All animal experiments were implemented in accordance with the guidelines for the use of animals as approved by the Animal Care and Use Committee, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (approval number UPM/FPSK/PADS/BR-UUH/00360). Fifteen female old Sprague Dawley rats aged 8 weeks were raised on regular chow diet by the breeder were fed regular chow ad libitum and free water access during the acclimatization week. The animal house was well-ventilated with a 12 h light/dark cycle at the ambient temperature of 25–30 °C, throughout the experimental period. The animals were then grouped (
n = 3) into high fat diet (HFD), regular chow (N), high fat diet with 50% GBR (GBR), high fat diet with high dose oryzanol extract (200 mg/kg body weight, OEHD) and high fat diet with low dose oryzanol extract (100 mg/kg body weight, OELD). Oryzanol at 100 and 200 mg/kg body weights were safe and produced metabolic changes in our earlier publications [
10], hence our choice of these concentrations in the present study. Male Sprague Dawley rats that had been fed with regular chow were mated with the female rats. The female rats were maintained on these diets during pregnancy and lactation. Oryzanol extract (high and low dose) were administered via intra- gastric gavage. The high Fat Diet (HFD) formulation was made up of 47.7% total carbohydrate, 16.1% protein, 31.1% fat, 2.5% fiber and 5.1% mineral and vitamin and prepared using 50% normal rat chow powder, 24% Mazola oil, 20% Nespray full-cream milk powder, 6% sugar and 50 g of starch, placed in an oven at 60 °C for 24 h, cut into smaller pieces and then used to feed the rats. The caloric compositions of the pellets given to each group are shown in Table
1. Food intake was set at 30 kcal/100 g body weight/day for all the groups (Table
1). Accordingly, the weights of the rats were measured weekly, and the total amount of feed given daily (grams) was reviewed every week based on the new weights of the rats. After delivery, male offsprings were chosen for follow up on the effects of the perinatal interventions on insulin resistance markers. At 4 weeks post-delivery, the male offsprings (
n = 6 per group) were weaned and maintained on regular chow (30 kcal/100 g body weight/day) for another 4 weeks. Energy efficiency of body weight (EE-BW) was calculated for the offsprings as weight gained in each group in g divided by energy intake in kJ [
14]. Dams were sacrificed via when they were approximately 16 weeks old, and after weaning off the offsprings. Weekly weights of the offsprings were taken for the 8 weeks, after which they were sacrificed. Prior to sacrifice, the animals were fasted overnight and subsequently dissected after light anesthesia to harvest the liver. Blood (10 mL/rat) was collected via cardiac puncture before dissection and centrifuged at 3000 rpm for 10 min at 4 °C to separate the serum. The liver was removed immediately, washed with ice-cold saline, dried with filtered paper, and then stored in RCL2® Solution (ALPHELYS, France) at −80 °C.
Table 1
Animal groups and diets
Normal | 0 | 100% normal rat pellet | 335 |
High fat diet | 50 | 50% normal rat pellet | 548 |
50% Germinated brown rice | 50 | 50% germinated brown rice | 554 |
Low dose OE | 100 | 100 mg/kg/day OE | 554 |
High dose OE | 100 | 200 mg/kg/day OE | 554 |