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litters were inspected twice per day, and in each instance, live pups were counted and weighed. the weights of these pups were used to adjust the feed to achieve an average weight of 18. the rates of live birth and postnatal viability were calculated in three daily intervals (8 a.m. to 12 noon, 12 noon to 4 p., and 4 p. to 8 p.). the body weight, the rate of weight gain, and the body weight gain coefficients were calculated for each pup in these three periods, using the following equation:
mice from the control, or the diet-supplemented groups were euthanized and killed by cervical dislocation at 20 and 27 weeks. during this time, the dams were housed singly in standard polycarbonate cages with wood shavings as bedding. food and water were provided *ad libitum*.
the stomachs from the dams and pups were removed. pieces of the stomach, which was cut into two equal parts, were fixed in 10% buffered formalin for histological analysis. the other portion of the stomach was frozen at -20 °c for later biochemical analysis. the sections were cut at 6 μm and stained with hematoxylin and eosin (h&e) staining to determine morphological changes. histological analysis was carried out with an optical microscope. morphometric analysis of the size and percentage of the gastric mucosa, glandular area, and mucous cell area (gma) were calculated using the image-pro plus image analyser (image-pro plus 6.0, media cybernetics, inc., usa).
for biochemical analysis, the stomach was finely ground, and the homogenate was obtained in 0.1 m tris-hcl buffer (ph 7.4). the homogenate was centrifuged at 12,000 g for 30 min. the supernatant was used to determine protein concentration, which was estimated using the bradford method [ 59 ].
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