Bifidobacterium agar

For three consecutive days, that is, on days 26, 27 and 28 of treatment, faecal samples were collected and stored at -20°C. For analysis, the samples were diluted in deionised water (1 mg/ml) and faecal pH was measured using a digital potentiometer (Q400AS; Quimis) (34) . A separate portion of faecal sample was dried in an oven (320-SE; Fanem) at 105°C for 24 h to determine faecal moisture (27, 35) .
Another portion of faecal samples collected from caecum was diluted (1:9) in sterile peptone water and inoculated (20 µl), using the microdrop technique (36) , on selective agar for counting Lactobacillus spp. (de Man, Rogosa and Sharpe (MRS); HiMedia), Bifidobacterium spp. (Bifidobacterium agar; HiMedia) and Enterobacteriaceae (MacConkey agar; HiMedia). Agar plates for counting Lactobacillus spp. and Bifidobacterium spp. were incubated under anaerobic conditions (Anaerobic System Anaerogen; Oxoid Ltd) and agar plates for counting Enterobacteriaceae were incubated under aerobic conditions, all at 37°C for 24-48 h. At the end of the incubation period, characteristic colonies on the selective media were counted, and the results were expressed as log of colony-forming units per g of faeces (log 10 CFU/g) (37) . Total lipid in faeces and liver was determined by cold extraction (38) .

Faecal samples were collected on the three consecutive days before euthanasia for quantification of excreted fat or counting of the faecal microbiota population in both dams and offspring on the 30th and 90th days. Part of the faecal samples from dams and pups was used to quantify total lipids by cold extraction using a previously described procedure (29) . The other part of the faecal samples was homogenised in peptone water (100 mg/ml) and serially diluted in the same diluent. In all, 20 µl aliquots of the respective dilutions were inoculated using a microdrop technique (30) in Bifidobacterium agar (HiMedia) to count Bifidobacterium spp.; agar Man, Rogosa and Sharpe (HiMedia) to count Lactobacillus spp.; agar MacConkey (HiMedia) to count Enterobacteriaceae and agar Bacteroides Bile Esculina (Acumedia) to count Bacteroides spp. (Acumedia). Incubation was performed under anaerobic conditions (Anaerobic System Anaerogen; Oxoid Ltd) for counting Bifidobacterium spp., Lactobacillus spp. and Bacteroides spp. and under aerobic conditions for counting Enterobacteriaceae. After an incubation period of 24-48 h, the number of colonies on each selective medium was counted, and the results were expressed as log colony-forming units/g (15) .