Screening And Preliminary Application Of Immunopotentiators For Cold-Water Fish

In recent years,the cold-water fish farming industry has developed rapidly in Guizhou province.But due to a series of problems such as increasing breeding scale and density,and lagging breeding management,some problems such as deteriorating water environment of cold-water fish farming and disease were increasingly prominent,which has severely restricted the healthy development of the cold-water fish farming industry.The current major control measure to prevent and control diseases of cold-water fish is using antibiotics and chemicals,which would cause drug residues and bacterial resistance if using irrationally.The immunopotentiator has become a research hotspot in the field of aquaculture disease prevention because it has the effect of strengthening the autoimmune system,and has the characteristics of high efficiency,no pollution and no residue.In this paper,the sturgeon,a representative of cold-water fish farmed in Guizhou Province,was selected as the target.In vitro screening and in vivo application were conducted to carry out screening and preliminary application research of cold-water fish immunopotentiators,in order to provide technical supports for the healthy development of the cold-water fish farming industry in Guizhou province.1.Immunopotentiators screening in vitro（1）Isolation condition optimization of leukocytes from peripheral blood from sturgeons:The peripheral blood of sturgeons were collected.The number and the viability of the isolated leukocytes were used as indicators.On the basis of single factor experiment,orthogonal experiment was designed to determine the optimal combination of factors in the separation conditions of peripheral blood leukocytes from sturgeons.Factors of L₉（3³）orthogonal experiment include percoll density（67%,72%and 77%）,centrifugation speed（400×g,600×g and 800×g）and centrifugation time（20 min,25 min and 30 min）.Through range analysis and variance analysis,the results showed that:When percoll density was 72%,centrifugation speed was 800×g and centrifugation time was 20 min,the number of leukocytes in peripheral blood of sturgeon was（4.13±0.12）×10⁶/m L,and the leukocyte activity rate was（99.10±0.11）%.That is to say,under this condition,the number and cell viability of the isolated leukocytes from peripheral blood of sturgeon are the best.（2）The sturgeon immunopotentiators screening in vitro:The peripheral blood leukocytes of sturgeon were prepared under the above optimized conditions,and treated with ten immunopotentiators at eight concentrations.Then the respiratory burst activity and phagocytic activity were detected.The results showed that:Compared with the control group,the SPO values,in groups ofβ-glucan,betaine,thymosin,curcumin,taurine,chlorogenic acid and chitooligosaccharide improved significantly or extremely significant（P<0.05 or P<0.01）.The STI values,in groups of thymosin,chlorogenic acid and chitooligosaccharide improved extremely significant（P<0.01）.The NBT_B%and NBT_C%values,in groups of thymosin,chlorogenic acid and chitooligosaccharide,were more than 100%.Compared with the control group,at partial concentration,some OD_510nmvalues of phagocytic activity,in groups ofβ-glucan,thymosin,taurine,soybean isoflavone,chlorogenic acid,chitooligosaccharide,levamisole,betaine,cod-liver oil and curcumin improved significantly or extremely significant（P<0.05 or P<0.01）,but only thymosin,chlorogenic acid and chitooligosaccharide,were more than 1.7.In summary,thymosin,chlorogenic acid and chitooligosaccharide can be selected as immunopotentiators for cold-water fish sturgeon.2.Preliminary application study of immunopotentiatorsThe three immunopotentiators（thymosin,chlorogenic acid,and chitooligosaccharide）were respectively added at the concentration of 200 mg/kg to the basal feed,basic feed group（control group）was set up at the same time.Samples were taken for detection on 0d,14d,28d and 42d,then sturgeons growth,tissue structure,biochemical index and immune index were detected and compared.The results showed that:（1）Growth performance index:Compared with the control group,thymosin,chlorogenic acid,and chitooligosaccharides have increased significantly or extremely significant（P<0.05 or P<0.01）the weight gain rate and specific growth rate,but have no significant（P>0.05）effect on condition factor,visceral index and spleen index.However chitooligosaccharide can reduce the liver index significantly（P<0.05）.