). At the end of the WLP (T1), all the dogs, with the exception of one that remained BCS 7, showed a reduction in BCS. In particular, the mean of BCS was 6.8 (BCS ranged between 5 and 8); 63.6% of the dogs showed a reduction of 1 point of BCS, 18.2% a decrease of 2 points, and 9.1% showed a decrease of 3 points. Besides, all owners emphasized many beneficial aspects related to the weight loss of their dogs. After 6 months of dietary treatment, 10 of the 11 owners reported that their dogs were more active, and the quality of their life was notably improved. The haircoat was shinier and softer in 7 of the 11 obese dogs. Other health benefits included a reduction in tachypnea (nine dogs) and breathing difficulties (one dog), and improvement of joint and locomotor problems (two dogs). It has already been observed that obese animals losing weight become more active and sociable (
Finally, the FM values, expressed in kilograms, and BF, expressed as a percentage, obtained using equations developed by Witzel et al. (
Morphometric equations in enrolled dogs.
In this study, the metabolic panel (
Biochemical profile in the control (CTR) group and obese group before and after the weight loss program (WLP).
Glucose | mg/dl | 95.09 ± 8.2 | 96.57 ± 9.1 | 91.91 ± 11.1 |
Insulin | mU/ml | 13.80 ± 2.3ab | 16.38 ± 5.6a | 12.58 ± 2.6b |
I:G ratio | – | 14.54 ± 2.8 | 17.08 ± 6.3 | 14.72 ± 5.3 |
Urea | mg/dl | 29.00 ± 6.9b | 46.16 ± 20.3a | 51.73 ± 12.5a |
Creatinine | mg/dl | 1.07 ± 0.22ab | 1.03 ± 0.37a | 1.26 ± 0.39b |
T-Chol | mg/dl | 252.54 ± 38.2 | 297.73 ± 114 | 240.81 ± 85.5 |
TG | mg/dl | 68.91 ± 21.4b | 194.8 ± 172.9a | 108.54 ± 97.7ab |
ALT | UI/L | 38.72 ± 7.2 | 56.4 ± 26.5 | 57.91 ± 20.83 |
ALP | UI/L | 93.45 ± 47.6 | 264.9 ± 364.6 | 216.27 ± 303.0 |
T-Bil | mg/dl | 0.313 ± 0.11 | 0.286 ± 0.19 | 0.237 ± 0.19 |
Na | mmol/L | 146.36 ± 3.10 | 144.00 ± 2.86 | 146.18 ± 2.32 |
K | mmol/L | 4.33 ± 0.35 | 4.27 ± 0.30 | 4.07 ± 0.24 |
TP | g/dl | 6.63 ± 0.42 | 7.00 ± 0.55 | 6.24 ± 0.26 |
Alb | g/dl | 3.44 ± 0.26 | 3.183 ± 0.52 | 3.097 ± 0.18 |
α1-glob | g/dl | 0.297 ± 0.10 | 0.307 ± 0.13 | 0.230 ± 0.06 |
α2-glob | g/dl | 0.780 ± 0.18c | 1.418 ± 0.34a | 1.022 ± 0.15b |
β1-glob | g/dl | 0.801 ±± 0.11 | 0.775 ± 0.11 | 0.632 ± 0.19 |
β2-glob | g/dl | 0.689 ± 0.05 | 0.827 ± 0.23 | 0.723 ± 0.10 |
γ-glob | g/dl | 0.600 ± 0.09 | 0.496 ± 0.17 | 0.512 ± 0.08 |
Biochemical parameters in enrolled dogs.
The elevated serum α 2-globulin fraction detected in the obese group at T0 could be related to the increased blood concentrations of acute phase proteins, suggestive of an inflammatory condition related to obesity condition (
Serum troponin levels were significantly greater in the obese dogs than in the CTR group, reducing with the weight loss (obese group at T0 vs. CTR group,
Cardiovascular biomarkers in the control (CTR) and obese groups before and after the weight loss program (WLP).
ET-1 | pg/ml | 41.30 ± 8.17 | 39.58 ± 13.27 | 36.84 ± 7.06 |
cTnI | ng/ml | 0.050 ± 0.017b | 0.118 ± 0.044a | 0.064 ± 0.018b |
Cardiovascular biomarkers in enrolled dogs.
The occurrence of endothelial dysfunction is well-known in human's obesity and is related to an increased cardiovascular risk (
The descriptive statistics of the examined cardiovascular parameters are shown in
Systolic blood pressure, heart rate, and echocardiographic findings in the control (CTR) and obese groups before and after the weight loss program (WLP).
