Effects Of Different Selenium Sources On Apoptosis Of Intestinal Epithelial Cells Of Broilers Induced By High Fluoride

Fluorosis has caused serious economic losses to the poultry industry due to the use of fluorinated mineral feed additives and the presence of naturally high fluoride water sources.The mechanism of fluorosis mainly causes the formation of excess reactive oxygen species,which exacerbates apoptosis.Selenium can scavenge reactive oxygen species by direct or indirect means..To investigate the alleviating effects of sodium selenite(SS)and selenomethionine(SM)on excessive apoptosis of duodenum and jejunum intestinal epithelial cells of broilers induced by high fluorine(HF)and its molecular mechanism,Lingnan Yellow broilers were selected as the research subjects.The effects of different selenium sources on HF-induced oxidative damage,apoptosis of intestinal mucosa,and reduction of performance in broilers were investigated by measuring the oxidative stress index of intestinal mucosa,observing the ultrastructure of intestinal epithelial cells,detecting the apoptosis level of intestinal epithelial cells and statistic of production performance.To investigate the role of nuclear factor erythroid 2-related factor 2(Nrf2)pathway in alleviating HFinduced excessive apoptosis of intestinal epithelial cells by different selenium in the way of measuring the expression of Nrf2 and its downstream antioxidant enzyme genes in intestinal mucosa.1.Effects of different selenium sources on oxidative damage and apoptosis of intestinal mucosa and inhibition of performance of broilers induced by high fluorideA total of 720 1-day-old healthy Lingnan yellow broilers were randomly divided into 4 groups(6 replicates per group and 30 broilers per replicate).The specific grouping as follows:①Control(CON)group:fed basal diet;②HF group:basal diet+sodium fluoride(NaF)(800 mg/kg F);③SS group:basal diet+fluoride Sodium(NaF)(800 mg/kg F)+SS(0.15 mg selenium(Se)/kg);④SM group:basal diet+sodium fluoride(NaF)(800 mg/kg F)+SM(0.15 mg selenium(Se)/kg).The feeding period lasted for 50 days.Feed and water were provided ad libitum during the feeding experiment.Broiler management was carried out according to the normal immunization procedures.Feed consumption,numbers of the dead chicken and temperature were recorded daily.At 50 days of age,daily gain,daily feed intake and feed conversion ratio were count and calculated.Then two roosters with similar weight were selected for slaughter per replicate,and intestinal samples were taken for analysis.The results showed that:(1)Oxidative stress index:compared with CON group,HF group significantly increased(P<0.05)the contents of reactive oxygen species(ROS),malondialdehyde(MDA),8hydroxydeoxyguanine(8-OHdG),Protein carbonyl and 3-nitrotyrosine(3-NT)in duodenal and jejunal mucosae of broilers.Compared with HF group,SS group significantly decreased(P<0.05)some oxidative stress indexes(MDA,8-OHdG and 3-NT in duodenal mucosa and ROS,8-OHdG,Protein carbonyl and 3-NT in jejunal mucosa).Compared with HF and SS groups,SM group significantly decreased(P<0.05)ROS,MDA,8-OHdG,Protein carbonyl and 3-NT levels in duodenal and jejunal mucosae.(2)Ultrastructural observation of intestinal epithelial cells:compared with CON group,intestinal mucosal microvilli were edema,sparse arrangement disorder,uneven length,partial lodging and shedding,mitochondrial swelling,and partial mitochondrial cristae fracture or disappearance.The morphology,arrangement,length and mitochondrial integrity of intestinal microvilli in SM group were similar to those in CON group.SS was less effective at relieving microvilli and mitochondrial damage to the intestinal mucosa than SM.(3)Mitochondrial membrane potential(MMP):Compared with CON group,HF group significantly decreased(P<0.05)the mucosal MMP in duodenum and jejunum.The MMP of SS and SM groups were significantly higher(P<0.05)than that of HF group,and SM group had the best effect.(4)Apoptosis related indexes:compared with CON group,HF group was significantly increased(P<0.05)Caspase-3 and apoptosis rates of duodenal and jejunal epithelial cells,and decreased(P<0.05)the contents of B-cell lymphoma 2(Bcl-2)in duodenal and jejunal epithelial cells.Compared with HF group,SS group was significantly(P<0.05)increased the level of Bcl-2,and decreased the level of Caspase-3 in duodenal and jejunal epithelial cells;SM group was significantly decreased(P<0.05)Caspase-3 contents and apoptosis rate of duodenal and jejunal epithelial cells,and increased(P<0.05)Bcl-2 contents of duodenal and jejunal epithelial cells.The Caspase-3,Bcl-2 and apoptosis rates of duodenal and jejunal epithelial cells were significant difference between SS and SM groups(P<0.05).