| Well-known immunostimulants such as yeast cell wall（YCW）and nucleotides（NT）are widely used in aquaculture.Most researchers used only one immunostimulant in their studies.Recently,many studies reported that combining two or more immuno-stimulants had a better synergistic effect.However,the effects of combination of dietary NT and YCW on fish have not been reported.Thus,we have investigated the effects of NT and YCW and their combination at the individual and cellular levels on the growth performance and immune response of grass carp,Ctenopharyngodon idella,respectively.The results are as follows:1.Effects of dietary nucleotide and yeast cell wall on growth performance,feed utilization,anti-oxidative and immune response of grass carpA 70-day feeding trial was conducted to study the effects of dietary nucleotide,yeast cell wall（containing 20%β-glucan）and their combination on growth performance,feed utilization and immune response of grass carp with an initial mean body weight of69.97±0.05 g.Four isonitrogenous（about 38%crude protein）and isolipidic（about 5%crude lipid）diets were formulated.Based on the control diet（CD）,the other three experimental diets were supplemented using 0.01%of NT,0.1%of the YCW and NT（0.01%）+YCW（0.1%）,respectively.And the health premix（HP）was used as the control additive.The results showed no significant differences in the survival of grass carp ranging from 94.44%to 97.78%among all groups（P>0.05）.Weight gain rate,muscle crude protein content,serum protein,trypsin and chymotrypsin activities in the midgut,lysozyme and Ig M in serum were significantly increased in the group fed with YCW when compared to the control（P<0.05）.The use of a combination of NT and YCW showed the highest weight gain rate,villus height and digestive enzymes activity in the midgut and innate immune parameters in serum among all treatments（P<0.05）.The expression ofβ-defensin,hepcidin,il-10 and tgf-β1 genes in the midgut significantly increased in fish fed with diets supplemented with NT+YCW,and the highest gene expressions were found in grass carp fed the NT+YCW diet.Moreover,fish fed with a combination of the NT+YCW showed a significant increase in the expression of tor and s6k1 genes in the liver（P<0.05）.Meanwhile,the m RNA levels of il-1βand tnf-αin the midgut significantly decreased（P<0.05）.Compared with the control group,the liver histology showed better development in the groups fed with dietary NT and/or YCW in-feed.In conclusion,the present results showed a combination of dietary NT+YCW produced synergetic improvements in the growth,feed utilization,immune response and histological structure of the midgut and liver of grass carp.2.Established an in vitro research platform for grass carp intestinal immunityIn the present study,the tissue culture method was used to culture the primary intestinal cells of grass carp.Intestinal tissue samples were used respectively to initiate the primary cell culture using Dulbecco’s Modified Eagle Medium（DMEM-Gluta MAX（?））,Leibovitz（L-15）and DMEM:Ham’s Nutrient F-12（1:1）（DMEM/F12）medium supplemented with 15%fetal bovine serum（FBS）,mouse epidermal growth factor（EGF）at 24℃and 5%CO2.However,intestinal tissue samples cultured in L-15and DMEM-Gluta MAX（?）medium were infected with bacteria during the experiment.Thus,only cells migrated from cultured tissues in DMEM/F12 medium were used in the present study.However,intestinal cell subcultures using DMEM/F12 exhibited slower adhesion and proliferation,and could not be cultured continuously.Therefore,the first generation of intestinal cells was used as the research object,using L-15 and DMEM Gluta MAX（?）medium for culture.Then the adherence,proliferation,and passage of the second generation of intestinal cells were observed.The results showed DMEM-Gluta MAX（?）medium was an appropriate medium for continuous culture.The intestinal cells passaged for 20 generations.The success of cell culture provided valuable materials for subsequent experiments.3.Effects of nucleotide andβ-glucan on the related signaling molecules of immune response,Nrf2/Keap1 signaling pathway and anti-apoptosis of grass carp intestinal cellThe results of the feeding trial