| Improvements in genetics and nutrition of the broilers have led to continuing improvement in feed conversion and carcass yield.However increased growth rate and fat have caused a greater incidence of metabolic disorders such as asides,sudden death syndrome,inefficiency immune functionand skeletal abnormalities.Some studies have demonstrated that energy restriction may have an obviously improving effects incarcass qualityand health condition.In mammals' study shows that metabolic alterations,as well as the metabolic and energetic programming of the immune response,are also critical components of immunometabolism.Mean while many layers of evolutionarily conserved interactions occur between immune response and metabolism.Proper maintenance of this delicate balance is crucial for health and has important implications for many pathological states such as obesity,diabetes,and other chronic non-communicable diseases.Calorie restriction(CR)without malnutrition is the most studied and robust non-genetic non-pharmacological experimental inter-vention for extending healthspan and lifespan in multiple animal models.Caloric restriction cound improve chicken disease resistance and immune function,but whose biological mechanism was unclear.In order to study the 15%,30% energy restriction on broiler chickens growth performance,immunological characteristic and gene expression in the spleen,in this study,mainly carried out from the following three parts.Experiment one: Effects of Energy Restriction on Growth Performance and Immunological Characteristic of broiler ChickensThe 72 female broilers Chickensat 22 day-old were fed in this experiment.The broilers were randomly assigned to 3 treatment groups: ad libitum(control group),15% feed restriction(trial group 1)and 30% feed restriction(trial group 2),24 in each group.Each bird raised in one cage,chickens were slaughtered after feed restriction for 7 days(28-day-old)and day21(42-day-old).Different levels of feed restriction on broiler chickens growth performance,immune organ index,peripheral blood lymphocytes were quantitatively evaluated.Following was the results:On experiment day 7: The results showed that the body weight,growth performance and ADG in control group was significantly higher compared with the experiment groups(p<0.05).And the body weight and ADG of 15% feed restriction group was significantly higher than that in 30%feed restriction group(p<0.05).The F/G among the three experiment groups were not significantly different(p >0.05).The bursa of Fabricius index,in 30% feed restriction group was significantly higher than that of 15% feed restriction group and the controls(p<0.05),and no obviously different between the 15% feed restriction group and the control group(p>0.05).Theserum immunoglobin Ig M levels of the two feed restriction groups was significantly higher than that of control group(p<0.05),but no obviously different between the two feed restriction groups(p>0.05).The peripheral CD3+ lymphocyte and CD4+ lymphocyte levels were higher in control group compared with that in the two feed restriction groups,but no statistics different(p>0.05).The peripheral blood CD8+ lymphocyte count in 30% feed restriction group was significantly higher compared with that in the control group and 15% feed restriction group(p<0.05),and no obviously different between the control group and the 15% feed restriction group respectively(p>0.05).The serum glucose,triglycerides and LDL levels in all three groups were no significant different(p>0.05).The serum total cholesterol concentration of 30% feed restriction group was significantly lower than that of the control group(p<0.05);the serum HDL in the feed restriction groups was significantly lower than that in the control group,and the 30% feed restriction group HDL was also lower than that of the 15% feed restriction group.On experiment day 21: Among the three groups,the final weight,growth performance and ADG in control group was significantly higher compared with the experiment groups(p<0.05),and the15% feed restriction group was significantly higher than that in 30% feed restriction group(p<0.05).The F/G was significantly higher in 30% feed restriction group compared with the control group and 15% restriction group(p<0.05),and no obviously different between 15%restriction group and controls(p>0.05).The spleen index in 30% feed restriction group was significantly higher than that of 15% feed restriction group and the controls(p<0.05).The bursa of Fabricius index and thymus gland index no obviously different among the three groups(p>0.05).The serum immunoglobin Ig M levels were not obviously different among the three experimental groups(p>0.05).Serum Ig G and Ig A levels were significantly higher compared to the control group(p<0.05),and no obviously different between the two feed restriction groups.The peripheral blood CD3+,CD4+ and CD8+ lymphocytes counts in the two feed restriction groups were all increased compared to the control group,but did not reach the statistics different level(p>0.05).).The serum glucose and LDL levels in all three groups were no significant different(p>0.05).The serum triglycerides concentration of 30% feed restriction group was significantly lower than that of 15% feed restriction group and the control group(p<0.05,but no different between the 15%feed restriction group and the control group.The results of the broiler chicken final weight,growth performance,immunological organ index evaluation,serum immunoglobin including Ig G,Ig A and Ig M concentrations,peripheral blood CD3+,CD4+,CD8+ lymphocyte counts,and serum glucose,triglyceride,total cholesterol,HDL and LDL level examination showed that the feed restriction treatment methods,both 15% or 30 %feed restriction,all modified the chicken's adipose tissue metabolism and obviously improved body immunological function.Experiment two: Transcriptional expression levels of chicken spleen are affected by energy restriction conditionWhile the 28-day-old and 42-day-old chickens were sacrificed,the spleen tissues werecollected as soon as possible.Total RNAs were extracted with the standard protocol.After measured the concentration of each sample,then frozen at-80 o C for future use.we performed transcriptome analyses for the chickens' spleen.The primary data showed that after 7 days energy restriction,in the chicken's spleen tissues,there were 26 genes expression significantly different in the 15% feed restriction group,and 70 gene RNA levels were changed significantly in 30%feed restriction group compared to the control group;after 21 days feed restriction,there were 141 gene expressions changed obviously in 15% feed restriction group and 108 gene expressions significantly different in 30% feed restriction group compared to the control group.We analyzed the obviously express changed genes in more detail.They were mostly related to two important categories,the main energy metabolism related protein enzymes and the most immune reaction related genes.Those genes including adipose,free fatty acids and lipids metabolic enzymes;cell immune reaction and immunological response related activation protein genes.T cells,B cells of lymphocytes,cytokines,phagocytosis related processing genes.Experiment three: The mechanism of the TNFSF13 B gene on immune regulation in broiler chickensTNFSF13B si RNA constructs transformed into UMNSAH/DF-1 cell line according our reported standard protocol.After harvested treated and control cells,total RNA and proteins were isolated respectively.Realtime RT-q PCR and Western blot were performed.The primary data showed that in si RNA treated cells TNFSF13 gene expression was significantly reduced compared with the control cells.NF?B and TGF-? gene m RNA expression were also obviously inhibited in the TNFSF13 B silenced UMNSAH/DF-1 cells by realtime RT-q PCR,and Western blot results indicated that NF-k B65 and TGF-?protein levels were significantly reduced compared to the control cells.This results revealed that feed restriction influenced broiler chicken's immunological responses may related to the TNFSF13 B gene expression,and downstream by modification the NF?B and TGF-?gene function.Both NF?B and TGF-?signal transduction pathways are play important roles in broiler chicken growth behavior,production,energy metabolism and immunology function. |
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