*2.4. Animals and Diet*

Thirty-two male Sprague Dawley rats of age between 8 and 10 weeks (180–230 g) were obtained from Animal and Research Centre (ARASC), Universiti Sains Malaysia, Kubang Kerian, Kelantan. Animals were housed in an individual cage with a constant temperature at 22–24 ◦C, given a 12 h light and dark cycle, and supplied with normal rat chow pellet with water ad libitum. Animals were handled according to guidelines provided from local Ethics Committee (USM/Animal Ethics Approval/ 2016/(98) (744)). Following an acclimatization period, rats were administered either a normal diet or high-fat diet (HFD). The normal diet is a standard Altromin pellet imported from Germany by Sterling Ascent, Malaysia. Obesity was established by feeding with a HFD using the previous method with slight modifications, consisting of 32 g of ghee (saturated fat from animal), 68 g of powdered normal rat chow, 300 mg of calcium, 100 UI of vitamin D3, and 12% cholesterol powder [22]. After a dough-like consistency formed, foods were shaped into small hand-balls and kept at 4 ◦C overnight to feed the rats in the next morning. Foods were prepared every two days to avoid lipid oxidation. The nutrient composition of normal and HFD is shown in Table 1.

**Table 1.** Content of nutrients in normal and high-fat diets.

