Nutrients

 

Metabolizable Energy

3000 KCal/kg

Dry Matter

%age 88.8

Crude Protein

23

Crude Fiber

4.12

Crude Fat

4.19

Phosphorous

0.33

Calcium

0.90

Dig. Methionine

0.45

Dig. Arginine

1.09

Dig. Valine

0.79

Dig. Lysine

1.10

Dig. Tryptophan

0.20

Dig. Threonine

0.78

Dig. Isoleucine

0.79

HF-diet for group 2

Normal basal diet + 15% Margarine

HF-diet for group 3

Normal basal diet + 30% Margarine

At the end of the trial, the study rats were anesthetized with a combination of medetomidine and ketamine (1 and 50 mg/kg, respectively) maintained with isoflurane. Animals were sacrificed, and blood samples were obtained in serum-collection vacutainers. Serum was extracted through centrifugation and analyzed for various biochemical tests.

Most often IBD (inflammatory bowel disease) affects a portion of the small intestine just before the large intestine; i.e., ileum. It also affects the colorectal region of the large intestine. Therefore, tissue samples from these regions of the intestine (ileum and colorectal regions) were collected to perform fat staining and immunohistochemistry (n = 6 rats per group).

Physical parameters

Weekly feed intake, defecation rate, and fecal pellet pH were monitored for each group using a pH meter (HANNA Instruments®; H12210). To measure the fecal defecation rate for each group, a daily recording of the fecal pellet number was performed, and then the weekly fecal defecation rate was calculated for each group from the collected data.

Body weight and organ-to-body weight ratio

The body weight for all animals was recorded for each group during the entire experimental period. The body weight ratios of the pancreas, intestine, and abdominal fat were estimated using the formula given below:

$$\mathrm= \frac}} \mathrm100$$

Biochemical analyses

Serum samples were analyzed to measure serum glucose, insulin, leptin, amylin, glucokinase, total cholesterol (TC), TGs, low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs) through relative commercial kits as given below:

Serum glucose: Bioclin® Glucose Monoreagent diagnostic kit, Berlin, Germany; detection limit range: 2-500 mg/dL; CV% < 3.1

Serum Insulin: Calbiotech Insulin ELISA®, CA, USA; detection limit range: 0.78-50 µIU/mL; CV% < 10.

Serum Leptin: Rat-LEP ELISA kit, E-EL-R0582, Thermo Fisher, Germany; detection limit range: 0.16~10 ng/mL; CV% < 10.

Serum Amylin: Rat-Islet Amyloid Polypeptide ELISA kit, E-EL-R2448, Thermo Fisher, Germany; detection limit range: 62.50-4000 pg/m; CV% < 10.

Serum glucokinase: Rat-GCK (glucokinase) ELISA kit, E-EL-R0426, Thermo Fisher, Germany; detection limit range: 0.63-40 ng/mL; CV% < 10.

Serum Total Cholesterol: Dia-Sys Diagnostic Systems USA, detection limit range 3-750 mg/dL; CV% < 10.

Serum Triglycerides: Dia-Sys Diagnostic Systems USA, detection limit range: 1000 mg/d; CV% < 10.

High-density and low-density lipoproteins: Randox, Randox Laboratories LTD, UK: detection limit range 20 to 129 mg/d; CV% < 10. The serum low-density lipoprotein (LDL) concentration was measured by using the formula:

$$\mathrm(\mathrm/\mathrm)=\mathrm-(\mathrm-\mathrm)5$$

Fat staining

After slaughter as per the recommended protocol, tissue samples of the ileal and colorectal regions were collected, and fat staining (through Sudan black staining) was performed to stain the granules of fat that accumulated in the enterocytes. The acidic groups of compound lipids containing phospholipids combine with Sudan black satin because of the slightly basic nature of the dye.

Immunohistochemistry

Immunohistochemistry was performed to determine the expression of I-FABP in the gut (ileum and colorectal regions). For this purpose, 5 μm sections of the intestine were fixed in formalin and embedded in paraffin, and then these intestinal sections were mounted on slides. The sections were deparaffinized, hydrated, and washed with phosphate buffer saline (PBS) at pH 7.4, and then, to block nonspecific binding sites, these sections were incubated with UltraCruz® Blocking Reagent (sc-516214) for blocking. Next, the primary antibody was incubated overnight with mouse anti-I-FABP antibody (sc-374482, Santa Cruz Biotechnology, USA) and then diluted with blocking reagent (1:200) at a temperature of 4 °C. Prepared slides were then washed and incubated after adding a secondary antibody, IgGκ BP-HRP anti-mouse antibody, sc-516102, Santa Cruz Biotechnology, Santa Cruz, USA, diluted with a blocking reagent (1:200) for a period of two hours. The visualization of the immunohistochemical reaction was performed by using the substrate, 3,3-di-amino-benzidine tetra-hydrochloride (DAB): sc-24982, Santa Cruz Biotechnology, and Immuno-Cruz® ABC kit, sc-516216. Dehydration was finally performed in a graded series of alcohol and xylene. The tissue sections were mounted with Ultra-Cruz™ mounting medium, sc-24941, Santa Cruz Biotechnology and cover-slipped.

Statistical analysis

The obtained results were then subjected to a two-way analysis of variance (ANOVA), considering both the effect of days and HF diet treatment, followed by Duncan’s multiple range (DMR) test, and the results are shown as the mean ± SE. GraphPad Prism and Co-Stat software were used for statistical analysis.