Research On The Damages Of Dietary Mercury Exposure To Laying Hens And Its Mechanism

In this study,we investigated the effects of dietary mercury exposure(added in the form of mercuric chloride)on laying performance,egg quality,histopathology,follicular atresia rate and serum hormone levels in laying hens.Meanwhile,we studied the molecular mechanisms of dietary mercury exposure on antioxidant systems in liver,kidney and ovary of laying hens.Based on the results of experiment in vivo,the granulose cells model and chicken embryo kidney cells model were established in vitro respectively.Furthermore,we studied the molecular mechanisms of mercuric chloride on progesterone secretion and apoptosis associated with PERK-ATF4-CHOP of endoplasmic reticulum stress.The main results were represented as followed:1 Effects of dietary mercury exposure on laying performance and egg quality of laying hensThe objective of this study was to determine the effects of dietary mercury exposure on laying performance and egg quality of laying hens.A total of 768 Hy-line brown laying hens aged 40 weeks were randomly allocated to four groups(8 pens per group and 24 hens per pen).The concentrations of mercury in four groups were 0.28,3.33,9.42,and 27.24 mg/kg.The trial period was 10 weeks,including 1 week of pre-feeding and 9 weeks of formal trial.Results revealed that dietary mercury exposure could significantly reduce laying rate(P<0.05)and the average egg weight(P<0.05),while the feed conversion ratio was significantly increased(P<0.05).Both average feed intake and eliminating ratio were no significant(P>0.05).Besides,the mercury concentrations of yolk,albumen,eggshell and the whole egg in 3.33,9.42 and 27.24 mg/kg mercury groups were significantly higher than those in 0.28 mg/kg mercury group.Both haugh unites and albumen height was negatively correlated with mercury concentration in yolk,albumen,and the whole egg.Meanwhile,both eggshell strength and eggshell thickness was also negatively correlated with mercury concentration in eggshell and the whole egg.2 Effects of dietary mercury exposure on hepatic and renal histopathology,follicular atresia rate of laying hensThe objective of this study was to determine the effects of dietary mercury exposure on hepatic and renal histopathology,follicular atresia rate of laying hens.Results showed that mercury could be deposited in heart,lung,spleen,liver,kidney,ovary,gizzard and proventriculus of laying hens.Meanwhile,with the increasing dietary dosage of mercury,accumulation of mercury in viscera was significantly increased(P<0.05),and histopathological damages in liver and kidney were more and more severe The levels of serum alanine aminotransferase(GPT)(P<0.05)and globulin(GLB)(P<0.05)in 27.24 mg/kg mercury groups were significantly higher than 0.28 mg/kg and 3.33 mercury groups.Meanwhile,aspartate aminotransferase(GOT)in 27.24 mg/kg mercury group was significantly higher than other groups(P<0.05).However,serum albumin and albumin to globulin ratio were significantly decreased(P<0.05)in 27.24 mg/kg mercury group compared with 0.28 mg/kg mercury group.Besides,blood urea nitrogen(BUN)(P<0.05),uric acid(UA)(P<0.05),and creatinine(CRE)(P<0.05)in 3.33,9.42,and 27.24 mg/kg mercury groups were all significantly increased compared with 0.28 mg/kg mercury group.In addition,the follicular atresia rate of laying hens was significantly increased after dietary mercury exposure.The follicular atresia rate was positively correlated with mercury concentration in ovary of laying hens(P<0·01).3 Effects of dietary mercury exposure on antioxidant systems in liver,kidney and ovary of laying hensThis study investigated the effects of dietary mercury exposure on antioxidant systems in liver,kidney and ovary of laying hens.Results revealed that the activities of superoxidative dismutase(SOD),catalase(CAT),glutathione reductase(GR)and glutathione peroxidase(GSH-Px),and glutathione(GSH)content were all significantly decreased(P<0.05),while malondialdehyde(MDA)content was significantly increased(P<0.05)after dietary mercury exposure in liver,kidney and ovary of laying hens.Meanwhile,after mercury exposure,the gene expressions of copper and zinc superoxide dismutase(CuZnSOD)(P<0.05),CAT(P<0.05),GR(P<0.05)and GSH-Px(P<0.05)were significantly decreased in liver,and the gene expressions of copper and zinc superoxide dismutase(CuZnSOD)(P<0.05),manganese superoxide dismutase(MnSOD)(P<0.05),CAT(P<0.05),GR(P<0.05)and GSH-Px(P<0.05)were also significantly decreased in kidney and ovary.The gene and protein expressions of Nrf2 were significantly decreased(P<0.05),while the gene and protein expressions of Keap1 were significantly increased(P<0.05)after dietary mercury exposure.In addition,positive relationships occurred between antioxidant enzyme activities and antioxidant enzyme gene expressions(P<0.05)except between SOD activity and manganese superoxide dismutase(MnSOD)gene expression in liver.Meanwhile,nuclear factor erythoid 2-related factor 2(Nrf2)gene expression was positively related to antioxidant gene expressions(P<0.05)and negatively connected with Kelch-iike ECH associated protein 