| Fish oil is the main source of fat for farmed fish.However,fish oil is prone to oxidatize,producing oxidizing substances that are harmful to aquatic animals.In this study,pearl gentian grouper（♀Epinephelus fuscoguttatus×♂Epinephelus lanceolatus）was selected as the research object,and the fresh fish oil group（containing 0%oxidized fish oil,named R）and the low-level oxidized fish oil group（containing 3%oxidized fish oil,named L）,moderately oxidized fish oil group（containing 6%oxidized fish oil,namred M）and highly oxidized fish oil group（containing 9%oxidized fish oil,named H）were set up for a 65-day feeding experiment to study the effect of dietary oxidized fish oil on growth performance,physiology and biochemistry,histopathology,nutritional composition,intestinal flora,and oxidative stress mechanism of important organs in pearl gentian grouper.The significance of this study lies in the use of transcriptomics,16S high-throughput sequencing and metabolomic techniques to further elucidate the effect of oxidized fish oil on the growth,immunity and quality of pearl gentian grouper.The specific results are as follows:（1）Effects of oxidized fish on growth performance,body composition and serum biochemistry of pearl gentian grouper.（1）Final body weight（FBW）,weight growth rate（WGR）,specific growth rate（SGR）and condition factor（CF）in M and H groups were significantly lower than those in R and L group（P<0.05）,while the feed conversion ratio（FI）of M and H groups was significantly higher than that of R and L groups（P<0.05）,the survival rate（SR）was not affected by dietary oxidized fish oil level（P>0.05）.（2）The moisture content of whole body in H group was significantly higher than that in R group（P<0.05）.The lipid content（CL）in H group was significantly lower than that in R group,L group and M group（P<0.05）.（3）Serum superoxide gasification enzyme（SOD）,catalase（CAT）,glutathione peroxidase（GSH-Px）activities and vitamin E content decreased significantly with increasing proportion of oxidized fish oil in diets（P<0.05）,while the content of malondialdehyde（MDA）in serum showed the opposite trend.（4）Oxidized fish oil significantly increased serum D-lactic acid（D-LA）and interleukin 10（IL-10）levels（P<0.05）,significantly decreased serum interleukin 6（IL-6）levels（P<0.05）,while serum endotoxin（LPS）,interleukin-8（IL-8）and tumor necrosis factor-α（TNF-α）levels were not affected by dietary oxidized fish oil level（P<0.05）.（2）The effect of oxidized fish oil on the gut health and flora spectrum of pearl gentian grouper.（1）SOD,GSH-Px and CAT activities,vitamin E content in foregut,midgut and hindgut decreased significantly with the increasing of dietary oxidized fish oil level（P<0.05）;MDA content in intestine showed the opposite trend（P<0.05）.（2）The intestinal protease of M and H group was significantly higher than that of R and L group（P<0.05）;The intestinal amylase and lipase in H group were significantly higher than those in R group（P<0.05）.（3）In H group,the foregut microvilli showed atrophy,the midgut microvilli were damaged,and the hindgut villi were inflamed.（4）There were a total of 18 differentially expressed genes in the gut transcriptome.According to GO enrichment,the functions of differential genes were mainly enriched in primary metabolic process,cellular biosynthesis process,organic matter biosynthesis process,cellular macromolecular metabolism process and protein metabolism process.There were two important pathways including the sulfur metabolism pathway and the P53 signaling pathway,involving the cytochrome c（Cyt2c）gene which was highly expressed under oxidative stress.（5）16S sequencing analysis showed that,compared with R group,the ACE index of H group was significantly decreased（P<0.05）,the Chao index was significantly decreased（P<0.05）,and the Simpson index and Shannon index were significantly decreased（P<0.05）.In the Line Discriminant Analysis Effect Size（LDAES）analysis,the marker flora of the fresh fish oil group was Deltaproteobacteria,s＿uncultured＿bacterium＿g＿Lachnosp-iraceae＿NK4A136＿group,g＿Lachnospiraceae＿NK4A136＿group and Veillonellaceae;In H group,Staphylococcus＿sciuri,Rubritaleaceae and Staphylococcaceae were the hallmark gut flora.From the results of 16S sequencing,it can be concluded that highly oxidized fish oil reduces the number of intestinal flora of pearl gentian grouper,and increases the intestinal flora of harmful bacteria.（3）Oxidized fish oil induced oxidative stress and impaired its function in the liver of pearl gentian grouper.（1）With the increase of the proportion of oxidized fish oil in diet,SOD,CAT and GSH-Pxactivities,vitamin E content in liver decreased significantly（P<0.05）;MDA content and total antioxidant capacity（T-AOC）in liver were significantly increased（P<0.05）.（2）IL-8 content in liver of M group was significantly lower than that in other three groups（P<0.05）;IL-10 and TNF-αcontents in liver increased significantly with the proportion of oxidized fish oil in diets（P<0.05）.（3）Serum total cholesterol（TC）and triglyceride（TG）contents were not affected by dietary oxidized fish oil level（P>0.05）;With the increase of the proportion of oxidized fish oil in diets,the content of low-density lipoprotein（LDL）in serum was significantly decreased（P<0.05）,while the content of high-density lipoprotein（HDL）in serum was significantly increased（P<0.05）.（4）Moisture and ash content in liver were not affected by dietary oxidized fish oil level（P>0.05）;The liver CP content of R group was significantly higher than that of L,M and H groups（P<0.05）,but there was no significant difference of liver CP content among L,M and H groups（P>0.05）.The CL content of H group was significantly higher than that of R group（P<0.05）,but there was no significant difference of CL content among R,L and M group（P<0.05）.