Research On The Reproductive Toxicity Of Dietary Cadmium Exposure To Laying Hens And Its Mechanism

Cadmium(Cd)is a common environmental and industrial pollutant,with obvious female reproductive toxicity.Cd exposure can not only damage the reproductive system of birds,reduce production performance,but also accumulate in eggs,which poses a potential threat to the embryonic development of birds,as well as food safety and human health.In this study,we investigated the effects of dietary Cd exposure on production performance and egg quality of laying hens.On this basis,we investigated the effects of Cd on the calcium homeostasis in body,as well as on the expression of the proteins related to eggshell formation in eggshell gland,to explore the possible mechanism of Cd affecting eggshell quality.We investigated the effects of Cd on the liver injury and lipid metabolism,to explore the molecular mechanism of Cd affecting yolk formation in laying hens.In addition,we investigated the effects of Cd on the ovarian damage and signaling pathways related to granulosa cell proliferation or apoptosis in laying hens,to explore the molecular mechanism of follicular atresia induced by Cd.The main results are represented as follows:1 Effects of dietary Cd exposure on the laying performance,egg quality and Cd residues in laying hensIn this study,we identified the effects of Cd on laying performance,egg quality and Cd residues in laying hens.A total of 480 laying hens at 38 weeks were randomly assigned into five groups,each of which included 6 replicates of 16 laying hens.Five groups are:(1)basal diet(control),(2)basal diet+7.5 mg/kg Cd(provided by CdCl2·2.5 H2O),(3)basal diet+15 mg/kg Cd,(4)basal diet+30 mg/kg Cd and(5)basal diet+60 mg/kg Cd.The concentrations of Cd in the diets of the five groups were 0.47,7.58,15.56,30.55 and 60.67 mg/kg.The trial period was 10 weeks,including one-week adaptation period.Feed intake,egg weight,and egg production were recorded during the experimental period.At the third and ninth weeks of the formal test,24 eggs(4 of each replicate)were selected from each group to determine the egg quality.At the end of experimental period,twelve birds per treatment(2 birds each replicate)were randomly selected for slaughter and sampling.The results showed that,the egg production rate(EPR)decreased significantly in 60 mg/kg Cd group as compared with other groups,and to be the lowest in 7-9 weeks(P<0.05).From the stage of 7-9 weeks,as compared with the control group,diet supplemented with 60 mg/kg Cd resulted in markedly decrease of the ADFI(P<0.05)and increase of feed conversion rate(FCR)(P<0.05).Compared with the control group,the albumen height,Haugh unit and eggshell strength were improved in the low levels of dietary Cd(7.5 mg/kg)after 3 weeks of treatment(P<0.05).Its positive impact on yolk color and eggshell thickness continued even in the ninth week.At the ninth week,the eggs were inferior in quality including albumen height,Haugh unit and eggshell strength to that at the third week,30 and 60 mg/kg Cd decreased the albumen height,Haugh unit and eggshell thickness significantly(P<0.05)compared with control group.Regression analysis results showed that the effects of Cd on EPR,ADFI,FCR,albumen height and Haugh unit were all time-and dose-dependent.With the increase of dietary Cd,the accumulation of Cd in eggshell,albumen,yolk and the whole egg increased significantly(P<0.05).The mainly accumulation site of Cd was the eggshell,but low in the albumen and yolk.The Cd content in the total egg exceeded the maximum permitted levels of Cd in fresh eggs when diet was supplemented with 9.725 mg/kg Cd or above.Results of Cd residues in tissues indicated that the kidney and liver were the main sites of Cd accumulation,followed by the bone,pancreas and lung.2 Effects of dietary Cd exposure on body calcium homeostasis and gene expression of matrix protein in eggshell glandThis study was conducted to evaluate the toxic effects of Cd on the kidney,bone and eggshell gland of laying hens,and to explore the possible mechanism of Cd affecting the calcium homeostasis and gene expression of matrix protein in eggshell gland in laying hens.Results showed that the renal function index of blood urea nitrogen(BUN),uric acid(UA),and creatinine were significantly increased in the 60 mg/kg Cd group compared with control group(P<0.05).High-dose Cd exposure decreased the activities of antioxidant enzymes and ATPase and induced the lipid peroxidation in the kidney and eggshell gland tissues(P<0.05).The levels of serum Ca were decreased significantly in 60 mg/kg Cd group(P<0.05).The activity of alkaline phosphatase(ALP)and the concentrations of bone ALP(BALP),1,25-(OH)2-D3 and calcitonin(CT)were decreased significantly,and the level of parathyroid hormone(PTH)were increased significantly in the groups of 30 and 60 mg/kg Cd(P<0.05).In the 30 or 60 mg/kg Cd groups,histological results presented shrinkage of glomeruli,enlargements of renal tubule and tubular fibrosis in kidney,and decrease of trabecular bone in tibia,and TRAP staining results of tibia showed osteoclast increased significantly(P<0.05).Cd induced endometrial epithelial cell proliferation accompany with upregulating the mRNA levels of progesterone receptor(PgR)and epidermal growth factor receptor(EGFR),downregulating the mRNA levels of estrogen receptor alpha(ER?)and interleukin 6(IL6).60 mg/kg Cd exposure induced inflammation of eggshell gland accompany with increasing the expression of complement C3 and pro-inflammatory cytokines tumor necrosis factor ?