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Molybdenum Toxicity in the Developing Chick Embryo
GROWTH-RATE SELECTION Hess, C. W. and M. A. Jull, 1948. A study of the inheritance of feed utilization efficiency in the growing domestic fowl. Poultry Sci. 27: 24-39. Hutt, F. B., 1949. Genetics of the Fowl. McGrawHill Book Company, New York. 590 pp. Lamoreux, W. F., and F. B. Hutt, 1939. Breed differences in resistance to a deficiency of vitamin Bi in the fowl. J. Agr. Res. 58: 307316. Lamoreux, W. F., and F. B. Hutt, 1948. Genetic resistance to deficiency of riboflavin in the chick. Poultry Sci. 27: 334-341. McDonald, M. W., 1957. Methionine supplements
1047
in chicken diets. II. A breed difference in growth response to DL-methionine. Australian J. Agr. Res. 8: 587-594. McDonald, M. W., 1958. Methionine supplements in chicken diets. III. The biochemical difference in sulphur-amino acid metabolism between White Leghorns and Australorps. Australian J. Agr. Res. 9: 161-169. Miller, E. C , J. S. O'Barr and C. A. Denton, 1960. The metabolism of methionine by single comb White Leghorn and Black Australorp chicks. J. Nutrition, 70: 42^16.
PAUL D. LEPOEE, R. F. MILLER AND H. S. SIEGEL Departments of Biochemistry and Nutrition and Poultry Husbandry, Virginia Polytechnic Institute, Blacksburg, Virginia (Received for publication July 28, 1961)
T
HE CHICK embryo has been widely used to investigate chemical toxicities because it develops rapidly under controlled environmental conditions through use of its complete, closed nutritional supply. In addition, large numbers of experimental animals are available at modest cost. Therefore, the developing chick embryo was considered in the study of molybdenum toxicity. Studies with growing chickens have indicated that the feeding of toxic quantities of molybdenum will result in depressed growth and anemia (Branion et al., 1958; Kratzer, 1962; Reid et al., 1958). At least some of these toxic effects were partially alleviated by the supplementation of the molybdenum diet with copper (Branion et al., 1958) or sulfate (Reid et al., 1958) salts. L. D.50 values for sodium molybdate injected into the yolk sac of chick embryos of 4 and 8 days were estimated by Ridgway and Karnofsky (1952) to be 0.80 mg. and 0.70 mg., respectively. These workers
observed no gross pathological effects when sub-lethal amounts of molybdenum were injected. Thus, it was of interest, not only to assess the toxic effects of molybdenum upon the developing chick embryo, but also to determine whether copper or sulfate salts were effective in reducing the molybdenum-inducted lethality. Fertile White Rock or White Leghorn eggs were incubated in the usual manner. After the appropriate period of incubation, the designated amount of chemical in 0.2 ml. of ion-free H 2 0 was aseptically injected into the yolk sacs of the embryonating eggs via the air cell. Molybdenum was1 injected as a solution of the sodium molybdate salt. Lethal effects of injected chemicals were ascertained by periodic candling. The embryonic mortality resulting from the various treatments was noted from one day post-treatment through the nineteenth day of incubation. At this time, all surviving embryos were sacrificed.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
Molybdenum Toxicity in the Developing Chick Embryo
1048
P. D. LEPOEE, R. F. MILLER AND H. S. SIEGEL
100
g >h_l <
1-
ac
o
WHITE ROCK o a fr
WHITE LEGHORN o—o—o—o—o—*—o-
90 R0 70 60 50
s o
/>— *
V 1 \v \X 23
4 5 6 7 8
9 10 110 1 2 3 4 5 DAY OF INCUBATION
AVV\ 67
8 9 10 II
FIG. 1. Mortality of chick embryos as influenced by age and level of injected molybdenum. 1.00 mg. molybdenum injected 0.50 mg. molybdenum injected 0.25 mg. molybdenum injected - • - • - • - • — 1.78 mg. NaCl injected (controls).
