The Effects Of Fructooligosaccharide On Growth, Immune Responses, Antistress In Two Megalobrama Fishes

As the global aquaculture rapidly develops,the environment changes which subsequently adversely affects the aquatic animals.Accordingly,the aquaculture industry has been limited by outbreaks of infectious,particularly those caused by viruses and bacteria.It has led to the production loss and impeded the sustainable development of this industry.One of the most common ways to prevent aquatic diseases is to administer antibiotics.However,recently,there are strict regulations on the use of antibiotics in aquaculture because of its seriously negative effects.Therefore,it is becoming increasingly important to use preventive approaches to control diseases through improved immunity,pathogen inhibition and so on.FOS is non-digestible carbohydrates that allow specific changes in the composition and/or activity of gastrointestinal microbiota which has a positive effect on the nutrition and health status of the host.As one of the most studied prebiotics in fish,it has a broad application prospect.Megalobrama is a typical herbivorous freshwater fish which belongs to the Cyprinidae family.It has a high potential for intensive aquaculture since it shares many admirable characteristics such as rapid growth,high disease resistance,good taste and high market value.the rapid expansion of triangular bream aquaculture resulted in several serious diseases,mainly skin ulceration syndrome,which is highly infectious and lethal to this species,and leads to significant economic loss.Bearing this in mind,the present study was conducted to investigate the mechanism of fructooligosaccharide to Megalobrama amblycephala and Megalobrama terminalis.The immustimulants could enhance the immunity,antioxidant capabilities and disease resistance.The data obtained here may give some instructions for solving the disease and stress problems.This study has six parts.1 Effects of dietary fructooligosaccharide levels and feeding modes on growth,immune responses,antioxidant capability and disease resistance to Aeromonas hydrophila of blunt snout bream(Megalobrama amblycephala)This study aimed to determine the effects of fructooligosaccharide(FOS)levels and its feeding modes on growth,immune response,antioxidant capability and disease resistance of blunt snout bream(Megalobrama amblycephala).Fish(12.5±0.5g)were subjected to three FOS levels(0,0.4%and 0.8%)and two feeding modes(supplementing FOS continuously and supplementing FOS two days interval 5 days)according to a 3X2 factorial design.At the end of 8-week feeding trial,fish were challenged by Aeromonas hydrophila with concentration of 1×105 CFU mL-1 and mortality was recorded for the next 96 h.Fish fed 0.4%FOS continuously(D2)and fish fed the basal diet for 5 days followed by 0.8%FOS for 2 days(D5)showed admirable growth performance.The highest plasma lysozyme,acid phosphatase and myeloperoxidase activities as well as complement component 3,total protein and immunoglobulin M(IgM)levels were all observed in fish fed D5.They were significantly higher(P<0.05)than those of the control group and/or fish fed 0.8%FOS continuously,but exhibited no statistical difference(P＞0.05)with that of fish fed D2.A similar trend was also observed in antioxidant capability as well as the expression of Leap-I and Leap-Ⅱ.Mortality showed an opposite trend with the immune response with the lowest rate observed in fish fed D5.The results indicated that diet supplementing FOS in appropriate levels and feeding modes could improve the growth,immune response and antioxidant capability of fish,as might consequently lead to enhanced disease resistance.It can be speculated that the basal diet for 5 days followed by 0.8%FOS for 2 days was most suitable for blunt snout bream.2 The effects of fructooligosaccharide on the immune response,antioxidant capability and HSP70 and HSP90 expressions in blunt snout bream(Megalobrama amblycephalaYih)under high heat stressThis study evaluated the effects of fructooligosaccharide(FOS)on the immune response,antioxidant capability and HSP70 and HSP90 mRNA expressions in blunt snout bream(Megalobrama amblycephala)under high heat stress.A total of 360 fish were randomly distributed into three groups(each with four replicates)and fed three levels of FOS(0,0.4%and 0.8%)for 8 weeks.After the feeding trial,20 fish per tank were exposed to high heat stress at 34 0C.At 3 and 6 hours after stress,both the plasma cortisol and glucose levels of fish fed 0.4%FOS were significantly lower than the levels observed for the control group(P<0.05).However,this also held true for the lactate levels of fish fed 0.4%FOS,except that