SABP | mmHg | 139.35 ± 15.09 | 145.64 ± 9.44 | 141.18 ± 14.27 |
HR | bpm | 118.54 ± 15.89 | 128.64 ± 22.81 | 130.45 ± 15.88 |
LA/AO | – | 1.44 ± 0.104 | 1.45 ± 0.119 | 1.44 ± 0.12 |
IVSd indexed | – | 0.423 ± 0.06b | 0.493 ± 0.06a | 0.450 ± 0.08a |
LVFWd indexed | – | 0.452 ± 0.04 | 0.485 ± 0.06 | 0.472 ± 0.06 |
IVSd/LVIDd | – | 0.254 ± 0.04b | 0.334 ± 0.05a | 0.282 ± 0.049b |
LVFWd/LVIDd | – | 0.263 ± 0.027b | 0.321 ± 0.05a | 0.287 ± 0.028 ab |
EDVI | ml/m2 | 54.53 ± 28.86 | 50.29 ± 28.49 | 50.63 ± 21.85 |
ESVI | ml/m2 | 21.74 ± 13.31a | 14.72 ± 9.39b | 16.33 ± 9.54a |
FS | % | 34.75 ± 5.64b | 41.27 ± 7.85a | 38.34 ± 5.74ab |
EF (A-L) | % | 64.84 ± 8.04b | 72.09 ± 10.48a | 69.48 ± 7.16ab |
AO-PV | m/s | 1.10 ± 0.218b | 1.41 ± 0.29a | 1.30 ± 0.132a |
PO-PV | m/s | 0.912 ± 0.190 | 0.994 ± 0.105 | 0.939 ± 0.103 |
E wave | m/s | 0.720 ± 0.09 | 0.793 ± 0.167 | 0.778 ± 0.157 |
A wave | m/s | 0.511 ± 0.102b | 0.714 ± 0.124a | 0.696 ± 0.110a |
E/A | – | 1.44 ± 0.227a | 1.15 ± 0.37b | 1.14 ± 0.301b |
DT | ms | 111.65 ± 13.33 | 120.93 ± 21.88 | 111.67 ± 7.17 |
In the obese group at T0, ECG revealed five animals with no respiratory sinus arrhythmia, two dogs with sinus tachycardia, one dog with features of left ventricular enlargement, two dogs with low R wave amplitude, and four dogs with features of myocardial microinfarcts. No ECG abnormalities were recorded in the CTR group. The mean electrical axis was in the normal range in both groups, and the heart rate was not significantly different between groups.
SABP values were not significantly different between groups. Only one obese dog was hypertensive (164 mmHg) at T0 and showed SABP values just below the cutoff limit (158 mmHg) after the WLP.
Regarding M-mode and 2D echocardiographic measurements, IVSd and LVFWd decreased in the obese group after the WLP (
Echocardiographic parameters in enrolled dogs.
Echocardiographic parameters in enrolled dogs.
The indexed LV end-systolic volume (ESVI) was greater in the CTR group compared to obese animals at T0 (
Echocardiographic parameters in enrolled dogs.
Regarding Doppler echocardiography, AO-PV was higher in the obese group at T0 (
A high proportion of obese people develops, over the years, cardiovascular pathologies (hypertension, coronary artery disease, atrial fibrillation, heart failure) with a shortened lifespan (
We performed the first approach of immune profile evaluation of obese dogs subjected to WLP basing on the peripheral blood leukocyte count. In this perspective, it appeared that obese group at T0 (before the start of the WLP) showed a higher (not statistically significant) leukocyte number than healthy weight dogs (CTR group) (
The number of Leucocytes, neutrophils and T cell subsets in enrolled dogs. The figure reports the evaluation on the number of
In a second approach, the evaluation of the number of T lymphocyte subpopulations was considered. In this context, the total number of CD3+ T cells appeared to be unmodified during WLP. CD3+ CD4+ T and CD3+ CD8+ T cell numbers appear to be in a decreasing trend after WLP in the obese group, although not statistically significant (
When the percentage of total CD3+ T lymphocytes is considered, a significant reduction between T0 and T1 was observed in dogs following the WLP. In contrast, subjects at T1 showed a lower CD3+ T cell percentage than the CTR dogs (
The asset of T cell subsets and the serum level of leptin in enrolled dogs.
