Comparative Effects of Selenium on Metabolism of Methylmercury by Chickens and Quail: Tissue Distribution and Transfer into Eggs1 j . L. SELL 2 Animal Science Department, North Dakota State University, Fargo, North Dakota 58102 (Received for publication October 5, 1976)
Poultry Science 56:939-948, 1977
It is well k n o w n t h a t Hg derived from alkyl c o m p o u n d s (i.e. m e t h y l m e r c u r y ) is deposited readily in eggs (Tejning and Vesterberg, 1 9 6 4 ; Kiwimae et al, 1 9 6 9 ; Campbell et al, 1 9 7 1 ; and Sell et al, 1 9 7 4 ) . T h e majority of this t y p e of Hg is f o u n d in egg w h i t e ( S m a r t and L l o y d , 1 9 6 3 ; Kiwimae et al, 1 9 6 9 ; and Campbell et al, 1 9 7 1 ) , associated p r e d o m i n a t e l y with t h e protein, ovalbumin (Sell et al, 1 9 7 4 ) . Recently, m u c h a t t e n t i o n has been focused o n t h e interaction b e t w e e n Se a n d Hg w h e r e b y Hg metabolism a p p a r e n t l y is modified ( G a n t h e r et al, 1 9 7 2 ; El-Bergearmi et al, 1 9 7 3 ; P o t t e r and M a t r o n e , 1 9 7 4 ; J o h n s o n and Pond, 1 9 7 4 ) . However, little information has been published concerning t h e effect of Se o n t h e transfer o f dietary Hg i n t o eggs. Stoewsand et al. ( 1 9 7 4 )
1
Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No. 713. This research was supported in part by Research Grant 1R01 FD 00752, United States Public Health Services, National Institutes of Health. 2 Present address: Department of Animal Science, Iowa State University, Ames, Iowa 50010.
presented d a t a which indicated t h a t Se reduced t h e m e t h y l m e r c u r y c o n c e n t r a t i o n of eggs produced b y Japanese quail fed this form of m e r c u r y . Emerick et al. ( 1 9 7 6 ) r e p o r t e d t h a t t h e eggs of chickens fed m e t h y l m e r c u r y and Se contained m o r e Hg t h a n t h o s e of h e n s fed m e t h y l m e r c u r y alone. T h e research r e p o r t e d here describes t h e influence of dietary Se on t h e transfer of dietary Hg i n t o eggs of chickens and Japanese quail. D a t a illustrating t h e modifying effects of Se on t h e distribution of Hg a m o n g tissues and a m o n g c o m p o n e n t s of eggs also are presented.
EXPERIMENTAL PROCEDURE Experiment 1. Single C o m b White Leghorn hens of a commercial strain were used. Sixteen hens were selected for t h e e x p e r i m e n t o n t h e basis of a high r a t e of egg p r o d u c t i o n at 32 weeks of age. T h e birds were k e p t in individual laying h e n cages e q u i p p e d with individual feeders and waterers. Each bird represented an e x p e r i m e n t a l u n i t for statistical purposes. F o u r hens were assigned r a n d o m l y t o each of four ration t r e a t m e n t s . T h e t r e a t m e n t s consisted of
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ABSTRACT Female chickens and Japanese quail in active egg production were used to determine the influence of selenium (Se as sodium selenite) on the metabolism of mercury (Hg as methylmercuric chloride). Ration treatments consisted of 0 and 8 p.p.m. Se in a complete factorial arrangement with 0 and 20 p.p.m. Hg. Rations were fed ad libitum during the 21-day experiments, and tracer doses of methyl-203-mercuric chloride ( 2 ° 3 Hg) were given orally daily by oral capsule to all females for the first seven days of each experiment. Egg production by chickens and quail was reduced by feeding 20 p.p.m. Hg. This effect of Hg was alleviated in quail but not in chickens by 8 p.p.m. Se. Se did not affect 2 ° 3 Hg excretion by chickens or quail, but the proportion of total 2 3 ° Hg doses retained by quail was twice that of chickens. Quail also deposited twice as much 2 3 " Hg (relative to dose) in eggs as did chickens. When fed simulutaneously with Hg, Se increased the proportion of 2 ° 3 Hg deposited in egg white and in whole eggs of chickens and quail. Se decreased 2 ° 3 Hg in egg yolk. Similarly, the percent of total egg 2 ° 3 Hg in egg white was increased by Se while that in egg yolk was reduced. Dietary Se increased the retention of 2 ° 3 Hg in liver and brain tissue of chickens and quail. The effect of SE on 2 * 3 Hg in red blood cells varied with level of Hg and with species of bird. The total Hg content of eggs and tissues, as determined on selected samples, closely paralleled 2 ° 3 Hg levels found. In general, the data indicate that major differences between chickens and quail exist with respect to the influence of SE on Hg metabolism.
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The hens were acclimated to the cages and basal diet for ten days and then the experiment was started. Concurrent with the initiation of feeding the four experimental rations, each hen was given 2 /xci of methylmercuric-203 chloride, (CH 3 2 0 3 HgCl; specific activity, 740 mci./g. of Hg; radiopurity in excess of 99%) by oral capsule, daily for the first seven days. The feeding of the experimental rations continued for an additional 14 days after cessation of radioisotope administration. Eggs produced by individual hens were collected, appropriately labeled, weighed, and refrigerated until prepared for analysis. Total excreta from each hen was collected by 7 day intervals; days 1 through 7, days 8 through 14, and days 15 through 21. The excreta samples were air-dried prior to analysis. Daily feed consumption records for individual hens were maintained and body weights were measured at the start and end of the experiment.
At the end of 21 days, blood samples were obtained from each hen by heart probe, and the plasma was separated from the packed cell volume (PCV). The hens were killed, and the livers and brains were excised, weighed, and frozen until analyses were performed. The remainder of the carcass, including feathers, was frozen, cut into small pieces with a band saw and ground with a grinding attachment for a Hobart Model A100 mixer. The homogenate of each ground carcass was sampled for analysis. Prior to determination of radioactivity, each egg was hard-cooked in boiling water, and the yolk, white and egg shell plus membranes were separated quantitatively. Samples of each component were analyzed for radioactivity. In a limited number of cases, samples of the calcareous shell and the shell membranes were analyzed separately. Radioactivity of samples of eggs, tissues and excreta was determined with a Nuclear-Chicago Model 1085 deep-well gamma counter equipped with a Nal (thallium activated) crystal. All data on radioactivity were corrected for natural decay. Selected samples of eggs and liver also were analyzed for total Hg by the method of Deitz et al. (1973). This procedure involved wet oxidation with a nitric-sulfuric acid mixture followed by cold vapor atomic absorption spectrophotometry.
