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. (51 (link)). In particular, the detection range was 2.3–173 mU/L with a detection limit of 1.15 mU/L. Absorbance was determined using a microplate reader (GDV programmable MPT Reader DV 990BV4, Agilent Technologies, Santa Clara, CA, USA) at 450 nm.
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 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).