Alternation of Physical Indexes and Plasma Biochemical Makers in Overweight Dogs Induced by High-Fat Diet Feeding

MATERIALS AND METHODS

Four female Beagles (3-8 years), maintained for examination in the common laboratories at Department of Veterinary Science of Nippon Veterinary and Life Science University and diagnosed clinically healthy and Body Condition Score (BCS) 3 which was determined on a five point scale: 1. very thin; 2. underweight; 3. ideal; 4. overweight; 5. obese, were used. This examination, approved by Nippon Veterinary and Life Science University Animal Research Committee, started from the 18th August and finished 17th September 2010.

All dogs were fed with high-fat diet for 1 month which is equivalent to 6 months in human term. At first Resting energy requirement (RER = 70xbody weight^0.75) were calculated for each dogs and multiplied it by moderate work coefficient (3.0) in order to calculate total amount of calories. We made a high-fat diet containing 30% higher calorie compared to usual diet (usual diet, 314.5 kcal/100 g; high-fat diet, 411.7 kcal/100 g). Nutrient profiles of the high-fat diet were ash 6.9%, crude fat 24.3%, crude fiber 1.86%, protein 22.9% and moisture 8.4%. Diet included corn, wheat flour, bean curd refuse, defatted soybean, fish flour, gluten meal, beer yeast, soy oil, beet pulp, poultry by product meal, vitamin E, salt, oxygen water, beef fat, zinc carbonate as detail ingredient composition. Dogs were fed the diet with a half amount of designated diet twice at 6:00 am and 2:00 pm and water was constantly available during the examination. BF was determined using morphometric measurement with simple calculations and non clinical laboratory equipment and correlated highly with dual- energy X-ray absorptiometry (Mawby et al., 2004). There are gender-specific and either gender formulas on morphmetric measurement. BF (%) was calculated according to gender-specific formula:

where, HS is length from hock joint to back knee and PC is length around bony pelvis.

Blood samples were taken from the fore limb vein of animals were obtained and collected into heparinized plastic tubes before breakfast at before and after high-fat diet feeding for one month. Plasma was recovered by centrifugation at 1200 g for 5 min at 4°C and subsequently stored at -25°C. Alkaline phosphatase(ALP), Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and Lactate Dehydorgenase (LDH) activities and Blood Urea Nitrogen (BUN), Creatinin (CRE), Glucose (GLU), Total Cholesterol (T-Cho), Total Protein and Triglyceride (TG) concentrations were measured using auto analyzer (AU680, Olympus Corporation, Tokyo) with the manufacturer’s reagents. Free fatty acid (FFA), Adiponectin (APN), Insulin (ISN), Cholesterol Ester Transfer Protein (CETP), Phospholipid Transfer Protein (PLTP), TNF-α and visfatin were determined with the following commercial kits: NEFA-C test Wako (Wako Pure Chemical Industries, Inc., Tokyo), Dog Adiponectin ELISA Kit and Mouse Visfatin/PBEF ELISA Kit (CircuLex Co., Ltd. Nagano), Lbis Dog Insulin kit (SHIBAYAGI Co., Gunma), CETP Activity Assay Kit and PLTP Activity Assay Kit (BioVision Research Products, Mountain View, CA), Canine TNF-α/TNF SF1A Immunoassay (R and D Systems, Inc., MN).

Data are presented as Means±SD. The Student’s t-test for paired groups was performed on the various data in order to determine significance using Graph Pad Prism software version4.0 (Graph Pad Soft ware, San Diego, CA). Differences were considered significant when the p value was <0.05. Pearson’s product moment correlation was used to identify significant correlations between BF and biochemical makers.

RESULTS

All dogs gained 2.8±0.5 kg as BW after the high-fat diet feeding. Three physical indexes, BW, BF (%) and PC increased significantly after the high-fat diet feeding (Table 1).

Table 1:	Changes in physical indexes and plasma biochemical markers in female beagles before and after high-fat diet feeding
Values are presented as Mean±SD. aIndicates significance when compared against before value (p<0.05 paired t-tes). ALP: Alkaline phosphatase, ALT: Alanine aminotransferase, APN: Adiponectin, AST: Aspartate aminotransferase, CETP: Cholesterol ester transfer protein, CRE: Creatinin, FFA: Free fatty acid, GLU: Glucose, ISN: Insulin, LDH: Lactate dyhydrogenase, PLTP: Phospholipid transfer protein, T-Cho: Total cholesterol, TG: Triglyceride, TP: Total protein, BUN: Blood urea nitrogen

Table 2:	Correlation between body fat (%) and biochemical markers in female beagles before and after high-fat diet feeding
Pearson’s product moment correlation coefficients, r, are displayed. BUN: Blood urea nitrogen, T-Cho: Total choresterol, ALT: Alanine aminotransferase, ALP: Alkaline phosphatase, ISN: Insulin

Dogs showed significant differences in GLU, BUN, AST, ISN and CETP as biomedical markers. BUN concentrations after the diet feeding were significantly lower than the basal concentrations values before the feeding but concentrations of CRE as a main maker of kidney failure were constant. Adiponectin as maker of which is one of adipocytokines activating insulin sensitivity was on a declining trend. Whereas Visfatin, TG, FFA, TP, ALP and LDH in obese dogs with fed high-fat foods also were on the rise and T-cho was stable. Table 2 shows the correlations between BF and some biomedical makers. BUN showed a strongly negative correlation with BF after gaining weight (before r = -0.59, after r = -0.75). ALT and T-Cho correlated positively with BF (%) in overweight dogs (T-Cho r = 0.61, ALT r = 0.97). ALP, TNF-α and insulin had a strongly positive correlations with BF (ALP, before r = 0.73 after r = 0.71; TNF-α, before r = 1.00 after r = 0.96; ISN, before r = 0.92 after r = 1.00).

DISCUSSION

CONCLUSION

High-fat diet feeding induced significant increases in physical indexes, BW, BF (%), PC and plasma glucose, AST, CETP and insulin levels as biochemical makers in female beagle dogs. In overweight dogs with high-fat diet feeding, plasma BUN concentrations decreased significantly. BF showed strongly positive correlation with plasma ALT, TNF-α and insulin levels in over weight dogs. BF was calculated by simple formula without specific laboratory equipment and appeared to be useful indicator to assess physical and metabolic condition of overweight dogs. The usefulness of body fat as a clinical index to assess metabolic condition of whole body should be farther studied in more breeds dogs (e.g. characterized with short legs: Miniature dachshund) with various physical conditions.

ACKNOWLEDGMENTS

This work was supported in part by the Strategic Research Base Development Program for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), 2008-2012 and Grant-in-Aid for Scientific Research (No. 21380195 to T. Arai.) from MEXT.