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Two studies were carried out to (a) characterize the gastrointestinal (GI) tract of bullfrog Rana (Lithobates) catesbeiana and (b) to evaluate the effects of total replacement of fish meal (FM) with soybean meal (SBM) on GI tract health. For characterization of the GI tract, oesophagus, stomach and intestine sections were sampled from 20 bullfrogs (80 g) fed a FM‐based diet. The results revealed that the GI tract of bullfrog is consisted of four tissue layers including mucosa, submucosa, muscularis and serosa, and that the intestine could be divided into duodenum, jejunum, ileum and rectum. Furthermore, the abundance and length of mucosal folds suggested that jejunum is the primary site of digestion and absorption of nutrients. In the second study, a diet containing 44% fish meal (FM) was formulated and used as a FM‐based diet, and a SBM‐based diet was prepared by entire substitution of FM with SBM. Sixty bullfrogs (35 ± 1 g) were divided into three replicates of the two groups and were fed the test diets to apparent satiation twice daily for 8 weeks. The changes in pH and morphology of GI tract in response to the shift in dietary protein source were evaluated. The results showed the enhancement of pH values in GI tract of the SBM group excluding cardia, jejunum and rectum. Also, SBM diet triggered significant decreases in the number, height and width of mucosal folds, while an opposite trend was observed for thickness of lamina propria. Moreover, the entire replacement of FM with SBM led to the separation of submucosa from muscularis and incidence of lesion in mucous epithelium. The findings in this study showed that jejunum is the main site of nutrients digestion and absorption. Also, it was demonstrated that entire replacement of FM with SBM adversely influenced the GI tract health in bullfrog.

Two consecutive feeding trials were conducted to evaluate the effects of Bacillus subtilis LCBS1 supplementation and substituting fish meal (FM) with fermented soybean meal (FSM) in terms of growth performance, feed digestibility, intestinal morphology, and microbial composition of bullfrogs. In trial 1, bullfrogs received either a soybean meal (SM)-based basal diet (control check, CK group) or probiotic diet (basal diet was supplemented with 1 × 107 CFU/g of B. subtilis LCBS1, BS group). Each diet was fed to three replicates of bullfrogs (29.61 ± 0.07 g) for 58 days. Evaluation of B. subtilis LCBS1 FSM as a FM replacer in bullfrog diet was conducted in trial 2. A diet containing 20% FM formed the basal diet, and 60% and 100% of the FM was replaced with SM or FSM (FM, SM60, FSM60, SM100, and FSM100 diets). Each diet was fed to three replicates of bullfrogs (23.3 ± 0.3 g) for 63 days. As a result, in trial 1, BS treatments significantly increased weight gain (WG) and whole-body protein deposition. Significant improvements in villus height (VH), muscularis thickness (MT), and digestibilities of dry matter (DM), crude protein (CP) and phosphorus (P) were observed in the BS group compared with the control group. Probiotic supplementation resulted in elevated IL-4 and IL-10 expression and decreased TNF-α and IL-17 expression. A remarkable decrease in serum D-lactate concentration and diamine oxidase activity was detected in the BS group. Proteobacteria, Fusobacteria, and Firmicutes were the three most abundant bacterial phyla in each group, while Enterobacter and Bacillus were the most abundant genera in the CK and BS groups, respectively. In trial 2, compared with bullfrogs that received the SM diets, bullfrogs fed either the FM or FSM diets showed greater WG, feed efficiency, hind leg index, whole-body CP content, and digestibilities of DM, CP, calcium, P, and phytate P, and increased VH, villus thickness, and MT. Serum urea nitrogen and ammonia concentrations were significantly higher in the SM group than the FM or FSM group. Overall, dietary B. subtilis LCBS1 supplementation improved growth and digestion in bullfrogs and attenuated the intestinal structural damage and microflora dysbacteriosis caused by SM-based diets. Substituting 60–100% of FM with SM impaired the growth performance, digestion ability, and jejunal morphology of bullfrogs, while replacing equivalent FM with FSM effectively ameliorated these traits.