（2）Observation on tissue structure:Compared with the control group,only sturgeons liver in thymosin group appeared cytoplasmic lysis and blurred cell boundary on 28d,and appeared nucleolysis,blurred cell boundary and vacuolation on 42d.All the three immunopotentiators increased the number of periarterial lymphatic sheaths and periellipsoid lymphatic sheaths,with a larger shape and a closer arrangement.Both chlorogenic acid and chitooligosaccharide can reduce the degree of vacuolar degeneration of intestinal villous epithelial cells,improve the villous damage,and increased villous length,V/C ratio and intraepithelial lymphocyte number significantly or extremely significant（P<0.05 or P<0.01）.Only the chlorogenic acid group’s crypt depth reduced significantly（P<0.05）.（3）Routine biochemical index:Compared with the control group,thymosin,chlorogenic acid and chitooligosaccharide can improved the serum total protein,albumin and globulin extremely significant（P<0.01）,but thymosin will reduce serum globulin significantly on 42d（P<0.05）,chlorogenic acid and chitooligosaccharide reduced uric acid,urea nitrogen,alanine aminotransferase and aspartate aminotransferase of serum significantly（P<0.05）,the thymosin will reduce serum urea nitrogen significantly on 14d（P<0.05）,but it also improved alanine aminotransferase levels significantly on 42d（P<0.05）.（4）Oxidation and Antioxidation index:Compared with the control group,in thymosin group,the concentration of RNS in serum and MDA in spleen were reduced significantly or extremely significant（P<0.05 or P<0.01）.The concentration of NO,SOD,CAT and GSH-Px in serum,liver and spleen were increased significantly or extremely significant（P<0.05 or P<0.01）.In chlorogenic acid group,the concentration of RNS and MDA in serum reduced significantly or extremely significant（P<0.05 or P<0.01）.The concentration of NO and SOD in serum and intestine were increased significantly or extremely significant（P<0.05 or P<0.01）,as well as the concentration of CAT and GSH-Px in serum,liver and intestine.In chitooligosaccharide group,the concentration of RNS in serum and MDA in serum and liver were reduced significantly or extremely significant（P<0.05 or P<0.01）.The concentration of NO and SOD in serum and intestine increased significantly or extremely significant（P<0.05or P<0.01）,as well as the concentration of CAT and GSH-Px in liver and intestine.（5）Cytokine index:Compared with the control group,in three groups,the concentration of TNF-α,IFN-γ,IL-1β,IL-2,IL-6 and IL-8 increased significantly or extremely significant（P<0.05 or P<0.01）.At the same time,the concentration of IL-10 also increased significantly or extremely significant（P<0.05 or P<0.01）in thymosin group and chitooligosaccharide group.（6）Other immune indexes:Compared with the control group,in three groups,the concentration of complement 3,complement 4,LZM,AKP and ACP in serum were increased significantly or extremely significant（P<0.05 or P<0.01）.In thymosin group,the concentration of complement 3 in spleen and LZM and ACP in liver and spleen and AKP in liver were increased significantly or extremely significant（P<0.05 or P<0.01）.In chlorogenic acid group,the concentration of complement 4 in liver and intestine increased significantly or extremely significant（P<0.05 or P<0.01）,as well as the concentration of LZM,AKP and ACP in intestine.In chitooligosaccharide group,the concentration of complement 3 in liver and complement 4 in liver and intestine increased significantly or extremely significant（P<0.05 or P<0.01）,as well as the concentration of LZM,AKP and ACP in intestine.In summary,in this study,through testing the respiratory burst activity and phagocytic activity of peripheral blood leukocytes isolated from sturgeon,three immunopotentiators for sturgeon（thymosin,chlorogenic acid and chitooligosaccharide）were screened from 10immunopotentiators generally used for animal.Through feeding and breeding experiment,from three aspects on serology,histology and immunology,the three screened immunopotentiators all have significant effects on improving immune resistance,which provided the scientific basis for the popularization and application of cold-water fish immunopotentiators.