1(Keapl)gene expression(P<0.05).Negative relationships occurred between Nrf2 and Keapl protein levels in liver,kidney and ovary of laying hens(P<0.05).4 Effect of mercury exposure on progesterone secretion in laying hensThis study investigated the effect of mercuric chloride exposure on progesterone(P4)secretion in laying hens.The results in vivo showed that the follicle-stimulating hormone(FSH),luteinizing hormone(LH)and P4 were all significantly decreased after dietary mercury exposure.The gene expressions of steroidogenic acute regulatory protein(StAR),cytochrome P450 cholesterol side-chain cleavage(P450scc)and 3β-hydroxysteroid dehydrogenase(3β-HSD),cyclic adenosine monophosphate(cAMP)-protein kinase A(PKA)pathway were further investigated to uncover the molecular mechanism at different dosages of mercury and different incubation times.Results revealed that 2,4 and 6 μM mercury exposure had significantly reduced P4 secretion at 12-h time point(P<0.05)compared with control group.Besides,cAMP level at 12-h and 24-h time points(P<0.05)and PKA level at 24-h time point(P<0.05)were significantly decreased after mercury exposure.Comared with control group,6μM mercury exposure had also significantly decreased cAMP level at 6-h time point(P<0.05),and PKA level at 12-h and 24-h time points(P<0.05).Meanwhile,StAR(P<0.05),P450scc(P<0.05)and 3β-HSD(P<0.05)at 24-h and 48-h time points were all significantly decreased after mercury exposure.The results of correlation analysis showed that there were significantly positive correlations among P4,cAMP,PKA,StAR,P450scc and 3β-HSD in ovarian granulosa cells of laying hens(P<0.05).5 Effect of mercury exposure on apoptosis in chicken embryo kindney cellsThis study investigated the effect of mercuric chloride exposure on apoptosis in chicken embryo kidney(CEK)cells.The results in vivo revealed that renal apoptosis were found in 9.42 and 27.24 mg/kg mercury groups.After that,the molecular mechanism of mercury inducing apoptosis was further investigated in vitro.The CEK cells were cultured in vitro and four treatment groups were set:control group(0 μM mercury),5 μM mercury group,10μM mercury group and 15λM mercury group Meanwhile,4-phenylbutyric acid(4-PBA),the endoplasmic reticulum stress(ERS)inhibitor,was used to investigate the mechanism of mercury-induced CEK cells apoptosis.Four other treatment groups were set:control group(0 μM mercury),4-PBA treatment group,15 μM mercury treatment group and 15 μM mercury + 4-PBA treatment group.Results revealed that compared with the control group,mercuric chloride exposure reduced the density and significantly reduced the activity of CEK cells(P<0.05)after 24-h incubation,while enhanced the nuclear concentrations of CEK cells.Compared with the control group,both the apoptosis rate and percentage reduction in mitochondrial membrane potential of CEK cells were significantly increased(P<0.05)after mercury exposure.Besides,the gene expression of Bax/Bcl-2(P<0.05)was significantly increased after mercuric chloride exposure.However,CEK cells activity in 15 μM mercury + 4-PBA treatment group was significantly higher than 15 μM mercury treatment group(P<0.05).The apoptosis rate in 15 μM mercury+4-PBA treatment group was significantly lower than 15 μM mercury treatment group(P<0.05).The gene expression of Bax/Bcl-2(P<0.05)in 15 μM mercury + 4-PBA treatment group was significantly lower than 15 μM mercury treatment group.In addition,the gene expressions of GRP78(P<0.05),ATF4(P<0.05)and CHOP(P<0.05)were significantly increased after mercuric chloride exposure.The protein expressions of GRP78(P<0.05),ATF4(P<0.05),p-PERK/PERK(P<0.05)and CHOP(P<0.05)were also significantly increased after mercuric chloride exposure.However,the gene expressions GRP78(P<0.05),ATF4(P<0.05)and CHOP(P<0.05)in 15 μM mercury + 4-PBA treatment group were all significantly lower than 15μM mercury treatment group.The protein expressions of GRP78(P<0.05),ATF4(P<0.05),p-PERK/PERK(P<0.05)and CHOP(P<0.05)in 15μM mercury + 4-PBA treatment group were all also significantly lower than 15 μM mercury treatment group.In conclusion,dietary mercury exposure significantly reduced laying performance and egg quality,and damaged liver and kidney histopathology,and significantly enhanced follicular atresia rate in laying hens.Dietary mercury exposure reduced downstream antioxidant gene expressions by means of inhibiting Nrf2-Keap1 pathway,significantly reducing antioxidant enzyme activity in liver,kidney and ovary,thus damaging the antioxidant system of liver,kidney and ovary in laying hens.Besides,we found that dietary mercury exposure significantly reduced serum FSH,LH,P4 levels in laying hens.In vitro,mercuric chloride exposure disturbed intracellular cAMP-PKA pathway and StAR,P450scc and 3β-HSD gene expressions,and then inhibited P4 secretion at time and dose dependent ways.In addition,we found that dietary mercury exposure induced renal apoptosis in laying hens.In vitro,mercuric chloride exposure could induce CEK cells apoptosis by activating intracellular PERK-ATF4-CHOP pathway associated with ERS,enhancing the Bax/Bcl-2 gene expression in mitochondria of CEK cells.