(TNF-?)significantly(P<0.05).In addition,the expression of genes related to the eggshell formation,including calbindin 1(CALB1),ovocalyxin-32(OCX32),ovocalyxin-36(OCX36),osteopontin(OPN),ovocledidin-17(OC17)in eggshell gland were decreased significantly(P<0.05).The ultrastructure of the eggshell showed that the palisade and mammillary layer thickness were decreased(P<0.05),and the outer surface of the eggshell became rougher in 30 and 60 mg/kg Cd exposure groups.3 Effects of dietary cadmium exposure on the damage and lipid metabolism in the liver of laying hensThe objective of this study was to determine the effects of Cd on histological changes,oxidative-and endoplasmic reticulum stress and lipid metabolism in the liver of layers.The results showed that,60 mg/kg Cd exposure decreased the antioxidant levels significantly(P<0.05).The results of immunofluorescence assay and RT-qPCR showed that exposure to 60 mg/kg Cd induced reactive oxygen species(ROS)production and endoplasmic reticulum stress in hepatocytes,accompany with upregulating the expression of cytochrome C,caspase 3,caspase 7,caspase 9,Grp78 and Chop(P<0.05).Histopathology and RT-qPCR results presented periportal fibrosis,bile duct hyperplasia and periportal inflammatory cells infiltration in the liver accompany with upregulating the expression of TNF-?,IL-6 and IL10 in 30 or 60 mg/kg Cd groups(P<0.05).Oil red O staining and RT-qPCR results showed that Cd induced lipid accumulation in hepatocytes through upregulating the expression of fatty acid synthase(FASN)and downregulating the expression of enzymes of ?-oxidation:acyl-CoA oxidase 1(ACOX1),carnitine palmitoyltransferase-1(CPT1)and peroxisome proliferator activated receptor ?(PPAR?)(P<0.05).Besides,the expression of ovum vitellogenin ?(VTG-?)and microsomal triglyceride transfer protein(MTP)were upregulated in 7.5 mg/kg Cd group,and apolipoprotein B(ApoB),VTG-? and apolipoprotein very-low density lipoprotein ?(apo-VLDL-?)were downregulated in 30 and/or 60 mg Cd/kg groups(P<0.05).4 Effects of cadmium on the follicular atresia and its regulation mechanismThis study was conducted to evaluate the toxic effects of Cd on the ovarian damage and follicular atresia through in-vitro experiments,and to investigate the mechanism of follicular granulosa cell proliferation and apoptosis induced by Cd through in-vivo experiments.Primary follicular granulosa cells were isolated and cultured and three groups were set:control group(0?M Cd),1?M Cd group and 15?M Cd group.The results from oxidative stress index(T-SOD,GSH-Px and MDA),nitric oxide(NO)content,T-NOS and ATPase activities,TUNEL assay and H&E staining indicated that excess Cd induced oxidative stress,granulosa cell apoptosis and follicular atresia in laying hens'ovary.1?M Cd induced the granulosa cell proliferation along with upregulating the phosphorylation of FoxO3a,Akt,ERK1/2,mTOR and p70S6K1 and gene expression of RSK1 and RHEB,and promoting cell cycle progression from phase G1 to S.In contrast,15?M Cd induced ROS generation and cell apoptosis along with downregulating the phosphorylation of ERK1/2,mTOR and p70S6K1 and the gene expression of RSK1 and RHEB and inducing the cell cycle arrest.Rapamycin pre-treatment completely blocked the phosphorylation of mTOR and p70S6K1 and cell cycle progression induced by 1 ?M Cd,and accelerated 15?M Cd induced cell apoptosis and cell cycle arrest.The results of microRNA sequencing showed that 15?M Cd induced differential expression of miRNA genes,which may regulate Akt/FoxO3a,ERK1/2 and mTOR signaling pathways and cell cycle progression by regulating the activity of cell cycle-related proteins and G proteins.In conclusion,Cd in the diet has significant reproductive toxicity after reaching a certain dose.Dietary Cd exposure reduced the production performance and egg quality significantly.Cd can induce damage to kidney,bone and eggshell gland,disrupt the secretion of calcium-regulating hormones and the balance of calcium,as well as inhibit the mRNA expression of proteins related to eggshell formation in eggshell glands,and thereby affecting the eggshell quality.Dietary Cd exposure can affect yolk formation by inducing liver damage,oxidative and endoplasmic reticulum stress,and disrupting lipid metabolism in the liver.In addition,dietary Cd exposure can induce the ovarian damage and follicular atresia in laying hens.In vitro,Cd treatment can regulate the proliferation or apoptosis of follicular granulosa cells through Akt/FoxO3a,ERK1/2 and mTOR signaling pathways,and thereby affecting the ovarian follicle atresia and egg production.