A Study On The Mechanism Of Zinc-methionine To Regulate Eggshell Quality In Laying Hens

To solve the problem of thin eggshell thickness and weak eggshell strength of laying hens after the peak period which seriously affected egg transportation,processing and economic benefits in poultry production,we determined the mechanism of dietary Zn-Methionine addition on the regulation of the shell quality in laying hens.Firstly,the effects of dietary Zn-Met supplementation on laying performance,egg quality,immunity and antioxidant capacity of laying hens were studied to determine the effects of Zn-Met on improving eggshell quality and its suitable addition levels.Starting from the characteristic of Zn-Met absorbed and transported in vivo,the mechanism of dietary Zn-Met addition on the regulation of shell quality in layers was studied in vivo and in vitro.The study enriched the metabolism theory of amino acid chelating zinc on egg birds as well as provided scientific basis for the rational application of organic trace element(amino acid chelating zinc)in layers’feedstuff.The main results of this study are shown as below:1 Study on the appropriate addition of Zn-Met in laying hens after the peak periodThe present study was to investigate the appropriate addition of Zn-Met compared with inorganic zinc in layers after the peak period.A total of 720 hens of Jinghong-1 strain at the age of 49 weeks were randomly divided into 6 groups with 6 replicates(20 hens per replicate).The control(80 mg/kg)was fed basal diet with zinc sulfate(ZnSO4);The 5 experimental groups(20,40,60,80,and 100 mg/kg,respectively)were fed basal diet with zinc-methionine(Zn-Met).The results showed that the feed intake and feed conversion ratio significantly decreased in the 80 mg/kg Zn-Met group compared with the control(P<0.05).In the pancreas,kidney and liver,the zinc content in the 80,100 mg/kg Zn-Met groups was significantly higher than that in control(P<0.05).Then the villus height and villus area in the duodenum,jejunum and ileum significantly increased in the 60 and 80 mg/kg Zn-Met groups compared with control group(P<0.05).Moreover,immunogloblin M(IgM)levels in the ileum and catalase(CAT)levels in the liver of the 80 and 100 mg/kg Zn-Met groups were significantly higher than those in control(P<0.05).and the levels of total antioxidant capacity(T-AOC)and glutathion peroxidase(GSH-Px)in serum,T-AOC,copper-zinc-superoxide dismutase(CuZnSOD)and GSH-Px in liver were significantly higher in the 60 mg/kg Zn-Met group than those in control(P<0.05).Total protein(TP)levels in serum significantly increased in 100 mg/kg Zn-Met group compared with control group(P<0.05).After 15 days in store,the egg weight in 60 mg/kg Zn-Met group,albumen height in the 80 and 100 mg/kg Zn-Met groups and haugh units in the 100 mg/kg Zn-Met group were significantly increased compared with the control(P<0.05).The decreased mammillary knob width and Ca2+in serum,the increased palisade layer thickness,carbonic anhydrase(CA)activity in eggshell gland(ESG)and alkaline phosphatase(AKP)levels in serum were found in the 80 mg/kg Zn-Met group(P<0.05).It was concluded that the appropriate dietary addition level of Zn-Met in laying hens was 60～80 mg/kg in current study.Compared with 80 mg/kg ZnSO4 group,80 mg/kg Zn-Met group can reduce feed conversion ratio,extend the shelf life of eggs,improve the intestinal morphology and the immune,antioxidant functions.2 Study on the mechanism of Zn-Met regulating eggshell quality of laying hens 2.1 The characteristic of Zn-Met absorbed and transported in the small intestine and liver of laying hensThe present study was to investigate the characteristic of Zn-Met absorbed and transported in the small intestine and liver of laying hens.Hyline grey layers(n=288,38 weeks old)were divided into 3 groups(6 replicates with 16 hens):0 mg Zn/kg,80 mg Zn/kg as Zn-Met,and ZnSO4.The results showed that compared with control group and ZnSO4 group,Zn-Met group significantly increased zinc content in the liver,duodenum and jejunum,as well as the villus height,villus area and villus height/crypt depth in jejunum,and significantly decreased the crypt depth(P<0.05).The mRNA expression of metallothionein(MT)in duodenum and metal-response element-binding transcription factor-1(MTF-1)in the liver of Zn-Met group was significantly higher than that of control group(P<0.05).Moreover,the mRNA expression level of B0 neutral amino acid transporter 1(B0AT1)in jejunum and L-type amino acid transporter-1(LAT1)mRNA in the duodenum of Zn-Met group was significantly increased(P<0.05).In conclusion,compared with the ZnSO4 group,Zn-Met group can enhance intestinal morphology in the jejunum and the expression of MT and LAT1 mRNA in