Notably, the Treg cells (CD3+ CD4+ CD25+ FoxP3+) appeared reduced in number (
No variation in B cell percentage was found in the before and after WLP, although it appears reduced in the obese group compared to the CTR dogs (
B cells, interleukin (IL)-6, and tumor necrosis factor (TNF)-α in enrolled dogs.
When serum proinflammatory cytokine (
Leptin and inflammatory cytokines in the control (CTR) and obese groups before and after the weight loss program (WLP).
Leptin | ng/L | 30.06 ± 5.64b | 120.40 ± 20.64a | 33.36 ± 12.04b |
IL-6 | pg/ml | – | 480.83 ± 46.65a | 412.60 ± 21.21b |
TNF-α | pg/ml | – | 98.30 ± 43.64a | 34.93 ± 14.63b |
One important limitation of this non-experimental study was the short term (6 months) of WLP, since the dogs were unfollowed until they achieved an ideal body condition. The average duration of a complete weight loss period, able to conduce dogs to a healthy weight, is 9 months, and some dogs need more than 12 months to reach the target (
Finally, the degree of weight loss achieved and, consequently, the immunological, biochemical, and cardiovascular findings observed may also be affected by timing and duration of obesity, which was not possible to ascertain accurately based on the information provided by the dog's owners.
In conclusion, in the present study, weight loss was associated by an improvement in both activity and global life quality of dogs. Furthermore, the metabolic status of obese dogs, with particular regard to dyslipidemia, improved after WLP.
Regarding the cardiovascular system, obese dogs can show systolic hypertension and LV remodeling, although probably the overweight condition has a less impact than humans. However, cardiovascular assessment should not be omitted in the evaluation of the clinical status of obese dogs. In our natural model of obesity, we observed only a partial recovery of the LV remodeling, although a greater effect cannot be ruled out if the dietary program had been longer.
Regarding the immunological status of obese dogs, our observations indicate that WLP, obtained in 6 months with a calorie-restricted diet, can modify the immune structure and, in some aspects, induce an overall homeostatic recovery of the canine immune response. The total number of leukocytes in obese dogs subjected to WLP tends to be similar to that observed in normal-weight subjects. Furthermore, WLP reduced the serum presence of proinflammatory cytokines, at least for those observed in the present trial (IL-6 and TNF-α). However, the evidence of a percentage increase in CD8+ T lymphocytes is worthy of interest, and further studies are needed to understand the potential homeostatic significance. Besides, the WLP significantly reduced the serum concentration of leptin, which has been very high at the beginning of the WLP, and after 6 months of treatment, it became similar to those observed in normal-weight dogs. Therefore, calorie restriction appeared to induce potentially beneficial effects, since it promoted a homeostatic recovery of the structure of the immune response.
The WLP for only 6 months was unable to normalize the levels of Treg, but we cannot exclude that a more prolonged treatment could have obtained this desirable effect. However, the reduction in serum leptin could also have induced the observed decreased proinflammatory cytokine production, as previously described. In this context, it is of fundamental relevance to suggest to the scientific veterinary community an in-depth study to understand the relationship between leptin and Treg cells in the dog, where, in humans, this aspect is particularly relevant and universally recognized as the cause of disease when altered.
This study opens new perspectives in the understanding of the relationship between diet and immune response in dogs, where the reference scientific literature is not sufficiently able to explain the effects of calorie restriction in the treatment of obesity in relation to the morbidities and pathophysiological aspects frequently associated to canine weight gain.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The animal study was reviewed and approved by Ethical Animal Care and Use Committee of the University of Naples Federico II. Written informed consent was obtained from the owners for the participation of their animals in this study.
LC, DP, GR, and GT contributed to the conception of the experimental design of the study. LC, DP, and PL participated in the clinical management of the dogs. DP performed the echocardiographic examinations. LC, NM, and PL performed the hematological and biochemical analysis, ELISA tests, and analyzed the data. AP, FC, VR, and AG performed the immunological research and analyzed the data. FF organized the database. AP and NM performed the statistical analysis. LC, DP, AP, GR, YA, PL, and GT wrote the manuscript. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Drs. Paolo Zarelli, Imma Autiero, Marzia Pasquarelli, and Maristella Filosa for their cooperation. We are also in debted to Gennaro Chierchia, Ciro Laperuta, Paolo Muzj, Antonio Sica, and Maria Ferrara for their technical assistance. Finally, we acknowledge Nestlé Purina Research and Development for providing the prescription dry dog food diet for the weight management necessary to the current study.