RESULTS AND DISCUSSION Experiments 1 and 2 were designed to obtain an indication of the lethal effect of a single injection of molybdenum as influenced by embryonic strain. In Experiment 1, 10 White Rock embryos, and in Experiment 2, 12 White Leghorn embryos were used for each treatment group. The dose levels and age range of embryos are depicted in the summarized data in Figure 1. Although there were differences in the responses of the two strains, a sharp increase in resistance to the lethal effects of the molybdenum was noted in 9 and 10 day embryos of both strains in those groups receiving 1.0 mg. of injected molybdenum. Embryos injected with 0.50 mg. of molybdenum exhibited a more variable response but in general were more resistant at and after the 8th day of incubation. The fatalities observed in the NaCl injected controls
were assumed to have resulted from a combination of natural causes and occasional faulty injections. In the course of these and other preliminary studies, it was observed that when 0.50 mg. of molybdenum was injected into the yolk sac of 4 day embryos, death consistently occurred on the 7th day of incubation. Therefore, in Experiment 3, the injection of a solution of both molybdenum and CuCl2 on the 4th day of incubation served as the basis for assessing whether copper exhibited a protective effect. The results are presented in Table 1. CuCl2 alone was partially lethal, even at levels of 0.11 and 0.43 mg. Some evidence of a protective effect of copper on the molybdenum induced lethality was noted, particularly in those groups receiving from 0.70 to 0.85 mg. of CuCl2.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
40 z o 30 >or GO 2 0 2 UJ 10
1049
MOLYBDENUM TOXICITY TABLE 1.—Embryonic mortality resulting from the yolk sat injection of CuCh alone and with the lethal dose of molybdenum Injection
^
1.79mg.NaCl 2 0.11 mg. CuCh 4 0.21 mg. CuCh 3 0.32 mg. CuCh 2,S,S 0.43mg. CuCh 4 0.80mg.CuCh 7 0.85mg. CuCh 10 1.70 mg. CuCh 12 1
Injection 0.50mg.Mo 0.50mg.Mo+0.11mg. CuCh 0.50mg.Mo+0.21mg. CuCh 0.50mg. Mo+0.32 mg. CuCU 0.50 mg. Mo+0.50mg. CuCh 0.50mg.Mo+0.70mg.CuCh 0.50 mg. Mo+0.80 mg. CuCh 0.50 mg. Mo + 0.85 mg. CuCh
TABLE 2.—Embryonic mortality resulting from the yolk sac injection of Na?SOi alone and with the lethal dose of molybdenum ^
12 12 10 12,12 12 7 10 7
12 embryos per group.
SUMMARY
Molybdenum injected into the yolk sac of developing chick embryos became less
^
1
Injection
2 3 1 1,1 3 2 5 2
0.50 mg. Mo 0.50 mg. Mo+1.98mg. NajSOj 0.50mg. Mo+2.96mg. Na s S0 4 0 50 mg. Mo+3.00mg. Na2SOi 0.50 mg. Mo+5.82mg. NazSOi 0.50mg. Mo+6.00mg. Na2S04 0.50 mg. Mo +9.00 mg. NajSOj
de°;
12 7 6 8 5 7 5
12 embryos per group.
TABLE 3.—Embryonic mortality resulting from the yolk sac injection of various salts alone and with the lethal dose of molybdenum Injection 8.0 mg. NaCl 8.0 mg. KC1 8.0 mg. K.2SO4 1
^
Injection 2 0 1
0.50 mg. Mo 0.50 mg. Mo+8.0 mg. NaCl 0.50 mg. Mo+8.0mg. KC1 0.50 mg. Mo+8.0mg. K2SO4
j^j 12 7 9 6
12 embryos per group.