Experiment 2. Japanese quail (Coturnix coturnix japonica) were used in this experiment. Sixteen quail, selected on the basis of active egg production, were kept in individual quail laying cages. The cages were equipped with individual feeders and waterers. The quail were approximately 14 weeks of age at the start of the trial. The ration treatments were the same as those tested with the chickens (0 and 20 p.p.m. Hg, 0 and 8 p.p.m. Se in a complete 3 Vitamin premix contributed the following per kg. factorial arrangement) except that the basal of ration: vitamin A, 8000 I.U.; vitamin D 3 , 4400 I.U., vitamin E, 8 I.U.; vitamin B, 2 ,13 Mg; riboflavin, diet was specifically formulated for quail. The 6.6 mg.; pantothenic acid, 6.6 mg.; niacin, 3.3 mg., composition of the basal diet was, expressed as percent of the ration: ground corn, 48.1; menadione bisulfite, 2.2 ;ug.; and choline, 660 mg. 4 Mineral premix contributed the following per kg. soybean meal (44% protein), 29.5; menhaden of ration: manganese, 30 mg. and sodium chloride, fish meal, 5.0; dehydrated alfalfa meal (17% 2.91 g. protein), 4.0; meat and bone meal, 5.0; ground s Vitamin premix contributed the following per kg. 1.0; aniration: vitamin A, 8000 i.U.; vitamin D 3 , 3500 I.U.; limestone, 4.5; dicalcium phosphate, 5 vitamin E, 6 I.U.; vitamin B 1 2 , 20 Mg-; menadione mal tallow, 2.0; vitamin premix , 0.3 mineral bisulfite, 100 fig.-, riboflavin, 8 mg.; pantothenic acid, premix 6 , 0.5; and methionine, 0.1. The basal 22 mg.; niacin, 75 mg.; and choline, 1045 mg. ration was found, by analysis, to contain about 6 Mineral premix contributed the following per kg. ration: manganese 70 mg.; zinc, 40 mg.; iron, 20 mg.; 0.03 p.p.m. of Hg and 0.22 of Se, and was fed to all quail for six weeks prior to the start of copper, 6 mg. and sodium chloride, 2.61 g.
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two levels of mercury (Hg), as methylmercuric chloride (0 and 20 p.p.m.) and two levels of selenium (Se), as sodium selenite (0 and 8 p.p.m.) in a complete factorial arrangement. The use of 20 p.p.m. Hg and 8 p.p.m. Se yielded a molar ratio of 1:1 for these elements. The basal diet consisted of the following, expressed as percent of the ration: ground corn, 64.0; soybean meal (44% protein), 22.4; meat and bone meal (50% protein), 2.0; ground limestone, 6.0; dicalcium phosphate, 2.0; animal tallow, 3.0; vitamin premix 3 , 0.25; mineral premix , 0.3; and, methionine, 0.05. The basal ration contained approximately 0.03 p.p.m. of Hg and 0.25 p.p.m. Se, by analysis, and the hens had been fed rations very similar to the basal ration for 12 weeks prior to start of the experiment.
SELENIUM AND EGG MERCURY
The data were analyzed statistically by analysis of variance (Snedecor, 1956).
RESULTS AND DISCUSSION
The average number of eggs produced by chickens during the 21-day trial was not affected by dietary Se (Table 1). Twenty p.p.m. of dietary Hg decreased egg production by an average of four eggs per hen although no abnormally shaped eggs, as described by Scott et al. (1975), were observed. Dietary Hg also reduced average hen-day feed consumption, especially when fed in combination with Se. The hens averaged about 1 550 grams in body weight at the start, and ration treatment did not affect this parameter. Egg production by quail was reduced slightly by 20 p.p.m. Hg even though feed consumption was not changed notably (Table 1). The concurrent presence of 8 p.p.m. Se in the Hg-containing diet appeared to alleviate the adverse effect of Hg on egg production of quail. Neither Hg nor Se affected body weights of the quail during the 21-day experiment. These comparative data coincide, somewhat, with the results obtained by Sell and Horani (1976) with immature chicks and quail. They reported that Hg depressed feed consumption and growth by chicks, and Se failed to offset these effects of Hg. In contrast, Hg exerted virtually no effect on feed consumption by quail but caused high mortality. Dietary Se alleviated the lethal effects of Hg in quail. A marked difference was observed between
TABLE 1. — The influence of dietary metbylmercury and selenium on egg production and feed consumption by chickens and quail
Ration treatment, p.p.m. Hg — p.p.m. Se Chickens 0-0 0-8 20-0 20-8
Quail 0-0 0-8 20-0 20-8
Average body weight (g.)
Eggs/ bird'
Feed/ bird-day1
20 20 16 16 1.6
97 98 92 79 3.8
1434 1566 1609 1481
18 19 16 19 1.2
26.6 27.2 28.5 26.5 1.0
210 210 203 210
1
Data are averages of four birds per treatment for the 21-day experiment.
2
Standard error of the means.
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the experiment. The quail were adapted to the cages for 14 days, then four quail were fed each of the four ration treatments. During the first seven days of the experiment, each quail was given 0.2 /lei. of CH 3 2 0 3 HgCl daily by oral capsule. As with the chickens, the experiment confined for 14 days after the administration of the last dose of radioisotope. Individual records were maintained on feed consumption and egg production throughout the trial and body weights were measured at the start and the end of the experiment. The eggs were weighed, separated into yolk, white and shell plus membrane. Total excreta of each quail was collected by seven day intervals during the 21-day experiment. The eggs and excreta of quail were handled for analysis as described for chickens in Experiment 1. At the end of 21 days, blood samples were taken, the quail were killed, and liver and brains excised. The carcasses, with feathers, were ground and homogenized prior t o sampling for analysis. All egg, tissue and excreta samples were analyzed for radioactivity as described in Experiment 1. A limited number of samples also were analyzed for total Hg by the method of Deitz et al. (1973).
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p p m H g . . o 0 20 20 ppm Se - 0 0 0 8 l I Chickens FIG. 1. Proportions of total excreta of chickens and quail.
0 0 2020 0 8 0 8 I I Quail 203
H g dose found in
203
H g in the bodies of the chickens and quail. It has been observed in mice that Hg from methylmercury was excreted at a rate directly proportional to the body burden (Ostlund, 1969; and Clarkson, 1972). Dietary Hg and/or Se did not affect the average weight of eggs or the relative weights of. whites, yolks and shells of eggs produced by chickens or quail. Scott et al. (1975) reported that feeding 20 p.p.m. Hg as CH 3 HgCl to chickens decreased egg weight markedly and reduced the amount of egg white slightly, but these effects were not observed in the current study. Ration treatment did change the proportion of the 2 0 Hg dose deposited in the major components of chicken eggs (Table 2). Dietary Se increased the percentage of total 2 0 Hg dose found in the egg white produced by chickens fed Hg. However, Se did not change the proportion of 2 0 Hg dose in egg white of chickens not fed Hg along with Se. The inconsistency of these effects of Se was reflected by a significant Hg X Se interaction. Selenium's effect on deposition of 2 0 3 H g in yolk of chicken eggs was more consistent. The proportion of Hg in yolk was significantly reduced by Se, irrespective of dietary Hg level. No significant effects of ration treatments were observed with respect to 2 0 3 H g in the shell plus membrane. The enhancing effect of Se on deposition of 2 3 ° Hg in egg white of chickens fed 20 p.p.m. Hg was significant (P
Hgdose'
White
Yolk
Shell
Total
White
Yolk
Shell2
50.53 50.96 47.27 53.84 3.12
5.12 4.20 5.93 5.09 0.32
0.73 0.50 0.88 1.05 0.60
56.38 56.00 54.08 59.98 3.07
89.61 91.00 87.41 89.76 0.90
9.09 7.50 10.97 8.50 0.84
1.30 1.50 1.62 1.74 0.11
N.S. 4 N.S. N.S.
N.S. 0.01 N.S.
N.S. N.S. N.S.
N.S. N.S. N.S.
N.S. 0.10 N.S.
N.S. 0.05 N.S.
