Effects Of Fasting And Coccidium Infection Stress On Performance And Cholesterol Metabolism In Broilers Showing Different TI Phenotype

Tonic immobility(TI)is an internal response related to fear and stress response.Birds can be assigned into short or long tonic immobility(STI or LTI)phenotype basised on of TI duration.Hypothalamic-pituitary-adrenal(HPA)axis controls stress hormones synthesis and secretion,and has profound effects on TI duration.Our previous studies showed that STI chickens showed better growth performance than LTI counterparts.In this study,in order to investigate the response of STI and LTI broilers facing to exogenous stressors,broiler chickens were challenged by fasting for 16 h or infectious treatment.1.Effects of fasting on appetite-associated factors and energy sensors expression in the hypothalamus of broilers showing STI and LTI durationIn order to segregated groups of STI and LTI phenotypes strictly,five hundred white-feather broilers were tested for TI on 11 d,18 d and 26 d,respectively.Finally,80 chickens demonstrating the shortest TI duration(29.6 ± 2.3 s)or the longest TI duration(246.2 ± 26.8 s)were classified into STI or LTI,respectively.The remaining chickens were excluded from the study.On 42 d,chickens were allocated into control group(feeding ad libitum for 16 h)or fasting group(food deprived for 16 h).Four groups were employed in this study:STI control chickens(STI-Con),STI fasting chickens(STI-Fas),LTI control chickens(LTI-Con)and LTI fasting chickens(LTI-Fas).Real-time PCR and western blot methods were used to determine the differential expression of key factors in hypothalamus involving in regulating of appetite and energy metabolism.The results showed that fasting significantly increased plasma total cholesterol(Tch),high density lipoprotein-cholesterol(HDL-C),non-esterified fatty acid(NEFA),nalonaldehyde(MDA),lactic dehydrogenase(LDH),creatine kinase(CK),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)concentrations compared to control broilers fed ad libitum(P<0.05).In contrast,the level of plasma glucose(Glu),triglyceride(TG),and total antioxidant capacity(TAOC)were decreased by fasting(P<0.05).LTI broilers had greater level of plasma low density lipoprotein-cholesterol(LDL-C)and glutathione peroxidase(GSH-Px)than STI(P<0.05).However,there was no significant interaction between TI phenotype and fasting treatment on the above plasma parameters.Real-time PCR results showed that only neuropeptide Y(NPY)mRNA expression showed a tendency to increase in STI broilers compared to LTI(P = 0.095).There was no significant difference of other appetite-associated factors including POMC,CART(cocaine and amphetamine regulated transcript),LEPR(leptin receptor),MCH(melanin-concentrating hormone)and MC4R(melanocortin receptor 4)expression in hypothalamus between LTI and STI broilers.Fasting down-regulated hypothalamic SREBP-1 and its target gene FAS mRNA expression(P<0.05),but up-regulated CPT1 gene expression in the hypothalamus(P<0.05).Additionally,levels of hypothalamic p-GR and p-AMPK protein expression were decreased by fasting(P<0.05).These results indicate that 16 h fasting results in a negative energy status and accompanied with significant changes of hypothalamic energy sensors and appetite-associated factors expression.However,there was no significant difference of TI phenotypes in response to fasting stress in broilers.2.Effects of coccidium infection on epithelial proliferation and cholesterol metabolism in the duodenum of LTI and STI broilersBroiler chickens were divided into LTI and STI groups according to the TI duration.On 32 d,20 chickens from each group were infected with coccidium.Then,chickens were randomly assigned to 4 groups including STI-Control,STI-Coccidium,LTI-Control and LTI-Coccidium,respectively.After 7 days,all broilers were slaughtered for samples and analysis.Our results showed that there was no significant effect of TI duration on growth performance and biochemical parameters in plasma from 32 to 39 d.Coccidium infection markedly affect plasma biochemical parameters,intestinal integrity and growth performance.However,there was no significant interaction between TI and coccidium infection on growth performance and plasma parameters.Therefore,in