duodenum,MTF-1 mRNA in liver and B0AT1 mRNA in jejunum,so as to improve the zinc content in the tissues and intestines,indicating that there may be other uptake pathway of Zn-Met except for zinc transporters.2.2 Study on the mechanism of Zn-Met regulating eggshell quality of laying hensBased on the previous feeding trial,the effects of Zn-Met on the apparent parameters and ultrastructure of eggshell,serum biochemical indexes and gene expression of shell matrix protein in laying hens were studied.Meanwhile,the content and distribution of calcium on the transversal surface of eggshell were analyzed by energy dispersive X-ray spectrometer,so as to explore the regulation mechanism of Zn-Met on the eggshell quality.Results showed that egg weight in the Zn-Met group was significantly higher than that in the control group while eggshell strength and eggshell thickness were significantly higher than that in the control group and the ZnSO4 group at the end of the experiment(P<0.05).Compared with the control group and ZnSO4 group,the Zn-Met group(P<0.05)had increased fiber diameter in inner surface,decreased mammillary knob width.Moreover,eggshell strength in Zn-Met group was negatively correlated with mammillary knob width(P<0.01,r2=0.5032).Compared with the control group and the ZnSO4 group,the Zn-Met group had larger relative atomic weight percentage of Ca,stronger signal intensity of Ca in linear distribution and the Ca was more evenly distributed in the transversal surface of eggshell.Zinc content in the egg yolk and serum of Zn-Met group was significantly higher than that of control group and ZnSO4 group(P<0.05).In egg white,eggshell.ovary,magnum and ESG of Zn-Met group,zinc content was significantly higher than that of control group(P<0.05).The CA and AKP activity in the serum,the concentration of Ca2+ and albumin(ALB)and the expression levels of CA and Ca-binding protein-d28k(CaBP-D28k)mRNA in ESG in the 80 mg/kg Zn-Met group were the highest among all treatments(P<0.05).In conclusion,Zn-Met can improve the microstructure of egg shell by increasing the activities of CA and AKP in serum,Ca2+concentration in ESG and mRNA expression levels of CA and CaBP-D28k,reduce the mammillary cone width,promote the deposition and distribution of calcium in egg shell,and thus enhance the thickness and strength of egg shell.2.3 Primary culture of endometrial epithelial cell model of laying hens in vitroIn this study,the uterus was removed from the laying hens at the peak period for primary culture of endometrial epithelial cell,and then digested using type I collagenase.Cells proliferation was detected by CCK 8 kit.The results showed that the endometrial epithelial cells digested by collagenase I had good attachment and growth in culture.The cultured cells reached the logarithmic growth stage at 2-5 days,and entered the plateau stage at 7 days after culture.The results suggested that the endometrial epithelial cells isolated by scraping and digested by type I collagenase could grow well.2.4 Effects of Zn-Met on the expression of eggshell quality-related genes in vitro The present study was conducted to investigate the effect of Zn-Met or ZnSO4 on the expression of eggshell quality-related genes in endometrial epithelial cells in vitro using the established endometrial epithelial cell model.On the 3rd day of cell culture,cells were cultured for 24 h or 48 h in DMEM treated with Zn-Met or ZnSO4(0,25,50,100,200 and 400μM,respectively).The results showed that cells had a optimal response in DMEM treated with 50 μM Zn(Zn-Met or ZnSO4)for 24 h.After treatment with 50 μM Zn for 24 h,intracellular Ca2+concentration and MT mRNA expression levels in the Zn-Met group were significantly higher than those in the control group(P<0.05).In conclusion,Zn-Met supplementation can increase intracellular Ca2+concentration and promote the expression of MT mRNA in endometrial epithelial cells in vitro.In summary,under the conditions of this experiment,dietary Zn-Met addition in laying hens can reduce the feed conversion ratio,extend the shelf life of eggs,and improve the immune and antioxidant functions,and the appropriate addition level of Zn-Met was 60～80 mg/kg;Zn-Met increases the zinc content in tissues and intestines by improving the intestinal morphology,enhancing the mRNA expression of zinc-related transporters in the small intestine and liver,and promoting the absorption of zinc;Zn-met improves the microstructure of eggshell,reduces the width of mastoid body,promotes the deposition and distribution of calcium in eggshell,and enhances the thickness and strength of eggshell by increasing the activities of CA and AKP in serum,Ca2+concentration in ESG and mRNA expression levels of CA and CaBP-D28k.