0.02 N.S. N.S.
All data are averages of four quail per treatment and include all eggs produced for 21 days.
2
Composed of approximately 92% calcareous portion and 8% shell membranes, by weight, and these components contained an average of 38 and 62% of the radioactivity of the shell plus membranes, respectively. 3
Standard error of the means.
4
See footnote 4, Table 2.
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closely with those observed by Sell et al. (1974) after methylmercury had been given to laying hens. The percentage of 2 0 3 H g in eggs of hens fed 8 p.p.m. Se were changed to about 90% and 9% for whites and yolks, respectively. Sell et al. (1974) found that the majority of Hg in egg white was bound to ovalbumin. It would be of interest to determine in future research whether or not Se influences Hg-binding to the various protein fractions of egg white. The effects of Se on Hg deposition and distribution in eggs of quail were not as marked as with chickens (Table 3). There were no significant treatment effects on the proportion of total 2 0 3 H g dose deposited in whites, shell plus membranes, or total eggs of quail. Se significantly decreased the proportion of 2 ° 3 Hg dose found in the yolks of eggs, irrespective of dietary Hg level. However, the magnitude of this decrease was relatively small. Stoewsand et al. (1974) presented data which indicated that dietary Se decreased the methylmercury content of quail eggs. However, there was no indication of this effect of Se in the current study, except for the slight reduction in 220033H g
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SELENIUM AND EGG MERCURY TABLE 4. — Influence of dietary methylmercury and selenium on the percent of0 found in the average eggs of chickens and quail
3
Hg dose
Average % of 2 ° 3 Hg dose' per White
per Yolk
per Egg
Chickens 0-0 0-8 20-0 20-8
1.087 1.076 1.092 1.418
0.230 0.116 0.253 0.138
0.160
0.029
1.330 1.202 1.368 1.570 0.153
Quail 0-0 0-8 20-0 20-8
2.807 2.682 2.954 2.834
0.284 0.221 0.371 0.268
3.132 2.947 3.380 3.157
Sv
0.287
0.036
0.283
1
Averages of four birds per treatment.
2
Standard error of the means.
cantly higher proportion of the 2 Hg dose in livers and total carcass than did those fed no Hg (Table 5). Mercury level had no significant effect on 2 ° 3 Hg in the brain. Dietary Se exerted a significant main effect whereby 2 Hg in brain was increased, irrespective of dietary Hg level. Se also increased radioactivity in liver, but only when fed together with 20 p.p.m. Hg. The carcass data indicate a similar
effect of Se on 2 0 3 H g retention but the Hg X Se interaction was not significant. Significantly more radioactivity was found in the cell pack of blood from chickens fed 20 p.p.m. Hg than in that of chickens not fed added Hg. As was observed with liver 2 0 3 H g , there was a significant Hg X Se interaction effect whereby Se decreased 2 0 3 H g in the cell pack of hens not fed Hg while it increased 2 ° 3 Hg in the cell pack
TABLE 5. • Influence of dietary methylmercury and selenium on the in certain tissues and blood of chickens
203
Hg found
D.P.M. pei •ml. blood 1 ment, p.p.m. Hg - p.p.m. Se 0-0 0-8 20-0 20-8
s-2 3 x Components of variance Hg Se HgX Se
Liver
Brain
Carcass
Plasma
Packed cell volume
0.865 0.308 0.985 1.440 0.148
0.020 0.042 0.020 0.032 0.0027
8.00 8.82 11.30 15.68 1.38
105 115 123 136 6
2170 755 2875 3964 121
0.01 3 N.S. 0.01
N.S. 0.01 N.S.
0.01 N.S. N.S.
N.S. N.S. N.S.
0.01 N.S. 0.05
% of total 2 ° 3 Hg dose1
1
Averages for four chickens per treatment.
2
Standard error of the means.
3
See footnote 3, Table 2.
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Ration treatment, p.p.m. Hg — p.p.m. Se
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significant reduction in the proportion of 203 H g dose found in brain while Se increased 203 H g in the same tissues, especially when no Hg was fed. There was also a significant Hg x Se interaction effect on 2 0 3 H g retained in the carcass. Se increased carcass Hg when no Hg was fed but decreased 2 0 Hg slightly when 20 p.p.m. Hg was in the diet. The amount of 203 H g in the cell pack from blood was increased by Se, particularly when no Hg was fed. Slight interaction effects also were observed with blood plasma—Se decreased plasma Hg when no Hg was fed but increased plasma 203 H g when fed with 20 p.p.m. Hg. Previously, Sell and Horani (1976) reported that Se markedly increased liver Hg of young quail fed 20 p.p.m. Hg but no significant effect of Se on brain Hg was noted. Similar observations were made in the current study with mature quail. However, Stoewsand et al. (1974) also using mature quail, found that Se decreased Hg levels in liver and brain. Thus, the effect of dietary Se on Hg retention in certain tissues of quail, as well as chickens, is subject to considerable variation due to factors not understood as yet. The influence of dietary Hg level on tissue 203 H g deserves comment, too. In both chickens and quail, dietary Hg exerted a significant effect on the proportions of 2 0 3 H g dose found in certain tissues. Also, significant interactions between Hg and Se occurred in some instances.
TABLE 6. — Influence of dietary methylmercury and selenium on the 2 ° 3 Hg found in certain tissues and blood of quail D.P.M. pei • ml. blood1 Ration treat]ment, p.p.m. Hg - p.p.m. Se 0-0 0-8
20-0 20 8
s-2 Components of variance Hg Se
HgX Se 1 2
Liver
Brain
Carcass
Plasma
Packed cell volume
0.220 0.480 0.300 0.670 0.056
0.050 0.150 0.033 0.035 0.0172
7.40 12.00 11.90 10.15 1.46
136 209 182 118
1164 2409 2109 2173
6
68
0.05 s 0.01 N.S.
0.01 0.01 0.01
N.S. N.S. 0.05
N.S. N.S. 0.05
% of total
Averages for four quail per treatment.
Standard error of the means. 3 See footnote 4, Table 3.