（5）In terms of liver fatty acid composition,vast majority of fatty acids were not affected by dietary oxidized fish oil level（P>0.05）.The content of hepatic eicosenoic acid（C20:1n9）in L group was significantly lower than that in R group（P<0.05）,but there was no significant difference compared with M and H group（P>0.05）.（6）Oxidative stress leads to the accumulation of fat in the liver,damages the structure of liver tissue,and changes the ultrastructure of liver cells.（7）liver transcriptomic profiles analysis showed that the liver functions primarily in metabolic processes under oxidative stress.Specifically,the liver responds to oxidative stress primarily through glycine,serine,and threonine metabolism,arginine biosynthesis,and glycerophospholipid metabolism.（8）Hepatic metabolic profile analysis showed that the differential metabolites in liver were phosphatidylcholine,phosphatidylethanolamine,and lysophosphatidylcholine,which upregulated under oxidative stress.The liver responded to oxidative stress through glycerophospholipid metabolism and cancer choline metabolism pathways.（4）The effect of oxidized fish oil on muscle structure and qulity of pearl gentian grouper.（1）With the increase of dietary oxidized fish oil level,SOD,CAT and GSH-Px activities,vitamin E content in muscle decreased significantly（P<0.05）;MDA content and T-AOC in muscle were significantly increased（P<0.05）.（2）The muscle fibers in the R group had uniform gaps,and the cross-sections were mostly polygonal.The muscle fibers in L group had large gaps and were loosely arranged.The muscle fibers in M group had large gaps and were loosely arranged,and the cross-sections of the muscle fibers were mostly oval.The cross-section of muscle fibers in H group was oval or oblong,with enlarged muscle fibers and larger muscle fiber gaps.（3）There was no significant difference of muscle moisture among R,L and M group（P>0.05）,but moisture content of H group was significantly higher than that of R and L group（P<0.05）.There was no significant difference of muscle CP content among treated groups（P>0.05）.The muscle CL content in H and M groups was significantly lower than that in R group（P>0.05）,while muscle CL content between R and L group was not significant difference（P>0.05）.The muscle ash content in H group was significantly lower than that in R group（P<0.05）,and there was no significant difference of ash content among R,L and M groups（P>0.05）.（4）Most amino acids in muscle were not affected by oxidized fish oil,but methionine（Met）,phenylalanine（Phe）,and histidine（His）were significantly affected by oxidized fish oil（P<0.05）.Met in R group was significantly higher than that in other three groups（P<0.05）,and Phe in R group was significantly higher than that in L and H groups（P<0.05）.His decreased significantly with the increasing of dietary oxidized fish oil（P<0.05）.Most fatty acids were not affected by oxidized fish oil（P>0.05）.（5）A total of 43,469unigenes were obtained from the muscle transcriptome,of which 76 were differentially expressed genes.There are 3 important pathways including vitamin B6metabolism,amino acid biosynthesis and P53 signaling pathway,of which the important pathways involve 5 different genes,including phosphatase,phosphoglycerate mutase 2（PGAM2）,S-adenosylmethionine Synthase isoform 2（SAM2）,shisa-5-like protein（SHISA5）,and growth arrest and DNA damage-inducing protein GADD45β-like protein（GADD45β）.（5）The effect of oxidized fish oil on the spleen function of pearl gentian grouper.（1）There was no significant difference in the spleen index among the L,M,H and R groups（P>0.05）,while the spleen index in the M group was significantly greater than that in the H group（P<0.05）.（2）The spleen SOD activity of R group and L group was significantly higher than that of M group and H group（P<0.05）;the CAT activity of R group and L group was significantly lower than that of H group（P<0.05）;GSH-Px activity increased significantly with the increase of dietary oxidized fish oil（P<0.05）;The vitamin E content in M and H group was significantly lower than that in R and L group（P<0.05）;The MDA content of R group and L group was significantly lower than that of H group（P<0.05）.（3）In R group,the boundary between red pulp and white pulp was obvious,lymphocytes were numerous and dense,arterioles were clear,and blood sinuses were full of blood cells;In L group,the boundaries of red and white pulp were blurred,the lymphocytes were numerous and dense,the arterioles were clear,and the blood sinuses were full of blood cells;In M group,the boundaries of red and white pulp were blurred,lymphocytes were sparse,small arteries were occluded,and blood sinuses began to decline;In H group,the boundaries of red and white pulp were further blurred,lymphocytes were sparse,arterioles were also blocked,and the number of blood cells in the blood sinus was further reduced.（4）Transcriptional profiles analysis showed that spleen responds to oxidative stress through platelet activation,FOXO and Notch signaling pathways involving amyloid beta precursor protein binding family B member 1 interacting protein（APBB1IP）gene,glucose-6-phosphatase（G6PC）gene,histone acetyl transfer Enzyme p300（EP300）gene,insulin gene and Notch 2 gene.In conclusion,oxidized fish oil induced oxidative stress in pearl gentian grouper and inhibited growth performance.Oxidative stress damaged intestinal tissue structure and altered intestinal transcriptional profile and intestinal flora.Oxidative stress damaged hepatic lipid metabolism and tissue structure,and changed liver transcriptional profile and metabolic profile;Oxidative stress damaged muscle fiber structure,and changed muscle nutritional composition and transcriptional profile;Oxidative stress damaged spleen tissue structure and changed transcriptional profile. |
PDF Full Text Request