Effects Of Small Peptides On The Growth And Nutrient Utilization Of Largemouth Bass (Micropterus Salmoides)

The major source of particle pollutants in aquaculture water is the faeces,which are from the high-density breed.If not addressed,it will lead to water pollution and be hazardous to the health of animals.One of the important ways to solve the above problems is to add green feed additives to the diet,which can improve the feed conversion ratio and reduce nutrient residues in the feces.Largemouth bass（Micropterus salmoides）is one of the main aquaculture species in China.A kind of proteolytic product is called small peptides,which were formed by 2-10 amino acids.It is a small molecule with a variety of biological functions that are easily digested and absorbed by the body.Previous studies in our research team have found that adding 2%small peptides to the diet could increase the protease activity of the digestive system small peptides supplementation could improve the growth and health of largemouth bass.However,it is unclear whether feeding strategies need to be changed to promote the digestion and absorption of nutrients by the body.Therefore,the growth experiment was conducted to investigate the effect of small peptides on the growth of largemouth bass.The optimal mathematical model of gastric evacuation for largemouth bass was constructed by comparing the fitting degrees of different models.Investigating the change of digestive enzyme activities after feeding in largemouth bass.LC-MS was used to analyze the mechanism of action and metabolic pathways of small peptides in the intestine of largemouth bass,and to investigate how small peptides affect the digestion and absorption of nutrients in the intestine.The defecation regularity of largemouth bass and the nutrition leaching of feces in water is a preliminary inquiry.The objective of this experiment was to optimize the feeding strategy of largemouth bass and provide effective technical support for standardized breeding.1.Effects of small peptides on growth,nutrient availability and fecal dissolubility of Largemouth bass（Micropterus salmoides）and Snakehead fish（Channa Argus）This study was conducted to investigate the effects of small peptides on the growth,nutrient utilization,and nutrient leaching from the faeces of Largemouth bass（Micropterus salmoides）and Snakehead fish（Channa Argus）.D0⁺and W0⁺group were the positive control group to meet the nutritional requirements of largemouth bass and Snakehead fish,D0^-and W0^-group were the negative control group with 3%and 2%dietary crude protein reduced respectively based on the D0⁺group and W0⁺group,the D2 and W2 group were the experimental group with 2%small peptides added respectively based on D0^-and W0^-group.Largemouth bass and snakehead fish with a body weight of（21.27±0.17）g and（12.18±0.03）g were fed with the above diets for 60days.Results showed that:the growth performance of snakehead fish in three groups was similar to largemouth bass,the final weight and weight gain rate in negative control group was slightly lower than the positive control group and experimental group,but there was no significant difference among the three groups（P>0.05）.The phosphorus retention in D0⁺group was significantly lower than the D0^-and D2 groups（P<0.05）,and the phosphorus excretion was significantly higher than the D0^-and D2 groups（P<0.05）.The lipid retention and phosphorus excretion in W0⁺group were significantly lower than the other two groups（P<0.05）.By fitting with the model,within 75min,the leaching of NH₃-N and TP from the faeces of largemouth bass increased firstly and then tended to be stable,and there was no significant difference in the fitting curve of NH₃-N among all groups(F<F_（17,17）,P>0.05).There was no significant difference in the fitting curve of TP(F<F_（17,17）,P>0.05).The maximum leaching concentration of NH₃-N in the D2 group was 0.23 mg/g,which is the lowest among the three groups.The maximum leaching concentration of NH₃-N in the W0^-and W2 groups was both 0.14 mg/g,which was lower than the 0.19 mg/g in the W0⁺group;The maximum leaching concentration of TP in the W0^-group was higher than that of the W0⁺group and the W2 group,which were both0.06 mg/g.The results show that the change of feed crude protein content（blow 3%）or2%small peptides adding in the feed of largemouth bass and snakehead fish have no obvious effect on growth performance,after reducing dietary crude protein,the protein retention of largemouth bass and snakehead fish tended to increase,and nitrogen excretion tended to decrease.Adding 2.0%small peptide after reducing crude protein could increase the phosphorus retention in largemouth bass and snakehead fish,and the phosphorus excretion showed a decreasing trend.Under laboratory conditions,within75min,the leaching of NH₃-N and TP from the feces of largemouth bass and snakehead in the first 30min showed an increasing trend,and after 30min showed a trend of stability.Like largemouth bass,snakehead fish had no significant difference in the fitting curve of