Study On Metallothionein Synthesis In Vivo, Metabolism And Its Action Mechanisms In Regulating Stress Resistance In Swine And Dairy Cattle

To approach the metabolism rule of metallothionein(MT) and its action mechanismsin regulating stress resistance in animals,several experiments were conducted with Duroc×Landrace×Yorkshire hybrid pigs (DLY pigs) , Wild×Duroc hybrid pigs (WD pigs) and Chinese-Holstein dairy cattle.The results are as below:1 MT synthesis in liver of animals was induced by different dosages of zinc solution which were infused into the animal's body by intraperitoneal injection for the pigs and intramuscular injection for the male calves and MT contents in liver of animals were mensurated by a MT colour reaction method. The total dosage of zinc which induced MT synthesis in liver effectively were 330mg/kg for the pigs and 185 mg/kg for the calves.MT yields synthesized in livers of the pigs and the calves were 7.82mg/g and 8.60mg/g respectively.And the structure of MT synthesized in livers of the animals,both the pigs and the calves,is so steady that it can be stored in below zero safely.2 A new indirect competitive ELISA was established to mensurate MT in the samples from the pigs and the catties. The suitable parameters for this ELISA were determined as shown below: the concentration of carrier antigen is 6.25μg/ml,the concentration of first antibody (mouse-anti swine) is 50μg/ml, and the diluent scale of HRP-immunoglobulin (goat-antimouse) is 1:8000 and the reaction time is 3hr. A linear regression equation of the criterion curve established in indirect competitive ELISA for determining MT is y=10⁵×2^{22.32880-0.39583x} and its correlation coefficient is R=0.9592. The sensitivity of this ELISA was 4.1ng/ml and the variation coefficient(VC) Between and within batches were 9.06%å’Œ12.35% respectively.All of these suggested that this method have quite high specificity, sensitivity and stability.And it is especially applicable to quick measurement for great batch of samples.3 MT dynamics in blood , feces and urine,and MT accumulation in tissues and organs of DLY pigs and WD pigs were also conducted by a isotopic tracing method using MT labeled with ¹²⁵I ¹²⁵I-MT which was affused through mouth..It was shown that MT was absorbted perfectly with a tardigrade mode and remained about 31 hours in the digestive tract of both DLY pigs and WD pigs . MT excreted from feces and urine of DLY pigs accounted for 7.91 and 21.45 percent of total MT affused through mouth while MT excreted from feces and urine of WD pigs accounted for 10.09 and 11.85 percent of total MT affused through mouth, respectively.There was a significant different (P<0.01) in MT excreted from urine between DLY pigs and WD pigs. MT accumulated in tissues and organs of DLY pigs and WD pigs accounted for 4.61 and 8.60 percent of total MT affused respectively. MT content in tissues and organs of DLY pigs shown the sequence as "small intestine > large intestine > stomach > shin > kidney > lung > liver > heart > meat > spleen>bone",and the sequence in WD pigs was "stomach > large intestine > small intestine > shin > kidney > lung > heart > liver > spleen > meat > bone".4 In this experiment, the effects of exogenous Zn-MT on anti-oxidative function and SOD gene expression in DLY pigs and DW pigs were whipped to be get stress were studied with RT-PCR. After Zn-MT injection, both SOD activity and GSH-PX activity in liver of the pigs increased significantly (P<0.05) and MDA content of in liver of the pigs decreased significantly(P<0.05, P<0.01). Six hours after Zn-MT injection, SOD gene expression levels in DLY pigs in groupâ…¡(injected 0.8mg Zn-MT /kgBW) and groupâ…¢(injected 1.6mg/kgBW) were higher significantly (P<0.05) than that in the control(injected 0 mg/kgBW). There were significant differences (P<0.05) in SOD gene expression levels in DLY pigs in groupâ…¡and groupâ…¢between six hours and three hours after Zn-MT injection. However, there was no significant difference (P > 0.05) in SOD gene expression levels in DW pigs between the and the control group in spite of the fact that SOD gene expression level in liver of the pigs in the trial groups were higher than the latter.5 SOD mRNA levels and SOD activities presented time-dependent and dose-dependent effects.The effects of Zn-MT on SOD activity, GSH-PX activity, MDA content and SOD gene expression in liver in DLY pigs were coincident with those in DW pigs.And there were significant differences (P < 0.05) in SOD activities and MDA contents affected by MT between DLY pigs and DW pigs,the impact of Zn-MT on DLY pigs was stronger than that on DW pigs.6 In this experiment, 28 lactating cows which were randomly allocated to group A,groupB, group C and group D,and supplemented with 0,6.0,12.0 and 16.0mg Zn-MT.capita^-1,respectively, by intravenous injection to approach the effects of exogenous Zn-MT on production performances , thermoresistance and SOD gene expression in dairy cattl. The results showed that the pulse rate, breathing rate, MDA content in serum of the cows in groupB, groupC and group D were lower (P < 0.05 or P < 0.01) than those in group A,while milk crop, MT contents in serum and milk, blood GSH-PX activity, erythrocyte SOD activity and SOD gene expression level of the cows in groupB, group C and group D were higher (P < 0.05 or P < 0.01) than those in group A. And the above of of the cows in group C and group D were better (P < 0.05 or P > 0.05 )than those in group B, but there were no significant differences (P > 0.05) in those traits above between group C and group D.Exogenous Zn-MT effects on the thermoresistance and SOD gene expression of dairy cattle mensurated on the 30^th day after injection were much better than those on the 1^st day (P < 0.05 or P<0.01) and the 15^th day (P < 0.05 or P > 0.05) ,and also better (P > 0.05) than those on the 45^th day.All of these suggested that exogenous Zn-MT should be an effective physiologically active substance for dairy cattle,and its effects on the thermoresistance and SOD mRNA expression of dairy cows,and presented time-dependent and dose-dependent effects.In conclution,the present study successfully induced MT synthesis in liver of pigs and calves, established an Indirect Competitive ELISA for measuring MT, and elucidated MT metabolism rule and dynamics in blood , feces and urine, tissues and organs of DLY pigs and WD pigs , investigated MT action in regulating stress resistance and anti-oxidation and its mechanisms in swine and dairy cattle. Therefore,MT can be used as an effective physiologically active substance for swine and dairy cattle.