a significant difference(P<0.05)was observed at 3 h.Plasma lysozyme,acid phosphatase(ACP)and alternative complement(ACH50)activities,as well as total protein,immunoglobulin M(IgM)and nitrogen monoxide(NO)contents,increased significantly(P<0.001),with maximum levels attained at 6 h for all factors except the ACP activity.Thereafter,these parameters all decreased significantly(P<0.001).In addition,fish fed 0.4%or 0.8%FOS obtained significantly (P<0.05)higher lysozyme,ACP and ACH50 activities as well as total protein,IgM and NO contents at 3 and/or 6 h after stress.The liver SOD and CAT activities of fish fed 0.4%FOS were both significantly(P<0.05)higher than those of the control group before and after stress,while the opposite was true for the MDA content.After stress,the HSP70 and HSP90 expressions of fish fed 0.4%FOS were both significantly(P<0.01)higher than those of the control group at 3,6 and 12 h(except the HSP90 at 12 h).Similar results were also observed in fish fed 0.8%FOS except that the differences in HSP70 expression were only significant(P<0.01)at 3 and 12 h.The results indicated that the supplementation of 0.4%FOS could increase the non-specific immunity,antioxidant capacity and HSP70 and HSP90 expression of blunt snout bream and enhance its resistance to high heat stress.3 Effects of fructooligosaccharide on immune response,antioxidant capability and HSP70 and HSP90 expressions of blunt snout bream(Megalobrama amblycephala)under high ammonia stressThis study aimed to determine the effects of fructooligosaccharide(FOS)on immune response,antioxidant capability and HSP70 and HSP90 mRNA expressions of blunt snout bream(Megalobrama amblycephala)under high ammonia stress.A total of 360 fish were randomly distributed into three groups(each with four replicates)and were fed three levels of FOS(0,0.4%and 0.8%)for 8 weeks.After the feeding trial,24 fish per tank were exposed to ammonia at 10 mg L-1.After stress,plasma cortisol and glucose levels of fish fed 0.4%FOS were all significantly lower than that of the control group at 6 and 3 h,respectively.Plasma lysozyme and alternative complement pathway(ACH50)activities as well as nitrogen monoxide(NO)levels all increased significantly with the maximum levels being attained at 6,6 and 3 h,respectively.Thereafter,these parameters all decreased significantly.In addition,fish fed 0.4%FOS showed higher immune parameters under stress compared to that of control group.In addition,liver superoxide dismutase and catalase activities of fish fed 0.4%FOS were both significantly higher than that of the control group before and after stress,while the opposite was true for malondialdehyde content.After stress,the expression of HSP70 and HSP90 of fish fed FOS were both significantly higher than that of the control group at 6 and 12 h,respectively.After 12 h post-stress,the cumulative mortality of fish fed FOS was significantly lower than that of the control.The results indicated that the supplementation of 0.4%FOS could increase the non-specific immunity,antioxidant capacity and HSP70 and HSP90 expression of blunt snout bream,and enhance its resistance to high ammonia stress.4 Combined effects of dietary fructooligosaccharide and Bacillus licheniformis on growth performance,body composition,intestinal enzymes activities and gut histology of triangular bream(Megalobrama terminalis)This study was conducted to investigate the effects of fructooligosaccharide(FOS)and Bacillus licheniformis(B.licheniformis)on growth performance,body composition,intestinal enzymes activities and gut histology of Megalobrama terminalis.Nine experimental diets was formulated to contain three FOS levels(0,0.3%and 0.6%)and three B.licheniformis levels(0,1 and 5×107CFU g-1)following a 3×3 factorial design.Accordingly,diets were named as 0/0,0/3,0/6,1/0,1/3,1/6,5/0,5/3 and 5/6(B.licheniformis/FOS).At the end of the 8-week feeding trial,weight gain(WG)and specific growth rate(SGR)of fish fed 6 g kg-1 FOS were both significantly(P<0.01 and P<0.05)higher than that of the other groups in terms of dietary FOS levels.Besides,WG and SGR of fish fed 1×107 CFU g-1 B.licheniformis were significantly(P<0.05)higher than that of the control group in terms of dietary B.licheniformis levels.In addition,a significant interaction(P<0.05)between dietary FOS and B.licheniformis was observed in finial weight,WG,SGR as well as the survival rate with the highest values all observed in fish fed diet 1/3.Hepatosomatic index,carcass lipid content,lipase activities as well as microvilli length increased significantly(P＜0.05)from 0 to 1×107 CFU g-1,but no significant difference(P>0.05)was observed in terms of dietary FOS levels.In addition,a