Study On The Effects And Mechanism Of Dietary Fluoride Level On The Intestinal Barrier Function And Eggshell Quality Of Laying Hens

Fluorine(F),known as the most electronegative and reactive element,is abundant in the earth's crust.It is necessary to maintain the health.Appropriate amount can promote growth,development,and reproduction,participate in bone metabolism,prevent caries,etc.,but excessive F had toxic effects on the body.It has long been reported that fluorosis of livestock and poultry was caused by excessive F in mineral feed,industrial fluorine pollution and natural high-F environment.Laying hens are more prone to chronic fluorosis due to the highest fluoride tolerance and the large amount of mineral feed.However,the current research on fluorosis mainly used rats and mice as animal models,and focused on bone toxicity,reproductive toxicity and neurotoxicity.Therefore,in this study,37-week-old Hy-Line Gray commercial hens were used to investigate:firstly,the effects and mechanism of dietary fluoride levels on the performance,egg quality and tissue damage;secondly,intestinal barrier function and cecum microbiota;finally,based on in vivo experiments,a model of endometrial epithelial cells of eggshell glands was established to further study the related mechanism of F reducing eggshell quality.The main content and results are as follows:1 Effects of dietary F levels on laying performance,hepatic and renal damage and antioxidant capacity of laying hensTotal of 384 Hy-Line Gray hens,37-week-old,were randomly divided into 4 groups(16 birds×6 replicates),and treated corn-soybean basal diet supplemented with 0(the control group),400(low fluoride group),800(medium fluoride group),and 1200(high fluoride group)mg F/kg feed(in the form of NaF).The trial lasted for 59 days,including a 10-d adaptation period and a 49-d experimental period.Results suggested that,dietary F levels at 800 and 1200 mg/kg significantly reduced laying rate(P<0.05),egg weight(P<0.05),ADFI(P<0.05),and increased FCR and mortality(P<0.05).Combined with the increase of F in serum and tissue,it was suggested that the fluorosis animal model of hens had been successfully established.Compared with the control group,the serum uric acid and alanine aminotransferase(ALT)activity in the 800 and 1200 mg/kg groups were increased(P<0.05),and the aspartate aminotransferase(AST)activity did not change,while the AST/ALT,glucose,total triglyceride,estradiol and progesterone levels were reduced(P<0.05).Under light microscope,in high-F group the hepatic lobule edge was blurred and the liver steatosis;the renal tubules were swollen,the lumen was narrowed and the glomerular was shrank;the serum and liver antioxidant capacity of the high-fluoride group was reduced(P<0.05),and the liver showed apoptosis.However,these was no difference in the 400 mg/kg group.2 Effects of dietary F levels on intestinal barrier function and cecum microbiota of laying hensThe intestinal morphology and ultrastructure were damaged by dietary high F intake,F residuals in duodenum responded to dietary F concentrations positively,and the activities of amylase,maltase and lactase were lower(P<0.05)in high-F group,than those in the control.The mRNA expression levels of jejunum and ileum zonula occludens-1(ZO-1),zonula occludens-2(ZO-2),claudin-1,claudin-4,secretory immunoglobulin A(sIgA)and Mucin 2,and slgA concentrations were decreased(P<0.05);the concentrations of serum diamine oxidase(DAO),and D-lactic acid(DL)and intestinal interleukin 1 beta(IL-1?),interleukin 6(IL-6),and tumor necrosis factor-alpha(TNF-?)were increased(P<0.05)in high-F group,compared with those in the control group.Compared with the control group,high F increased the diversity and abundance of cecum microbiota(P<0.05);changed the structure of the microbiota from the phylum to the genus level,and increased the relative abundance of Proteobacteria(P<0.05),Acidobacteria(P<0.05),Campylobacter(P<0.05)and Actinomycetes phylum(P<0.05);at the class level,increased the relative abundance of ?,? and ?