203
H g dose1
N.S. 0.05 0.10
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of hens fed 20 p.p.m. Hg. The influence of Se on 2 0 3 H g in livers of chickens fed 20 p.p.m. Hg corresponds closely with observations of Emerick et al. (1976) on laying hens but disagrees with data presented by Welsh and Soares (1974), Ansari and Britton (1974) and Sell and Horani (1976). Nevertheless, an effect of Se on Hg metabolism, as reflected by changes in liver Hg was apparent even though undefined variables seem to influence this effect. Emerick et al. (1976) found that brain Hg was increased by feeding 4 or 8 p.p.m. Se to hens fed 5 p.p.m. Hg as methylmercury. Sell and Horani (1976) were unable to detect any effect of dietary Se on Hg accumulation in brains of young chicks. The results of the current study with mature chickens agree with those of Emerick et al. (1976) but are at variance with those of Sell and Horani (1976). Perhaps, age and physiological development of chickens affected the modifying influence of dietary Se on brain Hg. Regardless, the increased brain Hg levels due to Se observed by Emerick et al. (1976) and in the current study were not associated with any overt signs of Hg toxicity. Dietary Se increased the percent of 2 °3 Hg dose found in quail livers, irrespective of dietary Hg level (Table 6). Dietary Hg, per se, also increased liver 2 ° 3 Hg, and there was no interaction effect on the parameter. Dietary Hg caused a
SELENIUM AND EGG MERCURY
T h e p r o p o r t i o n s of t h e t o t a l 2 0 3 H g doses t h a t were recovered in eggs, excreta and b o d y tissues were relatively high, ranging from 9 8 . 8 t o 1 0 2 . 5 % for chickens and from 9 3 . 0 t o 9 7 . 1 % for quail. These data indicate t h a t t h e vast majority of radioactivity was a c c o u n t e d for in t h e various fractions, and lends additional credence t o t h e information a b o u t 2 Hg metabolism which was o b t a i n e d . T h e total Hg c o n t e n t of eggs and tissues, as d e t e r m i n e d on selected samples, closely paralleled 2 0 3 H g c o n t e n t . F o r e x a m p l e , t h e whites of eggs p r o d u c e d on day 2 0 b y hens fed 20 p.p.m. Hg and n o Se contained 33.3 p . p . m . Hg while egg whites of hens fed 20 p . p . m . Hg plus 8 p.p.m. Se had 5 1 . 8 p . p . m . Hg. T h e ratio of 3 3 . 3 / 5 1 . 8 was 0.64 which c o r r e s p o n d e d well with a 2 0 3 H g activity ratio of 0.62 for t h e same t w o t r e a t m e n t groups. T r e a t m e n t effects on total Hg and Hg in livers were also similar. In quail liver, t h e ratio of total Hg for t h e 20 p.p.m. Hg—no Se g r o u p t o t h e 2 0 p . p . m . Hg—8 p.p.m. Se g r o u p was 0.42 while t h a t of 2 0 3 H g was 0 . 4 5 . Total Hg data also c o r r o b o r a t e d observations m a d e o n t h e basis of t h e 2 0 3 H g c o n t e n t of chicken livers and egg y o l k s with respect t o t r e a t m e n t effects. In general, t h e data o b t a i n e d from this research illustrate some i m p o r t a n t points. Unquestionably, dietary Se modifies t h e m a n n e r with which Hg, in t h e f o r m of m e t h y l m e r c u r y , is metabolized. C o n s e q u e n t l y , Hg deposition in eggs and Hg r e t e n t i o n in certain b o d y tissues of chickens and quail are changed. However, t h e magnitude and n a t u r e of these changes appear t o be subject t o species variation as well as s o m e , as y e t , poorly defined factors. Species variation is particularly intriguing since this makes generalizations a b o u t t h e i m p o r t a n t Se X Hg interrelationship t e n u o u s at this t i m e . Considerable m o r e research is n e e d e d o n species variation p h e n o m e n o n for t h e eventual elucida-
tion of m e c h a n i s m s whereby Se modifies Hg metabolism. ACKNOWLEDGEMENT T h e assistance of F a r o u k G. Horani, Sandra Matz and R o b e r t L. J o h n s o n in c o n d u c t i n g this research is gratefully acknowledged.
REFERENCES Ansari, M. S., and W. M. Britton, 1974. Effects of dietary selenium and mercury on 2 ° 3 Hg metabolism in chicks. Poultry Sci. 53:1134-1137. Campbell, L. D., G. C. Hodgson, A. Lutz and F. A. J. Armstrong, 1971. Dietary levels and mercury accumulation in chickens. Proc. Western Section, Amer. Soc. Anim. Sci. 23:152-155. Clarkson, T. W., 1971. The absorption of different chemical compounds of mercury from the gastrointestinal tract. Toxicol. Appl. Pharmacol. 19:409. Clarkson, T. W., 1972. The pharmacology of mercury compounds. Annual Rev. Pharmacol. 12:375—406. Deitz, F. D., J. S. Sell and D. W. Bristol, 1973. Rapid, sensitive method for determination of mercury in a variety of biological samples. J. Assoc. Offic. Anal. Chem. 52:378-382. El-Begearmi, M. M., C. Goudie, H. E. Ganther and M. L. Sunde, 1973. Attempts to quantitate the protective effect of selenium against mercury toxicity using Japanese quail. Federation Proc. 32:887. Emerick, R. J., I. S. Palmer, C. W. Carlson and R. A. Nelson, 1976. Mercury-selenium interrelationships in laying hens. Fed. Proc. 35:577. Friberg, L., and J. Vostal, 1972. Mercury in the Environment. Chemical Rubber Publishing Co., Cleveland, Ohio. Ganther, H. E., C. Goudie, M. L. Sunde, M. J. Kopecky, P. Wagner, Sand-Hwan Oh and W. G. Hoekstra, 1972. Selenium: Relation to decreased toxicity of methylmercury added to diets containing tuna. Science, 175:1122-1124. Johnson, S., and W. G. Pond, 1974. Inorganic vs.. organic Hg toxicity in growing rats: Protection by dietary Se but not Zn. Nutr. Rep. Int. 9:135-147. Kiwimae, A., A. Swensson, U. Ulfvarson and G. Westoo, 1969. Methylmercury compounds in eggs from hens after oral administration of mercury compounds. J. Agr. Food Chem. 17:1014—1016. Neathery, M. W., W. J. Miller, R. P. Gentry, P. E. Stake and D. M. Blackmon, 1974. Cadmium-109 and methylmercury-203 metabolism, tissue distribution and secretion in milk of cows. J. Dairy Sci. 57:1177-1183. Ostlund, K., 1969. Studies on the metabolism of methylmercury and dimethylmercury in mice. Acta Pharmacol, et Toxicol. 27, Supplement 1. Potter, S., and G. Matrone, 1974. Effect of selenite on the toxicity of dietary methylmercury and mercuric chloride in the rat. J. Nutr. 104:638—647. Scott, M. L., J. R. Zimmerman, S. Marinsky, P. A. Mullenhoff, G. L. Rumsey and R. W. Rice, 1975. Effects of PCB's, DDT and mercury compounds upon egg production, hatchability and shell quality in chickens and Japanese quail. Poultry Sci. 54:350-368.
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These observations serve t o emphasize t h e need for careful planning prior t o conducting research on t h e Hg X Se interrelationship. T h e use of trace doses of Hg only may yield information which would differ from t h a t obtained when substantial levels of " c o l d " Hg are given simultaneously. T h e data o n Hg found in liver and P.C.V. of t h e blood of chickens illustrate this p o i n t . Dietary Se decreased Hg in liver a n d P.C.V. w h e n n o Hg was added t o t h e feed. In contrast, Se increased 203 H g in b o t h t h e liver and P.C.V. when 20 p . p . m . Hg was fed.
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Sell, J. L., and K. L. Davison, 1975. Metabolism of mercury, administered as methylmercuric chloride of mercuric chloride by lactating ruminants. J. Agric. Food Chem. 23:803-808. Sell, J. L., W. Guenter and M. Sifri, 1974. Distribution of mercury among components of eggs following the administration of methylmercuric chloride to chickens. J. Agric. Food Chem. 22:248-251. Sell, J. L., and F. G. Horani, 1976. Influence of selenium on toxicity and metabolism of mercury in chicks and quail. Nutr. Rep. Int. 14:439-447. Smart, N. A., and M. K. Lloyd, 1963. Mercury residues in eggs, flesh and livers of hens fed wheat
treated with methylmercury dicyandiamide. J. Sci. Food Agr. 14:734-740. Snedecor, G. W., 1956. Statistical Methods, Iowa State University Press, Ames, Iowa. Stoewsand, G. S., C. A. Bache and D. J. Lisk, 1974. Dietary selenium protection of methylmercury intoxication of Japanese quail. Bull. Environ. Contam. Toxicol. 11:152-156. Tejning, S., and R. Vesterberg, 1964. Aklyl mercurytreated seed in food grain. Poultry Sci. 43:6—11. Welsh, S. O., and J. H. Soares, 1974. The bioavailability of mercury in the tissue of hens fed methylmercuric chloride. Fed. Proc. 33:660.