significant(P<0.05)interaction of FOS and B.licheniformis was observed in both protease and Na+,K+-ATPase activities with the highest value obtained in fish fed diet 1/3.The results indicated that the dietary applications of dietary FOS and B.licheniformis alone or in combination can significantly improve the growth performance,survival rate,intestinal enzymes activities as well as microvilli length of triangular bream.In addition,there is a significant interaction between dietary FOS and B.licheniformis.The best combination for this species is 0.3%FOS with 1×107 CFU g-1 B.licheniformis.5 Combined effects of dietary fructooligosaccharide and Bacillus licheniformis on innate immunity,antioxidant capability and disease resistance of triangular bream(Megalobrama terminalis)This study was conducted to investigate the effects of fructooligosacharide(FOS) and Bacillus licheniformis(B.licheniformis)and their interaction on innate immunity,antioxidant capability and disease resistance of triangular bream Megalobrama terminalis(average initial weight 30.5±0.5g).Nine experimental diets was formulated to contain three FOS levels(0,0.3%and 0.6%)and three B.licheniformis levels(0,1×107,5×107CFU g-1)according to a 3×3 factorial design.At the end of the 8-week feeding trial,fish were challenged by Aeromonas hydrophila(A.hydrophila)and survival rate was recorded for the next 7 days.The results showed that leucocyte counts,alternative complement activity as well as total serum protein and globulin contents all increased significantly(P＜0.05)as dietary B.licheniformis levels increased from 0 to 1×107CFU g-1,while little difference(P>0.05)was observed in these parameters in terms of dietary FOS levels.Both plasma alkaline phosphatase and phenoloxidase activities were significantly(P＜0.05)affected only by dietary FOS levels with the highest values observed in fish fed 0.6 and 0.3%FOS,respectively.Both immunoglobulin M content and liver superoxide dismutase(SOD)activity were significantly affected(P＞0.05)by both FOS and B.licheniformis.Liver catalase,glutathione peroxidase as well as plasma SOD activities of fish fed 1×107 CFU g-1 B.licheniformis were all significantly(P<0.05)higher than that of the other groups,whereas the opposite was true for malondialdehyde content.After A.hydrophila challenge,survival rate was not affected(P＞0.05)by either FOS levels or B.licheniformis contents,whereas a significant(P＜0.05)interaction between these two substances was observed with the highest value observed in fish fed 0.3%FOS and 1×107CFU g-1 B.licheniformis.The results of this study indicated that dietary FOS and B.licheniformis could significantly enhance the innate immunity and antioxidant capability of triangular bream,as well as improve its disease resistance.The best combination of these two prebiotics and/or probiotics was 0.3%FOS and 1×107CFU g-1 B.licheniformis.6 Cloning and expression of interleukin-6 gene in the liver of blunt snout bream(Megalobrama amblycephala)under ammonia stress and bacterial challengeIL-6 plays an important role as a prototypical proinflammatory cytokine in immune responses and inflammation in many cells and has been shown to affect macrophage functions.we identified IL-6 cDNA from blunt snout bream(Megalobrama amblycephala)and investigated its gene expression profiles under ammonia stress and bacterial challenge.The IL-6 sequence consisted of 1045 bp,including a 696 bp ORF which translated into a 232 amino acid(AA)protein.The protein contained a putative signal peptide of 24 AA in length.Gene expression by real time PCR analysis showed that the Il-6 gene is differentially expressed in various tissues under normal conditions and the highest IL-6 expression level was observed in the intestine tissue,followed by the liver,and then in the gills.Under ammonia stress,the expression profile of IL-6 mRNA level both in spleen and intestine increased significantly(P<0.05),with the maximum levels attained at 6h,12h respectively.Thereafter,they all decreased significantly(P<0.01)and returned to the basal value within 48 h.Whereas,in liver it slightly decreased at 3h firstly,and then significantly(P＜0.05)increased with the maximum levels attained 12h.Further expression analysis showed that the mRNA expression of IL-6 in spleen,intestine and liver of blunt snout bream all increased significantly(P<0.05),with maximum values attained at 6 h,3h,6h respectively after Aeromonas hydrophila(A.hydrophila)injection,and then decreased,which suggested that IL-6 was involved in the immune response to A.hydrophila.The cloning and expression analysis of the IL-6 provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions of blunt snout bream.