Preliminary Study About The Regulation Of Dietary Fatty Acid On Immunity And Fatty Acid Metabolism In Large Yellow Croaker (Larmichthys Crocea)

This study was conducted to investigate the effects of dietay fatty acid ongrowth, fatty acid composition, nonspecific immnity, disease resistance and relatedgene expression in large yellow croaker (Larmichthys crocea). Based on thesestudies, it was aimed to investigate mechamisms involved in the regulation of dietaryfatty acid on immunity and fatty acid metabolism of large yellow croaker. The resultsand conclusions are summarized as follows:1. Effects of dietary n-3highly unsaturated fatty acids on growth, nonspecificimmunity, expression of some immune related genes and disease resistance oflarge yellow croaker (Larmichthys crocea)The study was conducted to investigate the effects of dietary n-3highlyunsaturated fatty acid (n-3HUFA) on growth, nonspecific immunity, gene expressionand disease resistance of juvenile large yellow croaker (Larmichthys crocea). Sixisoproteic and isolipidic diets were formulated with graded levels of n-3HUFAranging from0.15%to2.25%of the dry weight and the DHA/EPA was approximatelyfixed at2.0. Each diet was randomly allocated to triplicate groups of fish in floatingsea cages (1.0×1.0×1.5m), and each cage was stocked with60fish (initial averageweight9.79±0.6g). Fish were fed twice daily (05:00and17:00) to apparent satiationfor58days. Results showed that moderate n-3HUFA level (0.98%) significantlyenhanced growth compared with the control group (P<0.05), while higher n-3HUFAlevels (1.37%,1.79%and2.25%) had detrimental effects on the growth though nosignificance was found (P>0.05). Nitro blue tetrazolium (NBT) positive leucocytespercentage of head kidney and serum superoxide dismutase (SOD) activity increasedwith increasing n-3HUFA from0.15%to0.60%, and decreased with further increaseof n-3HUFA from0.60%to2.25%(P<0.05). Serum lysozyme activity increasedsignificantly as n-3HUFA increased from0.15%to1.37%, and then decreased withn-3HUFA from1.37%to2.25%(P>0.05). There were no significant differences in phagocytosis percentage (PP) of head kidney leucocytes among dietary treatments(P>0.05). The hepatic mRNA expression of Toll-like receptor22(TLR22) andMyeloid differentiation factor88(MyD88) was increased in fish fed the diets withlow or moderate levels, while in kidney this increment was only found at specificsampling time. The cumulative mortality rate after parasite infection test withIchthyophthirius marinus decreased with n-3HUFA increased from0.15%to0.60%(P<0.05), and significantly increased with n-3HUFA from0.60%to2.25%(P<0.05).Results of this study suggested that dietary n-3HUFA could significantly influencefish growth, nonspecific immune responses, immune related gene expression anddisease resistance of large yellow croaker and low n-3HUFA levels (0.60%and0.98%)may benefit the immunity and growth by upregulating the mRNA expression ofcertain immune related genes.2. Effects of dietary docosahexaenoic to eicosapentaenoic acid ratio (DHA/EPA)on growth, nonspecific immunity, expression of some immune related genes anddisease resistance of large yellow croaker (Larmichthys crocea) following naturalinfestation of parasites (Cryptocaryon irritans)The study was conducted to investigate the effects of dietary docosahexaenoic toeicosapentaenoic acid ratio (DHA/EPA) on growth, nonspecific immunity, immunerelated gene expression and disease resistance of juvenile large yellow croaker(Larmichthys crocea) following natural infestation of parasites (Cryptocaryonirritans). Five isoproteic and isolipidic diets were formulated with graded ratios ofDHA/EPA (0.61,1.54,2.17,3.04and3.88) and the total amount of n-3highlyunsaturated fatty acids (n-3HUFA) was approximately fixed at1.0%of the dry weight.Each diet was randomly allocated to triplicate groups of fish in floating sea cages(1.0×1.0×1.5m), and each cage was stocked with60fish (initial average weight9.8±0.6g). Fish were fed