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Poultry Science 56:939-948, 1977
It is well k n o w n t h a t Hg derived from alkyl c o m p o u n d s (i.e. m e t h y l m e r c u r y ) is deposited readily in eggs (Tejning and Vesterberg, 1 9 6 4 ; Kiwimae et al, 1 9 6 9 ; Campbell et al, 1 9 7 1 ; and Sell et al, 1 9 7 4 ) . T h e majority of this t y p e of Hg is f o u n d in egg w h i t e ( S m a r t and L l o y d , 1 9 6 3 ; Kiwimae et al, 1 9 6 9 ; and Campbell et al, 1 9 7 1 ) , associated p r e d o m i n a t e l y with t h e protein, ovalbumin (Sell et al, 1 9 7 4 ) . Recently, m u c h a t t e n t i o n has been focused o n t h e interaction b e t w e e n Se a n d Hg w h e r e b y Hg metabolism a p p a r e n t l y is modified ( G a n t h e r et al, 1 9 7 2 ; El-Bergearmi et al, 1 9 7 3 ; P o t t e r and M a t r o n e , 1 9 7 4 ; J o h n s o n and Pond, 1 9 7 4 ) . However, little information has been published concerning t h e effect of Se o n t h e transfer o f dietary Hg i n t o eggs. Stoewsand et al. ( 1 9 7 4 )
1
Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No. 713. This research was supported in part by Research Grant 1R01 FD 00752, United States Public Health Services, National Institutes of Health. 2 Present address: Department of Animal Science, Iowa State University, Ames, Iowa 50010.
presented d a t a which indicated t h a t Se reduced t h e m e t h y l m e r c u r y c o n c e n t r a t i o n of eggs produced b y Japanese quail fed this form of m e r c u r y . Emerick et al. ( 1 9 7 6 ) r e p o r t e d t h a t t h e eggs of chickens fed m e t h y l m e r c u r y and Se contained m o r e Hg t h a n t h o s e of h e n s fed m e t h y l m e r c u r y alone. T h e research r e p o r t e d here describes t h e influence of dietary Se on t h e transfer of dietary Hg i n t o eggs of chickens and Japanese quail. D a t a illustrating t h e modifying effects of Se on t h e distribution of Hg a m o n g tissues and a m o n g c o m p o n e n t s of eggs also are presented.
EXPERIMENTAL PROCEDURE Experiment 1. Single C o m b White Leghorn hens of a commercial strain were used. Sixteen hens were selected for t h e e x p e r i m e n t o n t h e basis of a high r a t e of egg p r o d u c t i o n at 32 weeks of age. T h e birds were k e p t in individual laying h e n cages e q u i p p e d with individual feeders and waterers. Each bird represented an e x p e r i m e n t a l u n i t for statistical purposes. F o u r hens were assigned r a n d o m l y t o each of four ration t r e a t m e n t s . T h e t r e a t m e n t s consisted of
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ABSTRACT Female chickens and Japanese quail in active egg production were used to determine the influence of selenium (Se as sodium selenite) on the metabolism of mercury (Hg as methylmercuric chloride). Ration treatments consisted of 0 and 8 p.p.m. Se in a complete factorial arrangement with 0 and 20 p.p.m. Hg. Rations were fed ad libitum during the 21-day experiments, and tracer doses of methyl-203-mercuric chloride ( 2 ° 3 Hg) were given orally daily by oral capsule to all females for the first seven days of each experiment. Egg production by chickens and quail was reduced by feeding 20 p.p.m. Hg. This effect of Hg was alleviated in quail but not in chickens by 8 p.p.m. Se. Se did not affect 2 ° 3 Hg excretion by chickens or quail, but the proportion of total 2 3 ° Hg doses retained by quail was twice that of chickens. Quail also deposited twice as much 2 3 " Hg (relative to dose) in eggs as did chickens. When fed simulutaneously with Hg, Se increased the proportion of 2 ° 3 Hg deposited in egg white and in whole eggs of chickens and quail. Se decreased 2 ° 3 Hg in egg yolk. Similarly, the percent of total egg 2 ° 3 Hg in egg white was increased by Se while that in egg yolk was reduced. Dietary Se increased the retention of 2 ° 3 Hg in liver and brain tissue of chickens and quail. The effect of SE on 2 * 3 Hg in red blood cells varied with level of Hg and with species of bird. The total Hg content of eggs and tissues, as determined on selected samples, closely paralleled 2 ° 3 Hg levels found. In general, the data indicate that major differences between chickens and quail exist with respect to the influence of SE on Hg metabolism.
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The hens were acclimated to the cages and basal diet for ten days and then the experiment was started. Concurrent with the initiation of feeding the four experimental rations, each hen was given 2 /xci of methylmercuric-203 chloride, (CH 3 2 0 3 HgCl; specific activity, 740 mci./g. of Hg; radiopurity in excess of 99%) by oral capsule, daily for the first seven days. The feeding of the experimental rations continued for an additional 14 days after cessation of radioisotope administration. Eggs produced by individual hens were collected, appropriately labeled, weighed, and refrigerated until prepared for analysis. Total excreta from each hen was collected by 7 day intervals; days 1 through 7, days 8 through 14, and days 15 through 21. The excreta samples were air-dried prior to analysis. Daily feed consumption records for individual hens were maintained and body weights were measured at the start and end of the experiment.
At the end of 21 days, blood samples were obtained from each hen by heart probe, and the plasma was separated from the packed cell volume (PCV). The hens were killed, and the livers and brains were excised, weighed, and frozen until analyses were performed. The remainder of the carcass, including feathers, was frozen, cut into small pieces with a band saw and ground with a grinding attachment for a Hobart Model A100 mixer. The homogenate of each ground carcass was sampled for analysis. Prior to determination of radioactivity, each egg was hard-cooked in boiling water, and the yolk, white and egg shell plus membranes were separated quantitatively. Samples of each component were analyzed for radioactivity. In a limited number of cases, samples of the calcareous shell and the shell membranes were analyzed separately. Radioactivity of samples of eggs, tissues and excreta was determined with a Nuclear-Chicago Model 1085 deep-well gamma counter equipped with a Nal (thallium activated) crystal. All data on radioactivity were corrected for natural decay. Selected samples of eggs and liver also were analyzed for total Hg by the method of Deitz et al. (1973). This procedure involved wet oxidation with a nitric-sulfuric acid mixture followed by cold vapor atomic absorption spectrophotometry.