lethal as embryonic age advanced beyond 8 days. The molybdenum induced lethality of 4 day embryos can be partially eliminated by the concomitant injection of various salts. REFERENCES Branion, H. D., D. Arthur and I. Motzok, 1958. Interaction of dietary copper and molybdenum in rations fed to poultry. Poultry Sci. 37: 1181. Davies, R. C , A. A. Kurnick, B. L. Reid and J. R. Couch, 1957. Molybdenum and sulfate interaction of the growing chick. Poultry Sci. 36: 1111. Kratzer, F. H., 1952. Effect of dietary molybdenum upon chicks and poults. Proc. Soc. Exp. Biol. Med. 80: 483-486. Reid, B. L., R. E. Davies and J. R. Couch, 1958. Relationship of sulfate, copper and manganese to molybdenum in diet of the chick. Fed. Proc. 17: 294. Ridgway, L. P., and D. A. Karnofsky, 1952. The effects of metals on the chick embryo: toxicity and production of abnormalities in development. Ann. New York Acad, of Sci. 55: 203-215. Van Reen, R., K. F. Glassford and J. P. Zagrosky, 1959. The specificity of the molybdate-sulfate interrelationship in rats. J. Nutrition, 68: 243250.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
Experiment 4 investigated the protective effect of Na 2 S0 4 when injected with the lethal level of molybdenum on the 4th day of incubation (Table 2). From the data, one can conclude that the levels of NaS0 4 injected were essentially nontoxic and that the Na 2 S0 4 considerably reduced the fatalities caused by the injected molybdenum. There was no evidence, however, of an increase in the protection with increasing levels of injected Na 2 S0 4 . That the protection observed was not a specific sulfate effect was indicated by the results of Experiment 5 which is summarized in Table 3. Injections were again made on the 4th day of incubation. Some protective effect was exhibited also by KC1, NaCl and K 2 S0 4 . These results differ from those of Van Reen et al. (1959), which indicate that the alleviation of molybdenum toxicity in the growing rat was a specific effect of sulfate. In conclusion, these studies indicate that the molybdenum induced mortality of 4 day embryos may be reduced by the concomitant injection of various salts. Whether these salts act by a physical or chemical alteration of the molybdenum or yolk at the site of injection requires further investigation.
Injection lO.COmg. NaCl 1.48 mg. NajSOi 2.96mg. NasSOi 5.82 mg. NajS04 10.00 mg. Na2S04 11.64mg. Na2S04 23.28 mg.Na2S04 46.52 mg. Na2S04
1047
in chicken diets. II. A breed difference in growth response to DL-methionine. Australian J. Agr. Res. 8: 587-594. McDonald, M. W., 1958. Methionine supplements in chicken diets. III. The biochemical difference in sulphur-amino acid metabolism between White Leghorns and Australorps. Australian J. Agr. Res. 9: 161-169. Miller, E. C , J. S. O'Barr and C. A. Denton, 1960. The metabolism of methionine by single comb White Leghorn and Black Australorp chicks. J. Nutrition, 70: 42^16.