twice daily (05:00and17:00) to apparent satiation for58days. Results showed that specific growth rate (SGR) significantly increased from2.03%/d to2.26%/d (P<0.05) and then decreased with no significant differences(P>0.05). Serum lysozyme activity increased with increasing dietary DHA/EPA from0.61to3.04, and decreased with further increase of DHA/EPA from3.04to3.88(P<0.05). Nitro blue tetrazolium (NBT) positive leucocytes percentage of head kidneyshowed a similar changing trend with serum lysozyme activity with the highest value in the ratio of2.17, significantly higher than that in the control and ratio of3.88groups. Hepatic Toll-like receptor22(TLR22) and Myeloid differentiation factor88(MyD88) expression levels were significantly increased in fish fed higher DHA/EPA(3.04or3.88) at the early stage after natural infestation of parasites. At the later stageafter natural infestation of parasites, hepatic TLR22transcription was up-regulated infish fed moderate ratio of DHA/EPA (2.17). In kidney, the expression of TLR22wassignificantly up-regulated in fish fed moderate dietary DHA/EPA (2.17) at the earlystage after natural infestation of parasites. The13d cumulative mortality ratefollowing natural infestation of parasites decreased significantly with DHA/EPAincreased from0.61to3.04(P<0.05), and then increased with DHA/EPA from3.04to3.88(P>0.05). Results of this study suggested that fish fed moderate or higherDHA/EPA had higher growth, nonspecific immunity, immune related gene expressionand disease resistance following natural infestation of parasites and dietary DHA/EPAmay regulate fish immunity and disease resistance by altering the mRNA expressionlevels of TLR22and MyD88.3. Effects of conjugated linoleic acid (CLA) on growth, nonspecific immunity,antioxidant capacity, lipid deposition and related gene expression in juvenilelarge yellow croaker (Larmichthys crocea) fed soybean oil based dietsThe effects of conjugated linoleic acid (CLA) on growth performance,nonspecific immunity, antioxidant capacity, lipid deposition and related geneexpression were investigated in large yellow croaker, Larmichthys crocea. Fish (7.56(SEM0.60) g) were fed soybean oil based diets with graded levels of CLA (0,0.42,0.83,1.70%) for70days. Quantitative PCR was used to assess the effects of CLA ontranscription of inflammation and fatty acid oxidation related genes. Results showedthat growth in fish fed the diet with0.42%CLA was significantly higher. Also,phagocytic index and respiratory burst activity were significantly higher in treatmentsof0.42%and0.83%CLA, respectively. Hepatic total anti-oxidative capacity andcatalase activities increased significantly when CLA increased from0%to0.83%, andthen decreased with further increase of CLA. While hepatic maleic dialdehyde contentdecreased significantly as dietary CLA increased. Lipid concentration in whole bodyand muscle increased significantly with increasing dietary CLA. Transcription ofgenes related with inflammation (cyclooxygenase-2and IL-β) in liver and kidney and fatty acid oxidation (carnitine palmitoyl transferase Ⅰ and acyl CoA oxidase) inkidney decreased significantly as dietary CLA increased. PPARα and acyl CoAoxidase expression in liver decreased significantly as CLA increased from0.42%to1.70%. These results strongly suggested that dietary CLA could significantly affectgrowth performance, nonspecific immunity, antioxidant capacity, lipid deposition andtranscription of inflammation and fatty acid oxidation related genes of large yellowcroaker. This may contribute to understanding the mechanisms on the physiologicaleffects of dietary CLA in fish.4. Effects of dietary ratio of alfa-linolenic to linoleic acid (ALA/LA) on growth,nonspecific immunity, antioxidant capacity and related gene expression injuvenile large yellow croaker (Larmichthys crocea)Fish oil has seen a great shortage with the fast development of aquatic feedindustry. High inclusion of linoleic acid rich oils has deleterious effects on