Experiment 2. Japanese quail (Coturnix coturnix japonica) were used in this experiment. Sixteen quail, selected on the basis of active egg production, were kept in individual quail laying cages. The cages were equipped with individual feeders and waterers. The quail were approximately 14 weeks of age at the start of the trial. The ration treatments were the same as those tested with the chickens (0 and 20 p.p.m. Hg, 0 and 8 p.p.m. Se in a complete 3 Vitamin premix contributed the following per kg. factorial arrangement) except that the basal of ration: vitamin A, 8000 I.U.; vitamin D 3 , 4400 I.U., vitamin E, 8 I.U.; vitamin B, 2 ,13 Mg; riboflavin, diet was specifically formulated for quail. The 6.6 mg.; pantothenic acid, 6.6 mg.; niacin, 3.3 mg., composition of the basal diet was, expressed as percent of the ration: ground corn, 48.1; menadione bisulfite, 2.2 ;ug.; and choline, 660 mg. 4 Mineral premix contributed the following per kg. soybean meal (44% protein), 29.5; menhaden of ration: manganese, 30 mg. and sodium chloride, fish meal, 5.0; dehydrated alfalfa meal (17% 2.91 g. protein), 4.0; meat and bone meal, 5.0; ground s Vitamin premix contributed the following per kg. 1.0; aniration: vitamin A, 8000 i.U.; vitamin D 3 , 3500 I.U.; limestone, 4.5; dicalcium phosphate, 5 vitamin E, 6 I.U.; vitamin B 1 2 , 20 Mg-; menadione mal tallow, 2.0; vitamin premix , 0.3 mineral bisulfite, 100 fig.-, riboflavin, 8 mg.; pantothenic acid, premix 6 , 0.5; and methionine, 0.1. The basal 22 mg.; niacin, 75 mg.; and choline, 1045 mg. ration was found, by analysis, to contain about 6 Mineral premix contributed the following per kg. ration: manganese 70 mg.; zinc, 40 mg.; iron, 20 mg.; 0.03 p.p.m. of Hg and 0.22 of Se, and was fed to all quail for six weeks prior to the start of copper, 6 mg. and sodium chloride, 2.61 g.
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two levels of mercury (Hg), as methylmercuric chloride (0 and 20 p.p.m.) and two levels of selenium (Se), as sodium selenite (0 and 8 p.p.m.) in a complete factorial arrangement. The use of 20 p.p.m. Hg and 8 p.p.m. Se yielded a molar ratio of 1:1 for these elements. The basal diet consisted of the following, expressed as percent of the ration: ground corn, 64.0; soybean meal (44% protein), 22.4; meat and bone meal (50% protein), 2.0; ground limestone, 6.0; dicalcium phosphate, 2.0; animal tallow, 3.0; vitamin premix 3 , 0.25; mineral premix , 0.3; and, methionine, 0.05. The basal ration contained approximately 0.03 p.p.m. of Hg and 0.25 p.p.m. Se, by analysis, and the hens had been fed rations very similar to the basal ration for 12 weeks prior to start of the experiment.
SELENIUM AND EGG MERCURY
The data were analyzed statistically by analysis of variance (Snedecor, 1956).
RESULTS AND DISCUSSION
The average number of eggs produced by chickens during the 21-day trial was not affected by dietary Se (Table 1). Twenty p.p.m. of dietary Hg decreased egg production by an average of four eggs per hen although no abnormally shaped eggs, as described by Scott et al. (1975), were observed. Dietary Hg also reduced average hen-day feed consumption, especially when fed in combination with Se. The hens averaged about 1 550 grams in body weight at the start, and ration treatment did not affect this parameter. Egg production by quail was reduced slightly by 20 p.p.m. Hg even though feed consumption was not changed notably (Table 1). The concurrent presence of 8 p.p.m. Se in the Hg-containing diet appeared to alleviate the adverse effect of Hg on egg production of quail. Neither Hg nor Se affected body weights of the quail during the 21-day experiment. These comparative data coincide, somewhat, with the results obtained by Sell and Horani (1976) with immature chicks and quail. They reported that Hg depressed feed consumption and growth by chicks, and Se failed to offset these effects of Hg. In contrast, Hg exerted virtually no effect on feed consumption by quail but caused high mortality. Dietary Se alleviated the lethal effects of Hg in quail. A marked difference was observed between
TABLE 1. — The influence of dietary metbylmercury and selenium on egg production and feed consumption by chickens and quail
Ration treatment, p.p.m. Hg — p.p.m. Se Chickens 0-0 0-8 20-0 20-8
Quail 0-0 0-8 20-0 20-8
Average body weight (g.)
Eggs/ bird'
Feed/ bird-day1
20 20 16 16 1.6
97 98 92 79 3.8
1434 1566 1609 1481
18 19 16 19 1.2
26.6 27.2 28.5 26.5 1.0
210 210 203 210
1
Data are averages of four birds per treatment for the 21-day experiment.
2
Standard error of the means.
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the experiment. The quail were adapted to the cages for 14 days, then four quail were fed each of the four ration treatments. During the first seven days of the experiment, each quail was given 0.2 /lei. of CH 3 2 0 3 HgCl daily by oral capsule. As with the chickens, the experiment confined for 14 days after the administration of the last dose of radioisotope. Individual records were maintained on feed consumption and egg production throughout the trial and body weights were measured at the start and the end of the experiment. The eggs were weighed, separated into yolk, white and shell plus membrane. Total excreta of each quail was collected by seven day intervals during the 21-day experiment. The eggs and excreta of quail were handled for analysis as described for chickens in Experiment 1. At the end of 21 days, blood samples were taken, the quail were killed, and liver and brains excised. The carcasses, with feathers, were ground and homogenized prior t o sampling for analysis. All egg, tissue and excreta samples were analyzed for radioactivity as described in Experiment 1. A limited number of samples also were analyzed for total Hg by the method of Deitz et al. (1973).
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70-, (0
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n 1 - 7 days M 8-14 days • 15-21 days
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O20-I 10-
p p m H g . . o 0 20 20 ppm Se - 0 0 0 8 l I Chickens FIG. 1. Proportions of total excreta of chickens and quail.
0 0 2020 0 8 0 8 I I Quail 203
H g dose found in
203
H g in the bodies of the chickens and quail. It has been observed in mice that Hg from methylmercury was excreted at a rate directly proportional to the body burden (Ostlund, 1969; and Clarkson, 1972). Dietary Hg and/or Se did not affect the average weight of eggs or the relative weights of. whites, yolks and shells of eggs produced by chickens or quail. Scott et al. (1975) reported that feeding 20 p.p.m. Hg as CH 3 HgCl to chickens decreased egg weight markedly and reduced the amount of egg white slightly, but these effects were not observed in the current study. Ration treatment did change the proportion of the 2 0 Hg dose deposited in the major components of chicken eggs (Table 2). Dietary Se increased the percentage of total 2 0 Hg dose found in the egg white produced by chickens fed Hg. However, Se did not change the proportion of 2 0 Hg dose in egg white of chickens not fed Hg along with Se. The inconsistency of these effects of Se was reflected by a significant Hg X Se interaction. Selenium's effect on deposition of 2 0 3 H g in yolk of chicken eggs was more consistent. The proportion of Hg in yolk was significantly reduced by Se, irrespective of dietary Hg level. No significant effects of ration treatments were observed with respect to 2 0 3 H g in the shell plus membrane. The enhancing effect of Se on deposition of 2 3 ° Hg in egg white of chickens fed 20 p.p.m. Hg was significant (P
Hgdose'
White
Yolk
Shell
Total
White
Yolk
Shell2
50.53 50.96 47.27 53.84 3.12
5.12 4.20 5.93 5.09 0.32
0.73 0.50 0.88 1.05 0.60
56.38 56.00 54.08 59.98 3.07
89.61 91.00 87.41 89.76 0.90
9.09 7.50 10.97 8.50 0.84
1.30 1.50 1.62 1.74 0.11
N.S. 4 N.S. N.S.
