Effects Of Feeding Frequency And Feed Protein Source On Growth Performance Of Grass Carp

Soybean meal is a kind of high quality plant protein raw material,and widely used in grass carp feed.But with the international trade dispute and the rapid development of aquaculture industry in China,it is urgent to find suitable protein material to replace soybean meal.The utilization efficiency of Soybean meal,Clostridium ethanol protein,Tenebrio molito,Chlorella,Cottonseed protein concentrate was evaluated by two-factor orthogonal test(3×5)at three feeding frequencies,The amino acid composition in muscle,free amino acid content in liver,hepatic and intestinal tissue structure and intestinal microbial diversity of grass carp were analyzed at feeding frequency for maximum growth performance.The main findings were as follows:1.Utilization of five non grain protein sources by grass carp at different feeding frequenciesA two-factor orthogonal experiment(3×5)was used to evaluate the utilization efficiency of Soybean meal,Clostridium ethanol protein,Tenebrio molito,Chlorella and Cottonseed protein concentrate for grass carps at three feeding frequencies.Five isonitrogenous and isoenergetic feeds were prepared with five raw materials as a single protein source,Grass carps with an initial body weight of 80g were fed at feeding frequency(2,3 and 4 times/d)for 8 weeks in a pond cage(1m×1.5m×1.5m),To investigate the growth performance,whole fish,muscle nutrient composition and serum biochemical indexes of grass carp.The results showed that grass carp in tenebrio molito test group(2 times/day)had the lowest growth performance;soybean meal(4 times/day)and clostridium ethanol protein(3 times per day and 4 times per day)groups showed the highest growth performance;Feeding frequency had interaction with protein source on final weight,feed conversion ratio,protein efficiency ratio and weight gain rate of grass carp(P<0.05).With the increase of feeding frequency,the overall end body weight and protein efficiency of grass carp tended to increase,but there were slight differences among protein source groups.Regardless of feeding frequency,The weight gain rate and protein efficiency rate in tenebrio molito and cottonseed concentrate protein groups were significantly lower than those in soybean meal,clostridium ethanol protein and chlorella groups(P<0.05).The feed conversion ratio showed an opposite trend(P<0.05).At different feeding frequencies,Five protein sources had no significant effects on body indexes(hepatosomatic index,viscerosomatic index and condition factor)and basic nutrients of whole fish and muscle of grass carp(P>0.05).Feeding frequency had significant effects on alkaline phosphatase and glucose in serum of grass carp(P<0.05),The alkaline phosphatase activity of 2 times/d was significantly higher than that of 3 times/d and 4 times/d(P<0.05),The glucose content of fish fed 4 times/d was significantly higher than that of fed 2 times/d and 3 times/d(P<0.05).The activities of alanine aminotransferase and aspartate aminotransferase in serum of grass carps in tenebrio molito group were significantly higher than those in soybean meal group and chlorella group(P<0.05).The results showed that increasing feeding frequency could improve the utilization efficiency of protein source(except chlorella)of grass carp(initial body mass 80g).When feeding frequency was 2 times/d,the utilization efficiency of grass carp to Chlorella reached the peak.Feeding frequency was 3 times/d,and the utilization efficiency of cottonseed protein concentrate and mealworms reached the peak.When feeding frequency was 4 times/d,the utilization efficiency of soybean meal and ethanol clostridium protein reached the maximum.The utilization capacity of five protein source feeds for grass carps was as follows:clostridium ethanol protein=soybean meal=chlorella>cottonseed protein concentrate>mealworm.2.Effects of Different protein sources on liver tissue structure,liver amino acid and muscle free amino acid composition of Grass carpIn order to explore the reasons for the difference in utilization efficiency of five different protein sources for grass carps,the effects of feeding frequency(4 times/d)on liver tissue structure,liver amino acid and muscle free amino acid composition of grass carp with maximum growth performance were analyzed.The results show that:The liver texture of grass carp in soybean meal,clostridium ethanol protein and chlorella group was basically intact;A large number of vacuoles appeared in the liver of grass carp of cottonseed protein group,and the liver cells were slightly swollen,with obvious vacuoles locally and nuclear deviation;A large number of vacuoles were found in the liver of grass carp in the tenebrio molito group,and the hepatocytes were severely enlarged,Compared with the normal hepatocytes,the vacuoles became bigger and round,and the nuclei were shifted more obviously.The contents of free amino acids in liver of clostridium ethanol protein group and cottonseed protein group were higher than those of tenebrio molito group and chlorella group.The free amino acid content in Tenebrio molito diet group was the lowest,and that in soybean meal diet group was the highest(P<0.05).There was no significant difference in total essential amino acid content in muscle of grass carp among experimental groups(P>0.05).The non-total essential amino acid content in control group was the highest and significantly higher than that in other four groups(P<0.05).These results showed that mealworm and cottonseed protein concentrate as a single protein source had a great negative effect on liver health of grass carp,and liver free amino acid concentration was positively correlated with growth performance of grass carp.3.Effects of different protein sources on intestinal morphology and intestinal microbe of grass carpIn order to explore the reasons for the difference in utilization efficiency of five different protein sources for grass carps,Intestinal tissue structure and intestinal microflora diversity of grass carp with maximum growth performance were detected and analyzed at feeding frequency(4 times/d).The results show that:The villus height of clostridium ethanol protein group was significantly higher than that of control group(P<0.05),and that of tenebrio molito group was significantly lower than that of control group(P<0.05);Ace,Chaol index and OTU values of clostridium ethanol protein were the highest,while those of tenebrio molito group were the lowest;Fusobacteriota,Fimicutes,Proteobacteria and Bacteroidota form the core microbial community.clostridium ethanol protein increased the abundance of Fimicutes and decreased the abundance of Proteobacteria.The tenebrio molito reduced the abundance of Fusobacteriota and Bacteroidota,and increased the abundance of Proteobacteria.There was no significant change among other groups.At the genus level,Cetobacterium and Firmicutes are the main microbial genera.clostridium ethanol protein increased the abundance of Firmicutes,while tenebrio molitos reduced the abundance of Cetobacterium.