the further study we only focused on investigating the effect of coccidium infection on intestinal integrity,cell proliferation,cholesterol metabolism and the related mechanism.Coccidium-challenged(CC)chickens showed significantly higher lesion score in the duodenum tissue(P<0.01)compared to control(CON).The number of oocysts in per gram feces(OPG)was higher in CC group(P<0.01),which was absent in CON chickens.CC chickens showed a tendency to increase of food intake(P = 0.06)compared to CON.However,the mean body weight gain of CC group was less than CON,yet did not reach the statistical significance.Coccidium infection increased the level of GSH-PX enzyme activity and UA,which suggested that the increasing of oxidative stress;the level of total cholesterol(Tch)and HDL in plasma was significantly reduced by CC infection(P<0.01)compared to CON.Moreover,stress hormone corticosterone concentration in plasma was doubled in CC chickens and was significantly higher than that in CON(P<0.05).HE staining and the ultrastructure of the duodenum epithelium showed that the destruction of the small intestinal structural integrity and severe cellular damages,while the control chickens exhibited integrity and normal epithelia morphology and structure of duodenum.Cell proliferation in the intestine is commonly occurred during infection and inflammation to replace damaged enterocytes,and cholesterol as an essential constituent of cell membrane,is required for cell proliferation and growth.Here we found that coccidium-challenged chickens showed severe damages in intestinal structure,a significant increase of cell proliferation,and an activation of genes expression involved in the innate immune response.Compared to CON,CC chickens showed a marked decrease of cholesterol(Tch)level in the circulating system(P<0.01),but a significant increase in local duodenum epithelium(P<0.05).Coccidium-challenged did not change the genes expression of cholesterol metabolism in duodenum.However,the LDLR protein expression showed an increasing tendency(0.05<P<0.1)and CYP27A1 protein expression showed decreased(P<0.05)in enterocytes.Coccidium-challenged greatly increased the level of miRNA1699?miRNA7477-5p?miRNA1451-5p(P<0.05)and miRNA1608(P<0.01)which targeted CYP27A1 gene in the duodenum.In addition,in vitro validation showed miRNA1608 and miRNA 1699 binded the CYP27A1 3'UTR precisely.Increase of LDLR protein combined with a significant decrease of CYP27A1 protein expression in duodenum epithelium may contribute to intestinal cholesterol accumulation in CC chickens.Moreover,we found miRNAs targeting to CYP27A1 gene participating in post-transcriptional regulation.Hence,these results provide a new insight for the intervention of epithelial proliferation and cholesterol metabolism in the gastrointestinal tracts.3.Effect of coccidium infection on cholesterol metabolism in liver and hypothalamus of broiler chickensLiver is the central site for cholesterol metabolism in mammals and avian species.It's well known that the balance of cholesterol content in the brain is essential for maintaining physiological function of the neuronal cells.In healthy status,the BBB(blood brain barrier)effectively prevents the exchange of brain tissue and plasma lipoproteins,thus,peripheral cholesterol can't enter the brain through the BBB.In order to further investigate the mechanism underlying the significant decrease of plasma cholesterol,the changes of cholesterol metabolism in liver and hypothalamus were analyzed in this study.The results showed that the concentration of cholesterol and triglyceride in the liver and hypothalamus was not changed by coccidium-challenged.Coccidium-challenged significantly decreased mRNA expression of ABCG5,ABCG8,LDLR,SREBP1 and GR in the liver compared to control group(P<0.05).However,the levels of LDLR and HMGCR,CYP27A1 protein expression were not altered by CC infection in the liver and hypothalamus.These results indicated that the metabolism of cholesterol in the liver and hypothalamus still keep the homeostasis after CC infection.The significant decrease of cholesterol in the circulating system may be caused by the markedly increase of corticosterone synthesis because cholesterol is the precursor of corticosterone.