PAUL D. LEPOEE, R. F. MILLER AND H. S. SIEGEL Departments of Biochemistry and Nutrition and Poultry Husbandry, Virginia Polytechnic Institute, Blacksburg, Virginia (Received for publication July 28, 1961)
T
HE CHICK embryo has been widely used to investigate chemical toxicities because it develops rapidly under controlled environmental conditions through use of its complete, closed nutritional supply. In addition, large numbers of experimental animals are available at modest cost. Therefore, the developing chick embryo was considered in the study of molybdenum toxicity. Studies with growing chickens have indicated that the feeding of toxic quantities of molybdenum will result in depressed growth and anemia (Branion et al., 1958; Kratzer, 1962; Reid et al., 1958). At least some of these toxic effects were partially alleviated by the supplementation of the molybdenum diet with copper (Branion et al., 1958) or sulfate (Reid et al., 1958) salts. L. D.50 values for sodium molybdate injected into the yolk sac of chick embryos of 4 and 8 days were estimated by Ridgway and Karnofsky (1952) to be 0.80 mg. and 0.70 mg., respectively. These workers
observed no gross pathological effects when sub-lethal amounts of molybdenum were injected. Thus, it was of interest, not only to assess the toxic effects of molybdenum upon the developing chick embryo, but also to determine whether copper or sulfate salts were effective in reducing the molybdenum-inducted lethality. Fertile White Rock or White Leghorn eggs were incubated in the usual manner. After the appropriate period of incubation, the designated amount of chemical in 0.2 ml. of ion-free H 2 0 was aseptically injected into the yolk sacs of the embryonating eggs via the air cell. Molybdenum was1 injected as a solution of the sodium molybdate salt. Lethal effects of injected chemicals were ascertained by periodic candling. The embryonic mortality resulting from the various treatments was noted from one day post-treatment through the nineteenth day of incubation. At this time, all surviving embryos were sacrificed.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
Molybdenum Toxicity in the Developing Chick Embryo
1048
P. D. LEPOEE, R. F. MILLER AND H. S. SIEGEL
100
g >h_l <
1-
ac
o
WHITE ROCK o a fr
WHITE LEGHORN o—o—o—o—o—*—o-
90 R0 70 60 50
s o
/>— *
V 1 \v \X 23
4 5 6 7 8
9 10 110 1 2 3 4 5 DAY OF INCUBATION
AVV\ 67
8 9 10 II
FIG. 1. Mortality of chick embryos as influenced by age and level of injected molybdenum. 1.00 mg. molybdenum injected 0.50 mg. molybdenum injected 0.25 mg. molybdenum injected - • - • - • - • — 1.78 mg. NaCl injected (controls).
RESULTS AND DISCUSSION Experiments 1 and 2 were designed to obtain an indication of the lethal effect of a single injection of molybdenum as influenced by embryonic strain. In Experiment 1, 10 White Rock embryos, and in Experiment 2, 12 White Leghorn embryos were used for each treatment group. The dose levels and age range of embryos are depicted in the summarized data in Figure 1. Although there were differences in the responses of the two strains, a sharp increase in resistance to the lethal effects of the molybdenum was noted in 9 and 10 day embryos of both strains in those groups receiving 1.0 mg. of injected molybdenum. Embryos injected with 0.50 mg. of molybdenum exhibited a more variable response but in general were more resistant at and after the 8th day of incubation. The fatalities observed in the NaCl injected controls
were assumed to have resulted from a combination of natural causes and occasional faulty injections. In the course of these and other preliminary studies, it was observed that when 0.50 mg. of molybdenum was injected into the yolk sac of 4 day embryos, death consistently occurred on the 7th day of incubation. Therefore, in Experiment 3, the injection of a solution of both molybdenum and CuCl2 on the 4th day of incubation served as the basis for assessing whether copper exhibited a protective effect. The results are presented in Table 1. CuCl2 alone was partially lethal, even at levels of 0.11 and 0.43 mg. Some evidence of a protective effect of copper on the molybdenum induced lethality was noted, particularly in those groups receiving from 0.70 to 0.85 mg. of CuCl2.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
40 z o 30 >or GO 2 0 2 UJ 10
1049
MOLYBDENUM TOXICITY TABLE 1.—Embryonic mortality resulting from the yolk sat injection of CuCh alone and with the lethal dose of molybdenum Injection
^
1.79mg.NaCl 2 0.11 mg. CuCh 4 0.21 mg. CuCh 3 0.32 mg. CuCh 2,S,S 0.43mg. CuCh 4 0.80mg.CuCh 7 0.85mg. CuCh 10 1.70 mg. CuCh 12 1
Injection 0.50mg.Mo 0.50mg.Mo+0.11mg. CuCh 0.50mg.Mo+0.21mg. CuCh 0.50mg. Mo+0.32 mg. CuCU 0.50 mg. Mo+0.50mg. CuCh 0.50mg.Mo+0.70mg.CuCh 0.50 mg. Mo+0.80 mg. CuCh 0.50 mg. Mo + 0.85 mg. CuCh
TABLE 2.—Embryonic mortality resulting from the yolk sac injection of Na?SOi alone and with the lethal dose of molybdenum ^
12 12 10 12,12 12 7 10 7
12 embryos per group.