-Proteobacteria(P<0.05)and Ktedonobacteria(P<0.05);at the genus level,increased the relative abundance of Lactococcus and Shigella(P<0.05),and the relative abundance of Lactobacillus is reduced(P<0.05);Lactobacillus was biomarkers in the control group,while Chloroflexi,Gammaproteobacteria,Escherichia-Shigella,Streptococcaceae,and Enterobacter,which are known to include(potential)pathogens of poultry and humans,were biomarkers of the high-F group;F level at 1200 mg/kg significantly reduced(P<0.05)the contents of total short-chain fatty acids(SCFAs),acetic,butyric,isobutyric,and isopentanoic acid in cecum,and enhanceed the contents of pentanoic acid(P<0.05).3 Effect of dietary F levels on egg quality and its mechanismThis study mainly focused on the effects of dietary low(400 mg/kg)and high(1200 mg/kg)F levels on egg quality,eggshell ultrastructure and the damage of eggshell gland.The results showed that,the F concentrations of the eggs in the F-treated group were higher(P<0.05);on the 21st day of the experiment,the high F level decreased the albumen height,eggshell strength,and eggshell thickness;on day 49,the yolk color(P<0.05),eggshell strength(P<0.05)and eggshell thickness(P<0.05)were decreased,and on day 49 the eggshell strength is lower than those on the 21st day;under the scanning electron microscope,the surface of the eggshell in the high F group is rough,the whole eggshell is thin,and the space between the mammillary knobs became larger;The data of eggshell ultrastructure indicated that the total eggshell thickness,effective layer thickness,and effective layer ratio of the high F group were decreased significantly(P<0.05),while the mammillary layer thickness,mammillary layer ratio,and mammillary layer/effective layer ratio were increased significantly(P<0.05);serum alkaline phosphatase activity in the high F group was increased(P<0.05),phosphorus and 25-hydroxyvitamin D3 contents were decreased(P<0.05).The F content of the eggshell gland in the F-treated group was increased(P<0.05);high F level damaged the inner structure of eggshell gland,the villi was shorter and less,and the apoptosis was obvious;the mRNA expression levels of NCX1 and CA2 in the eggshell gland in the high F group was increased(P<0.05),and the mRNA expression of OC116,CX32,OCX36 and SERCA2 was decreased(P<0.05);in the F-treated group,OPN and CaBP-d28k mRNA expression was decreased(P<0.05).4 Establishment of eggshell glandular epithelial cell model and study on the mechanism of F on eggshell structureThe primary eggshell glandular endometrial epithelial cells were isolated and cultured in vitro,and the logarithmic growth phase was determined on the 2nd-5th day.The cell test used 0,0.25,and 1 mM F concentrations to treat cells for 12 h as the control group,low-F group,and high-F group,respectively.The results showed that,the calcium ion concentration in the cytoplasm,the mRNA expression levels of NCX1,SERCA2,CaBP-d28k,and TRPV6 in the high F-treated group were increased(P<0.05);the F treatment reduced(P<0.05)the mRNA expression level of OPN;low F concentrations up-regulated(P<0.05)OCX32 mRNA expression,while high F concentrations down-regulated OCX32(P<0.05)and up-regulated OCX36(P<0.05)mRNA expression.In summary,(1)dietary F level at 800 and 1200 mg/kg decreased the laying performance and egg quality;(2)increasing F in tussue destroyed the structure of small intestine,liver,kidney and eggshell gland,promoted the apoptosis,and decreased the antioxidant capacity;(3)high F changed the structure,and increased the diversity of cecum microbiota,via increasing the relative abundance of harmful bacteria,and reducing the relative abundance of beneficial bacteria and total SCFAs content,aggravated the intestinal barrier function;(4)in vivo and vitro,high F changed the ultrastructure of eggshells and reduced the quality of eggshell by regulating the mRNA expression of calcium transport proteins and shell matrix proteins.