N.S. 0.01 N.S.
N.S. N.S. N.S.
N.S. N.S. N.S.
N.S. 0.10 N.S.
N.S. 0.05 N.S.
0.02 N.S. N.S.
All data are averages of four quail per treatment and include all eggs produced for 21 days.
2
Composed of approximately 92% calcareous portion and 8% shell membranes, by weight, and these components contained an average of 38 and 62% of the radioactivity of the shell plus membranes, respectively. 3
Standard error of the means.
4
See footnote 4, Table 2.
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closely with those observed by Sell et al. (1974) after methylmercury had been given to laying hens. The percentage of 2 0 3 H g in eggs of hens fed 8 p.p.m. Se were changed to about 90% and 9% for whites and yolks, respectively. Sell et al. (1974) found that the majority of Hg in egg white was bound to ovalbumin. It would be of interest to determine in future research whether or not Se influences Hg-binding to the various protein fractions of egg white. The effects of Se on Hg deposition and distribution in eggs of quail were not as marked as with chickens (Table 3). There were no significant treatment effects on the proportion of total 2 0 3 H g dose deposited in whites, shell plus membranes, or total eggs of quail. Se significantly decreased the proportion of 2 ° 3 Hg dose found in the yolks of eggs, irrespective of dietary Hg level. However, the magnitude of this decrease was relatively small. Stoewsand et al. (1974) presented data which indicated that dietary Se decreased the methylmercury content of quail eggs. However, there was no indication of this effect of Se in the current study, except for the slight reduction in 220033H g
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SELENIUM AND EGG MERCURY TABLE 4. — Influence of dietary methylmercury and selenium on the percent of0 found in the average eggs of chickens and quail
3
Hg dose
Average % of 2 ° 3 Hg dose' per White
per Yolk
per Egg
Chickens 0-0 0-8 20-0 20-8
1.087 1.076 1.092 1.418
0.230 0.116 0.253 0.138
0.160
0.029
1.330 1.202 1.368 1.570 0.153
Quail 0-0 0-8 20-0 20-8
2.807 2.682 2.954 2.834
0.284 0.221 0.371 0.268
3.132 2.947 3.380 3.157
Sv
0.287
0.036
0.283
1
Averages of four birds per treatment.
2
Standard error of the means.
cantly higher proportion of the 2 Hg dose in livers and total carcass than did those fed no Hg (Table 5). Mercury level had no significant effect on 2 ° 3 Hg in the brain. Dietary Se exerted a significant main effect whereby 2 Hg in brain was increased, irrespective of dietary Hg level. Se also increased radioactivity in liver, but only when fed together with 20 p.p.m. Hg. The carcass data indicate a similar
effect of Se on 2 0 3 H g retention but the Hg X Se interaction was not significant. Significantly more radioactivity was found in the cell pack of blood from chickens fed 20 p.p.m. Hg than in that of chickens not fed added Hg. As was observed with liver 2 0 3 H g , there was a significant Hg X Se interaction effect whereby Se decreased 2 0 3 H g in the cell pack of hens not fed Hg while it increased 2 ° 3 Hg in the cell pack
TABLE 5. • Influence of dietary methylmercury and selenium on the in certain tissues and blood of chickens
203
Hg found
D.P.M. pei •ml. blood 1 ment, p.p.m. Hg - p.p.m. Se 0-0 0-8 20-0 20-8
s-2 3 x Components of variance Hg Se HgX Se
Liver
Brain
Carcass
Plasma
Packed cell volume
0.865 0.308 0.985 1.440 0.148
0.020 0.042 0.020 0.032 0.0027
8.00 8.82 11.30 15.68 1.38
105 115 123 136 6
2170 755 2875 3964 121
0.01 3 N.S. 0.01
N.S. 0.01 N.S.
0.01 N.S. N.S.
N.S. N.S. N.S.
0.01 N.S. 0.05
% of total 2 ° 3 Hg dose1
1
Averages for four chickens per treatment.
2
Standard error of the means.
3
See footnote 3, Table 2.
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Ration treatment, p.p.m. Hg — p.p.m. Se
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significant reduction in the proportion of 203 H g dose found in brain while Se increased 203 H g in the same tissues, especially when no Hg was fed. There was also a significant Hg x Se interaction effect on 2 0 3 H g retained in the carcass. Se increased carcass Hg when no Hg was fed but decreased 2 0 Hg slightly when 20 p.p.m. Hg was in the diet. The amount of 203 H g in the cell pack from blood was increased by Se, particularly when no Hg was fed. Slight interaction effects also were observed with blood plasma—Se decreased plasma Hg when no Hg was fed but increased plasma 203 H g when fed with 20 p.p.m. Hg. Previously, Sell and Horani (1976) reported that Se markedly increased liver Hg of young quail fed 20 p.p.m. Hg but no significant effect of Se on brain Hg was noted. Similar observations were made in the current study with mature quail. However, Stoewsand et al. (1974) also using mature quail, found that Se decreased Hg levels in liver and brain. Thus, the effect of dietary Se on Hg retention in certain tissues of quail, as well as chickens, is subject to considerable variation due to factors not understood as yet. The influence of dietary Hg level on tissue 203 H g deserves comment, too. In both chickens and quail, dietary Hg exerted a significant effect on the proportions of 2 0 3 H g dose found in certain tissues. Also, significant interactions between Hg and Se occurred in some instances.
TABLE 6. — Influence of dietary methylmercury and selenium on the 2 ° 3 Hg found in certain tissues and blood of quail D.P.M. pei • ml. blood1 Ration treat]ment, p.p.m. Hg - p.p.m. Se 0-0 0-8
20-0 20 8
s-2 Components of variance Hg Se
HgX Se 1 2
Liver
Brain
Carcass
Plasma
Packed cell volume
0.220 0.480 0.300 0.670 0.056
0.050 0.150 0.033 0.035 0.0172
7.40 12.00 11.90 10.15 1.46
136 209 182 118
1164 2409 2109 2173
6
68
0.05 s 0.01 N.S.
0.01 0.01 0.01
N.S. N.S. 0.05
N.S. N.S. 0.05
% of total
Averages for four quail per treatment.
Standard error of the means. 3 See footnote 4, Table 3.
