Knowledge of how novel antigens or dietary stimuli affect stomach development and function in pigs remains limited. This study aimed to investigate stomach characteristics, parietal cell numbers, and the expression of genes essential to the functioning of the fundic and pyloric gland regions at weaning compared to seven days post-weaning and to examine whether maternal probiotic supplementation or piglet dietary tryptophan (Trp) levels influence these stomach parameters. This study has a 2 × 3 factorial design, with 48 sows assigned to one of two diets: basal or basal supplemented with
Bacillus subtilis and
Bacillus amyloliquefaciens. Their litters received creep diets containing 0.22, 0.27, or 0.33% standardized ileal digestible (SID) Trp. In total, 96 pigs were sacrificed for gastric sampling, 48 on the day of weaning and 48 on day 7 post-weaning. At 7 days post-weaning, pigs had an increased number of parietal cells and expression of parietal cell activity and digestive enzyme (
PGA5 and
CHIA) genes in the fundic gland region (
p < 0.05), although the expression of signaling molecules involved in the regulation of acid secretion was unchanged in the fundic gland region (
p > 0.05) and reduced in the pyloric gland region (
p < 0.05), compared to the day of weaning. Overall, maternal probiotic supplementation had a significant impact on gene expression in the fundic gland region of the offspring, elevating several genes related to parietal cell activity (
CLIC6,
HRH2,
KCNE1,
KCNQ1,
CHRM3, CCKBR, and
SSTR2) (
p < 0.05). Additionally, there were time × maternal interactions, where certain acid secretion pathway (
ATP4A and
HDC), chitinase enzyme (
CHIA), and ghrelin (
GHRL) genes were increased in offspring from probiotic sows compared to control sows at weaning (
p < 0.05), but not at 7 days post-weaning (
p > 0.05). Maternal probiotic supplementation did not influence growth performance pre-weaning or during the 7-day post-weaning period. There was a limited effect of creep Trp level or maternal × creep interactions on performance, gene expression, or parietal cell counts. Low pre-weaning creep intake may have confounded this analysis. In conclusion, maternal probiotic supplementation accelerated the maturation of the offspring’s stomach, particularly in terms of the expression of genes linked to acid secretion from parietal cells.
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