growth andhealth of marine fish species. However, it is still unclear whether these negativeeffects could be eliminated by enhancing dietary alfa-linolenic to linoleic acid ratio(ALA/LA). Thus, this study was conducted to investigate the effects of dietaryALA/LA on growth performance, nonspecific immunity, antioxidant capacity, lipiddeposition and related gene expression in juvenile large yellow croaker, Larmichthyscrocea. The control diet (FO) was formulated with30%fish meal and9%fish oil.Then, four other diets were formulated by replacing78%fish oil in the FO withvegetable oil to acquire graded ratios of ALA/LA (0.03,0.45,0.90and1.51), whichwere named R-0.03, R-0.45, R-0.90and R-1.51, respectively. Fish were fed twicedaily (05:00and17:00) for70days. Results showed that experimental fish hadsignificantly lower weight gain rate (WGR) and worse nonspecific immunity after78%fish oil was replaced by sunflower oil. However, the negative effects of LA onWGR and nonspecific immunity could be completely eliminated as dietary ALA/LAincreased to0.45and0.90, respectively. Anti-oxidative capacity in liver was increasedto some extent after78%fish oil was replaced by vegetable oil. Hepatic superoxidedismutase and catalase activities increased significantly when ALA/LA increasedfrom0.03to0.45, and then decreased with further increase of this ratio. While hepaticmaleic dialdehyde content first decreased with dietary ALA/LA increasing from0.03 to0.90, and then increased as this ratio increased to1.51(P>0.05). Transcription ofgenes related with inflammation (cyclooxygenase-2, IL-1β and TNF-α), fatty acidoxidation (carnitine palmitoyl transferase Ⅰ and acyl CoA oxidase), and fatty acidsynthesis (fatty acid synthase and malic enzyme) in liver and kidney was significantlyincreased in R-0.03, and then decreased to comparable levels in FO as dietaryALA/LA reached equal to or above0.45. These results strongly suggested that dietaryALA could exert some similar properties like n-3HUFA at least from the points ofinflammation, fatty acid oxidation and synthesis. This may contribute tounderstanding the mechanisms on the physiological effects of dietary ALA/LA in fish.5. Molecular cloning, characterization, and nutritional regulation of fatty acyl Δ6desaturase and elovl5elongase of large yellow croaker, Larmichthys croceaIn this study, the full-length cDNA of fatty acyl Δ6desaturase and elovl5elongase were first cloned from the large yellow croaker, Larmichthys crocea. Thefatty acyl Δ6desaturase was2049bp, with a107bp5′-UTR, a604bp3′-UTR, and anORF of1338that specified a protein of445amino acids. The fatty acyl elovl5elongase was1551bp, including a5′-terminal untranslated region (UTR) of120bp, a3′-terminal UTR of546bp and an open reading frame (ORF) of885bp encoding apolypeptide of294amino acid residues. Sequence comparison and phylogeneticanalysis showed that the two enzymes belong to the fatty acyl Δ6desaturase andelovl5elongase family, respectively. Tissue distribution analysis revealed that theLcElovl5expression level was higher in liver, brain and gill while lower in stomach,intestine, heart and spleen. Then two58d feeding experiments were conducted toinvestigate the effects of dietary n-3long chain polyunsaturated fatty acid (n-3HUFA)levels (0.15,0.60,0.98,1.37,1.79and2.25%) and docosahexaenoic toeicosapentaenoic acid ratio (DHA/EPA)(0.61,1.54,2.17,3.04and3.88) on thehepatic expression levels of LcElovl5in large yellow croaker juveniles. The resultsshowed that the transcriptional levels of LcElovl5decreased significantly with theincreasing dietary n-3LC-PUFA (P<0.05). The LcElvol5transcript levels wereup-regulated by1.77-fold,1.41-fold, and1.41-fold in the level of0.15%,0.60%and0.98%n-3LC-PUFA treatments compared with the control group (2.25%n-3LC-PUFA), respectively. The hepatic expression levels of LcElovl5transcriptshowed an increasing tendency in response to the increased dietary DHA/EPA though no significance was observed among dietary groups (P>0.05).