203
H g dose1
N.S. 0.05 0.10
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of hens fed 20 p.p.m. Hg. The influence of Se on 2 0 3 H g in livers of chickens fed 20 p.p.m. Hg corresponds closely with observations of Emerick et al. (1976) on laying hens but disagrees with data presented by Welsh and Soares (1974), Ansari and Britton (1974) and Sell and Horani (1976). Nevertheless, an effect of Se on Hg metabolism, as reflected by changes in liver Hg was apparent even though undefined variables seem to influence this effect. Emerick et al. (1976) found that brain Hg was increased by feeding 4 or 8 p.p.m. Se to hens fed 5 p.p.m. Hg as methylmercury. Sell and Horani (1976) were unable to detect any effect of dietary Se on Hg accumulation in brains of young chicks. The results of the current study with mature chickens agree with those of Emerick et al. (1976) but are at variance with those of Sell and Horani (1976). Perhaps, age and physiological development of chickens affected the modifying influence of dietary Se on brain Hg. Regardless, the increased brain Hg levels due to Se observed by Emerick et al. (1976) and in the current study were not associated with any overt signs of Hg toxicity. Dietary Se increased the percent of 2 °3 Hg dose found in quail livers, irrespective of dietary Hg level (Table 6). Dietary Hg, per se, also increased liver 2 ° 3 Hg, and there was no interaction effect on the parameter. Dietary Hg caused a
SELENIUM AND EGG MERCURY
T h e p r o p o r t i o n s of t h e t o t a l 2 0 3 H g doses t h a t were recovered in eggs, excreta and b o d y tissues were relatively high, ranging from 9 8 . 8 t o 1 0 2 . 5 % for chickens and from 9 3 . 0 t o 9 7 . 1 % for quail. These data indicate t h a t t h e vast majority of radioactivity was a c c o u n t e d for in t h e various fractions, and lends additional credence t o t h e information a b o u t 2 Hg metabolism which was o b t a i n e d . T h e total Hg c o n t e n t of eggs and tissues, as d e t e r m i n e d on selected samples, closely paralleled 2 0 3 H g c o n t e n t . F o r e x a m p l e , t h e whites of eggs p r o d u c e d on day 2 0 b y hens fed 20 p.p.m. Hg and n o Se contained 33.3 p . p . m . Hg while egg whites of hens fed 20 p . p . m . Hg plus 8 p.p.m. Se had 5 1 . 8 p . p . m . Hg. T h e ratio of 3 3 . 3 / 5 1 . 8 was 0.64 which c o r r e s p o n d e d well with a 2 0 3 H g activity ratio of 0.62 for t h e same t w o t r e a t m e n t groups. T r e a t m e n t effects on total Hg and Hg in livers were also similar. In quail liver, t h e ratio of total Hg for t h e 20 p.p.m. Hg—no Se g r o u p t o t h e 2 0 p . p . m . Hg—8 p.p.m. Se g r o u p was 0.42 while t h a t of 2 0 3 H g was 0 . 4 5 . Total Hg data also c o r r o b o r a t e d observations m a d e o n t h e basis of t h e 2 0 3 H g c o n t e n t of chicken livers and egg y o l k s with respect t o t r e a t m e n t effects. In general, t h e data o b t a i n e d from this research illustrate some i m p o r t a n t points. Unquestionably, dietary Se modifies t h e m a n n e r with which Hg, in t h e f o r m of m e t h y l m e r c u r y , is metabolized. C o n s e q u e n t l y , Hg deposition in eggs and Hg r e t e n t i o n in certain b o d y tissues of chickens and quail are changed. However, t h e magnitude and n a t u r e of these changes appear t o be subject t o species variation as well as s o m e , as y e t , poorly defined factors. Species variation is particularly intriguing since this makes generalizations a b o u t t h e i m p o r t a n t Se X Hg interrelationship t e n u o u s at this t i m e . Considerable m o r e research is n e e d e d o n species variation p h e n o m e n o n for t h e eventual elucida-
tion of m e c h a n i s m s whereby Se modifies Hg metabolism. ACKNOWLEDGEMENT T h e assistance of F a r o u k G. Horani, Sandra Matz and R o b e r t L. J o h n s o n in c o n d u c t i n g this research is gratefully acknowledged.
REFERENCES Ansari, M. S., and W. M. Britton, 1974. Effects of dietary selenium and mercury on 2 ° 3 Hg metabolism in chicks. Poultry Sci. 53:1134-1137. Campbell, L. D., G. C. Hodgson, A. Lutz and F. A. J. Armstrong, 1971. Dietary levels and mercury accumulation in chickens. Proc. Western Section, Amer. Soc. Anim. Sci. 23:152-155. Clarkson, T. W., 1971. The absorption of different chemical compounds of mercury from the gastrointestinal tract. Toxicol. Appl. Pharmacol. 19:409. Clarkson, T. W., 1972. The pharmacology of mercury compounds. Annual Rev. Pharmacol. 12:375—406. Deitz, F. D., J. S. Sell and D. W. Bristol, 1973. Rapid, sensitive method for determination of mercury in a variety of biological samples. J. Assoc. Offic. Anal. Chem. 52:378-382. El-Begearmi, M. M., C. Goudie, H. E. Ganther and M. L. Sunde, 1973. Attempts to quantitate the protective effect of selenium against mercury toxicity using Japanese quail. Federation Proc. 32:887. Emerick, R. J., I. S. Palmer, C. W. Carlson and R. A. Nelson, 1976. Mercury-selenium interrelationships in laying hens. Fed. Proc. 35:577. Friberg, L., and J. Vostal, 1972. Mercury in the Environment. Chemical Rubber Publishing Co., Cleveland, Ohio. Ganther, H. E., C. Goudie, M. L. Sunde, M. J. Kopecky, P. Wagner, Sand-Hwan Oh and W. G. Hoekstra, 1972. Selenium: Relation to decreased toxicity of methylmercury added to diets containing tuna. Science, 175:1122-1124. Johnson, S., and W. G. Pond, 1974. Inorganic vs.. organic Hg toxicity in growing rats: Protection by dietary Se but not Zn. Nutr. Rep. Int. 9:135-147. Kiwimae, A., A. Swensson, U. Ulfvarson and G. Westoo, 1969. Methylmercury compounds in eggs from hens after oral administration of mercury compounds. J. Agr. Food Chem. 17:1014—1016. Neathery, M. W., W. J. Miller, R. P. Gentry, P. E. Stake and D. M. Blackmon, 1974. Cadmium-109 and methylmercury-203 metabolism, tissue distribution and secretion in milk of cows. J. Dairy Sci. 57:1177-1183. Ostlund, K., 1969. Studies on the metabolism of methylmercury and dimethylmercury in mice. Acta Pharmacol, et Toxicol. 27, Supplement 1. Potter, S., and G. Matrone, 1974. Effect of selenite on the toxicity of dietary methylmercury and mercuric chloride in the rat. J. Nutr. 104:638—647. Scott, M. L., J. R. Zimmerman, S. Marinsky, P. A. Mullenhoff, G. L. Rumsey and R. W. Rice, 1975. Effects of PCB's, DDT and mercury compounds upon egg production, hatchability and shell quality in chickens and Japanese quail. Poultry Sci. 54:350-368.
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These observations serve t o emphasize t h e need for careful planning prior t o conducting research on t h e Hg X Se interrelationship. T h e use of trace doses of Hg only may yield information which would differ from t h a t obtained when substantial levels of " c o l d " Hg are given simultaneously. T h e data o n Hg found in liver and P.C.V. of t h e blood of chickens illustrate this p o i n t . Dietary Se decreased Hg in liver a n d P.C.V. w h e n n o Hg was added t o t h e feed. In contrast, Se increased 203 H g in b o t h t h e liver and P.C.V. when 20 p . p . m . Hg was fed.
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treated with methylmercury dicyandiamide. J. Sci. Food Agr. 14:734-740. Snedecor, G. W., 1956. Statistical Methods, Iowa State University Press, Ames, Iowa. Stoewsand, G. S., C. A. Bache and D. J. Lisk, 1974. Dietary selenium protection of methylmercury intoxication of Japanese quail. Bull. Environ. Contam. Toxicol. 11:152-156. Tejning, S., and R. Vesterberg, 1964. Aklyl mercurytreated seed in food grain. Poultry Sci. 43:6—11. Welsh, S. O., and J. H. Soares, 1974. The bioavailability of mercury in the tissue of hens fed methylmercuric chloride. Fed. Proc. 33:660.
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