Effects Of Soybean Meal Replacement Fish Meal On Bullfrog Rana (Lithobates) Catesbeiana Growth,Intestinal Health,Intestinal Phytase Activity And Gene Diversity

A feeding trial was conducted to investigate the effect of replacement fish meal by soybean meal on growth performance,apparent digestibility,digestive enzyme activity,intestinal health,the phytase activity and the genetic diversity of phytase in intestinal mucous of bullfrog.(1)Effects of replacement fish meal by soybean meal on growth performance and feed utilization rate of bullfrogBullfrogs with an initial body weight of(40 ± 0.3 g)were randomly distributed into 15 fiberglass tanks.There were 10 bullfrogs each tank..Each diet was randomly assigned to 5 tanks for 30 days to determine the growth performance and feed utilization of bullfrog.The results showed that there was no significant difference in the survival rate among all groups.Weight gain,feed efficiency and protein efficiency of bullfrogs in SM50 and SM100 groups were significantly lower than FM group(P<0.05).And the similar tendency was showed for apparent digestibility of dry matter,protein gross energy and calcium in all groups.The apparent digestibility of phosphorus of bullfrogs in SM100 was significantly lower than that in FM and SM50 groups(P<0.05),and there was no significant difference between FM and SM50 groups.Bullfrog in SM100 group showed lowest protease activity in the jejunum.The lipase activities in the ileum of SM50 and SM100 groups were significantly lower than that of the FM group,and the amylase activities in the ileum of SM50 and SM100 groups were significantly higher than that of the FM group(P<0.05).(2)Effects of replacement fish meal by soybean meal on intestinal health of bullfrogThe intestinal histomorphology,the expression of intestinal pro-inflammatory factors and the diversity of intestinal mucous microorganisms of bullfrog were studied after the culture experiment.The results showed that the height of the intestinal villi,the thickness of the muscular layer and the height of the columnar epithelium were significantly reduced with the increase of fish meal being replaced by soybean meal(P<0.05).The expression levels of intestinal pro-inflammatory factors IL-1β,IL-17,IL-8,and TNF-α were significantly increased with the increase of the replacement(P<0.05).Diversity of intestinal microorganisms decreased after soybean meal replaced fish meal.More than 99% bacteria is Fusobacterium,Firmicutes and Proteobacteria on the intestinal mucos of bullfrog.It was increased in the abundance of pathogens(Edwardsiella,Enterobacteriaceae and Vibrio)and decreased in the abundance ofprobiotics(Bacillus and Lactobacillus)in intestinal mucus of bullfrogs fed whole soybean meal.(3)Effects of replacement fish meal by soybean meal and oral antibiotics on the intestinal phytase activity of bullfrogThe experimental diets were same as the experiment one.There were 9 groups in this experiment(FM,SM50,SM100,FM+A,SM50+A,SM100+A,FM+A+S,SM50+A+S and SM100+A+S).Each group had 3 replicates.Each diet was randomly assigned to 9 tanks for 30 days.After feeding trial,the bullfrogs in FM,SM50 and SM100 groups were conducted to investigate intestinal phytase activity,the bullfrogs in FM+A and SM50+A,SM100+A groups were conducted to investigate intestinal phytase activity after oral antibiotics for 4 days under feeding condition,and the bullfrogs in FM+A+S,SM50+A+S and SM100+A+S groups were conducted to investigate intestinal phytase activity after oral antibiotics for 4 days under fasting condition.The results showed that the phytase activity in ileum mucous of bullfrog was higher than that in jejunum mucous under the same feeding conditions without antibiotics,the average values were 39.76 and 25.52 nmol/mgprot/min,respectively.Replacing fish meal by soybean meal enhanced the phytase activity in jejunum and ileum of bullfrog.Antibiotics under satiety decreased the activity of phytase in ileum and jejunum mucous of bullfrogs in SM50+A and SM100+A groups(P < 0.05).Protein substitution level and administration of antibiotics had significant interaction on intestinal phytase activity of bullfrog.There was no significant effect of soybean meal substitution level on intestinal phytase activity in bullfrog(P > 0.05)under the same condition of antibiotics.Phytase activity in jejunum and ileum mucous of bullfrog was significantly lower in starvation than that in satiety(P < 0.05).Feeding conditions and protein substitution levels had significant interaction on intestinal phytase activity(P < 0.05).So it could be concluded that the phytase activity in intestine of bullfrog was related to intestinal microorganisms.The phytase activity was 16.46-26.41 nmol/mgprot/min in the intestinal mucous of bullfrog fed with antibiotics.(4)The genetic diversity of phytase in intestinal mucous of Rana catesbeiana in SM100groupThe phytase produced by Bacillus belongs to Beta-folding phytase(BPP).The BPP phytase gene primers were designed consider the conserved regions of phytase gene of Bacillus spp.At last we got a pair of available primers which length was 522 bp,and the optimum Tm value was57℃.The Diversity of phytase genes were studied in the genomic DNA of the bullfrog ileum,Bacillus subtilisand and Enterobacter cloacaeas.Bacillus subtilisand and Enterobacter cloacaeas was come from the bullfrog intestine.11 phytase gene sequences were obtained from Bacillus subtilis DNA.10 phytase gene sequences were obtained from Enterobacter cloacae DNA.6 phytase gene sequences were obtained from the genomic DNA of the bullfrog ileums.The above sequences were compared with known amino acid sequences of phytase from Bacillus.The results showed that the sequences were located in the functional region of phytase and contained the functional sites [D/A] [STA] DDPA [I/V] W [I/V/L] T [N/D/L] K.Therefore,it can be inferred that BPP phytase gene has intraspecific polymorphism and interspecific diversity.