NH₃-N and TP leaching from faeces in water after feeding different diets(F<F_（17,17）,P>0.05).2.Gastric evacuation and changes of digestive enzyme activities in the gastroin-testinal tract of largemouth bass when fed diet with supplemental small peptidesIn order to investigate the effects of the small peptides additive on gastric evacuation rate and digestive enzyme activities along the gastrointestinal tract of largemouth bass,The largemouth bass with a body weight of（85.58±1.81）g fed by the three diets were selected and 80 largemouth bass in each group were placed into their respective small fish cages according to groups and time periods,with 10 largemouth bass in each fish cage.The gastric evacuation rates,fitting degree of the three mathematical models to the gastric emptying curve of each group and the changes of digestive enzyme activities of largemouth bass were compared after feeding the above three diets in different time periods.The results showed that the dry weight of stomach contents were better fitting to the mathematical models,the gastric evacuation rates and the optimal model of the largemouth bass were not significantly affected by varying protein levels and supplementation of small peptides in the diet,and the gastric emptying changes were curve digestive pattern（slow-fast-slow）during the gastric evacuation process with not entirely same trend.The linear,exponential,and square root models could fit the gastric emptying curve of largemouth bass well.The exponential model was the best model to simulate the gastric emptying pattern of the D0⁺group,and the square root model was the best model to simulate the gastric emptying pattern of the D0^-and D2groups.During 30 hours,the theoretical time of 50%and 80%gastric evacuation was8.14h and 19.04h in D0⁺group,11.28h and 21.4h in D0^-group,10.21h and 19.67h in D2group,according to the best model calculation in each group.The activities of digestive enzymes（amylase,protease and lipase）in the gastrointestinal tract in the three groups were firstly increased and then decreased during gastric evacuation,and the activities of amylase,lipase and protease in the gastrointestinal tract of D0⁺group reached the highest value at 9h after ingestion（P<0.05）.The activity of pepsin in D0^-and D2 groups reached the highest value at 9h after ingestion（P<0.05）,and the activities of amylase,lipase and protease in gastrointestinal tract reached the highest value at 12h after ingestion（P<0.05）.Largemouth bass may have four relative fecal peaks during the day.Peaks in defecation overlapped between groups.The results indicated that the change of dietary protein content（within 3%of the range）and the addition of 2%dietary small peptides could result in change of digestive enzymes activities,so it was not necessary to adjust the feeding frequency to keep the gastric emptying time relatively constant.Combined with the aquaculture practice,the feeding interval of largemouth bass was suggested to be 8-21h,twice a day.Under laboratory conditions,four relative defecation peaks were observed within 24h after feeding,ranging from 2-6h,7-10h,11-13h,and 18-22h after the first feeding.Therefore,we recommend removing the feces from the water after the two feedings in 3-6 hours and 2-5 hours before the first feeding the next day.3.Metabolic profile of intestinal contents in largemouth bass after ingestion of small peptide-containing dietsTo further investigate the effects of small peptides on intestinal metabolic profile of largemouth bass,the intestinal contents of largemouth bass were detected using LC-MC non-targeted metabolomics technique.Metabolomics analysis showed that there were significant differences between D2 group and D0⁺group in 7 metabolic pathways（P<0.05）,including Neuroactive ligand-receptor interaction,Aminoacyl-tRNA biosynthesis,ABC transporters,Primary bile acid biosynthesis,Intestinal immune network for IgA production,Vascular smooth muscle contraction and Purine metabolism.There were 10metabolic pathways significantly different between group D2 and group D0^-（P<0.05）.They are Arachidonic acid metabolism,Primary bile acid biosynthesis,Neuroactive ligand-receptor interaction,Retinol metabolism,Intestinal immune network for IgA production,ABC transporters,Linoleic acid metabolism,Steroid hormone biosynthesis,Glycerophospholipid metabolism and Aminoacyl-tRNA biosynthesis.According to the screening results and database comparison,23 different metabolites were identified between D2 group and D0⁺group,and 35 different metabolites were identified between D2 group and D0^-group.Compared with the other two groups,small peptide supplementation could down-regulate the contents of L-histidine,L-isoleucine,L-valine and homocysteine in intestinal tract,and up-regulate the contents of primary bile acids and Lipoxin A₄.These results indicate that the addition of small peptides can promote the absorption of amino acids to a certain extent,promote the intestinal production of relevant immune active substances,and reduce the harmful intermediate metabolites.