SUMMARY
Molybdenum injected into the yolk sac of developing chick embryos became less
^
1
Injection
2 3 1 1,1 3 2 5 2
0.50 mg. Mo 0.50 mg. Mo+1.98mg. NajSOj 0.50mg. Mo+2.96mg. Na s S0 4 0 50 mg. Mo+3.00mg. Na2SOi 0.50 mg. Mo+5.82mg. NazSOi 0.50mg. Mo+6.00mg. Na2S04 0.50 mg. Mo +9.00 mg. NajSOj
de°;
12 7 6 8 5 7 5
12 embryos per group.
TABLE 3.—Embryonic mortality resulting from the yolk sac injection of various salts alone and with the lethal dose of molybdenum Injection 8.0 mg. NaCl 8.0 mg. KC1 8.0 mg. K.2SO4 1
^
Injection 2 0 1
0.50 mg. Mo 0.50 mg. Mo+8.0 mg. NaCl 0.50 mg. Mo+8.0mg. KC1 0.50 mg. Mo+8.0mg. K2SO4
j^j 12 7 9 6
12 embryos per group.
lethal as embryonic age advanced beyond 8 days. The molybdenum induced lethality of 4 day embryos can be partially eliminated by the concomitant injection of various salts. REFERENCES Branion, H. D., D. Arthur and I. Motzok, 1958. Interaction of dietary copper and molybdenum in rations fed to poultry. Poultry Sci. 37: 1181. Davies, R. C , A. A. Kurnick, B. L. Reid and J. R. Couch, 1957. Molybdenum and sulfate interaction of the growing chick. Poultry Sci. 36: 1111. Kratzer, F. H., 1952. Effect of dietary molybdenum upon chicks and poults. Proc. Soc. Exp. Biol. Med. 80: 483-486. Reid, B. L., R. E. Davies and J. R. Couch, 1958. Relationship of sulfate, copper and manganese to molybdenum in diet of the chick. Fed. Proc. 17: 294. Ridgway, L. P., and D. A. Karnofsky, 1952. The effects of metals on the chick embryo: toxicity and production of abnormalities in development. Ann. New York Acad, of Sci. 55: 203-215. Van Reen, R., K. F. Glassford and J. P. Zagrosky, 1959. The specificity of the molybdate-sulfate interrelationship in rats. J. Nutrition, 68: 243250.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
Experiment 4 investigated the protective effect of Na 2 S0 4 when injected with the lethal level of molybdenum on the 4th day of incubation (Table 2). From the data, one can conclude that the levels of NaS0 4 injected were essentially nontoxic and that the Na 2 S0 4 considerably reduced the fatalities caused by the injected molybdenum. There was no evidence, however, of an increase in the protection with increasing levels of injected Na 2 S0 4 . That the protection observed was not a specific sulfate effect was indicated by the results of Experiment 5 which is summarized in Table 3. Injections were again made on the 4th day of incubation. Some protective effect was exhibited also by KC1, NaCl and K 2 S0 4 . These results differ from those of Van Reen et al. (1959), which indicate that the alleviation of molybdenum toxicity in the growing rat was a specific effect of sulfate. In conclusion, these studies indicate that the molybdenum induced mortality of 4 day embryos may be reduced by the concomitant injection of various salts. Whether these salts act by a physical or chemical alteration of the molybdenum or yolk at the site of injection requires further investigation.
Injection lO.COmg. NaCl 1.48 mg. NajSOi 2.96mg. NasSOi 5.82 mg. NajS04 10.00 mg. Na2S04 11.64mg. Na2S04 23.28 mg.Na2S04 46.52 mg. Na2S04