(5)Production performance:Compared with CON group,the 50 d average weight,21-50 d and 1-50 d average daily gain(ADG)of broilers in the HF group were significantly reduced(P<0.05),and the feed conversion rate(FCR)of 21-50 d and 1-50 d was significantly increased(P<0.05).Compared with HF group,the 50 d average weight,21-50 d and 1-50 d ADG of broiler chickens in SS group and SM group were significantly improved(P<0.05),and the FCR of 21-50 d and 1-50 d was reduced(P<0.05),and SM group had the best effect,and reached a significant difference level with the SS group(P<0.05).2.Study on the antagonistic effect of Nrf2 pathway on the high-fluoride-induced excessive apoptosis of broiler intestinal epithelial cells by different selenium sourcesOn the basis of the above experiments,we further investigated whether different selenium sources can alleviate the HF-induced excessive apoptosis of intestinal epithelial cells of broilers induced by affecting Nrf2 signaling pathway.Experimental design,grouping and sampling were the same as above.The results showed that:(1)Nrf2 gene expression:Compared with CON group,HF group significantly decreased(P<0.05)the relative expressions of Nrf2 mRNA and Nrf2 nucleoprotein in duodenal and jejunal mucosae.Compared with HF group,SS group significantly increased(P<0.05)the jejunal mucosa’s Nrf2 mRNA and duodenal mucosa’s Nrf2 nucleoprotein levels;SM group significantly increased(P<0.05)the relative expression levels of Nrf2 mRNA and Nrf2 nucleoprotein in the duodenal and jejunal mucosae.The relative expressions of Nrf2 mRNA in duodenum and jejunum mucosae and the relative expressions of Nrf2 nucleoprotein in jejunal mucosa in SM group were significantly higher(P<0.05)than those in SS group.(2)mRNA level of antioxidant enzyme genes downstream of Nrf2:Compared with CON group,HF group significantly decreased(P<0.05)Heme oxygenase-1(HO-1),NAD(P)H:Quinone oxidoreductase(NQO1),catalase(CAT),Copper zinc superoxide dismutase(CuZn-SOD)and manganese superoxide dismutase(Mn-SOD)mRNA relative expression levels in duodenal and jejunal mucosae.Compared with HF group,SS group significantly increased(P<0.05)mRNA relative expression levels of some antioxidant enzyme genes(HO-1,CAT and Mn-SOD in duodenal mucosa and HO-1,NQO1,CuZn-SOD and Mn-SOD in jejunal mucosa);SM group significantly increased(P<0.05)mRNA relative expression levels of HO-1,NQO1,CAT,CuZn-SOD and MnSOD in duodenal and jejunal mucosae.Compared with SS group,SM group significantly increased(P<0.05)the relative expression levels of HO-1,NQO1,CAT,CuZn-SOD and Mn-SOD mRNA in the duodenal and jejunal mucosae.(3)The expression of antioxidant enzymes downstream of Nrf2:Compared with HF group,the levels of CAT,total superoxide dismutase(T-SOD)in duodenal mucosa and NQO1,HO-1 and TSOD in jejunal mucosa in SS group were significantly increased(P<0.05);the levels of NQO1,HO-1,CAT and T-SOD in duodenal and jejunal mucosae in SM group were significantly increased(P<0.05).Compared with SS group,the levels of NQO1,HO-1,CAT and T-SOD in duodenal and jejunal mucosae in SM group were significantly increased(P<0.05),but there was no significant difference in the level of T-SOD in duodenal mucosa(P>0.05).In conclusion,results of our study indicated that HF could accelerate ROS production,which led to the oxidation of biological macromolecules and subsequently resulted in oxidative stress,caused mitochondrial damage and promoted apoptosis in the duodenal and jejunal mucosae,and ultimately reduced performance of broilers.Dietary SM supplementation could activate the Nrf2 pathway and promoted the gene expression of its downstream antioxidant enzymes,thereby antagonized the HF-induced intestinal oxidative stress,and finally reduced the apoptosis rate of intestinal epithelial cells and improved the performance of broilers.Our results also confirmed the superiority of SM in inducing the Nrf2 pathway in the jejunum and mitigating performance degradation in broilers when compared to SS.The application of the findings could provide a theoretical basis for the excavation of the protective effect of the SM intestinal mucosal barrier and the improvement of feed nutrient utilization.