implied that the possible reason for the improved immunity of grass carp might be because of the immuno-stimulating properties of NT and BG.Because BG is the main component of YCW in the present study.In order to further explore if NT and BG are involved in immunity,we have investigated the effects of NT,BG and their combination on the related signaling molecules of immune response,anti-apoptosis,and the Nrf2/Keap1 signaling pathway of grass carp intestinal cell in an in vitro experiment.The experiment included three parts:In part l:6 levels of NT（0,10,30,50,70 and 100μg/m L）and 6 BG levels（0,10,30,50,70 and 100μg/m L）were applied in the intestinal cell culture,respectively.The intestinal cells were treated with different levels of NT and BG for 6 h and 12 h,respectively and then analyzed.The results showed that the levels of NT（0,10,50 and100μg/m L）and BG（0,10,50 and 100μg/m L）at 6 h of administration could meet the requirements for subsequent experiments.Part 2 was designed using 4 NT levels（0,10,50 and 100μg/m L）and BG levels（0,10,50 and 100μg/m L）with 3 replicates for each one.The intestinal cells were treated for6 h.The results are as follows:The lysozyme,il-10 and nrf2 levels in a group that received10μg/m L of NT were significantly increased when compared to the control（P<0.05）.The use of 10,50 and 100μg/m L of NT significantly increased the tnf-αand inos genes expression（P<0.05）.Compared with the control group,the 50μg/m L of NT had significantly increased the cat,sod1 and tgf-βgenes expression（P<0.05）.The use of 50μg/m L of NT significantly increased nrf2 and keap1 expression as well（P<0.05）.The100μg/m L of NT had no significant effects on il-10 and tgf-βexpression（P<0.05）,and significantly down-regulated the lysozyme gene expression（P<0.05）.Moreover,compared with the control group,10 and 50μg/m L BG groups had significantly up-regulated the cat,sod1,tgf-β,il-1β,tnf-αand inos genes expression（P<0.05）,and significantly increased nrf2 and keap1 genes expression（P<0.05）.The use of 100μg/m L of BG had no significant effects on il-10,tgf-β,il-1βand tnf-αgenes expression（P<0.05）,and significantly reduced the lysozyme gene expression（P<0.05）.The bcl-xl and bcl-2m RNA levels in a group treated with 50μg/m L of NT and groups treated with 10 and 50μg/m L BG were significantly higher than those in the control（P<0.05）.The bcl-xl level showed no significant difference when 100μg/m L of NT and 100μg/m L of BG were applied（P>0.05）.In part 3 we used three levels of BG（0,10 and 50μg/m L）and at each level,three levels of NT（0,10 and 50μg/m L）were added（3×3 factorial design）.The intestinal cells were treated for 6 h.The results are as follows:the interaction between NT and BG showed significant effects on all the indices of grass carp intestinal cells（P<0.05）.Compared with the control group,the use of 10μg/m L or 50μg/m L of NT separately significantly up-regulated the expression of cat,sod1,tgf-β,tnf-α,inos,nrf2 and keap1genes（P<0.05）.The use of 10 and 50μg/m L of BG alone up-regulated the expression of cat,sod1,il-1β,tnf-αand inos genes（P<0.05）,and significantly up-regulated nrf2and keap1 genes expression（P<0.05）.Moreover,the bcl-xl and bcl-2 levels with the use of 50μg/m L of NT and 10 and 50μg/m L of BG were significantly increased（P<0.05）.Combination of 50μg/m L NT and 50μg/m L BG had the highest level of the related signaling molecules of the immune response,anti-apoptosis,and the Nrf2/keap1 signaling pathway when compared to other groups.Thus we can see that the present results showed that the application of NT and BG and their combination in the grass carp cell culture could improve the related signaling molecules of the immune response,anti-apoptosis,and the Nrf2/Keap1 signaling pathway.In summary,the effects of the combination of dietary NT and YCW could enhance the growth performance and the immune response of grass carp at both the individual and cellular levels,and reveals the immune mechanism of NT and YCW.This can provide a more theoretical basis and academic reference for the aquaculture field with suitable and effective immunostimulants combinations. |
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