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The toxicity of zinc to pregnant sheep
ENVIRONMENTAL
RESEARCH
The
20,
l-13
Toxicity J.K.
(197%
of Zinc to Pregnant CAMPBELL*
AND C.F.
Sheep
MILLS
Received November 7, 1978 The effects on pregnant sheep of cadmium and excess dietary zinc, separately and in combination, were investigated, Zinc induced severe copper deficiency in the ewes and caused a high incidence of abortions and stillbirths. Feed consumption, weight gain. and efficiency of feed utilization were all reduced in sheep receiving 750 mg Znkg diet. Supplementary copper prevented the development of copper deficiency but failed to prevent the adverse effect of high zinc on weight gain, feed consumption, effkiency of feed utilization, and lamb viability. The possible mechanisms involved in these effects are discussed. No adverse effect on reproductive performance resulted from the inclusion of 3 mg Cd/kg diet.
INTRODUCTION
Zinc is a relatively nontoxic element, but toxicity has been reported to arise in sheep and cattIe when dietary concentrations exceed 900- 1000 mg/kg (Ott ef ai., 1%6a, b). These experiments by Ott and his collaborators varied in duration from a few days up to 12 weeks and the dietary zinc concentrations employed ranged from 500 to 4000 mg/kg dry matter (D.M.). In sheep, toxic symptoms appeared at a zinc intake of approximately 50 mg Zn/kg body weight per day. Higher dose rates, up to 240 mg/kg body weight per day, caused acute toxicity. Although detailed studies have not been carried out on the mechanisms underlying zinc intoxication, there is evidence that at high zinc intakes antagonistic effects on copper, iron, and calcium metabolism can arise. High levels of dietary zinc have been shown to induce copper deficiency in rats (Grant-Frost and Underwood, 1958; Campbell and Mills, 1974) and to interfere with the metabolism of calcium (Cox ef al., 1969) and iron (Grant-Frost and Underwood, 1958). Zinc concentrations of up to 5000 mg/kg in the diet of pregnant rats induced hypocuprosis in the fetuses and caused a high incidence of stillbirths and fetal resorptions (Ketcheson et af., 1969). In contrast, Kinnamon (1963) reported no resorption in rats receiving similar levels of zinc. Little is known of the toxicity of zinc to human subjects but it has been reported (Kumar, 1976) that of four women dosed orally with zinc (300 mg Zn/d) during pregnancy, three gave birth prematurely and the child of a fourth was stillborn. Cadmium is also a metabolic antagonist of copper and has been shown to inhibit the accumulation of copper by the developing fetus (Mills and Dalgarno, 1972). Cadmium also had adverse effects upon the absorption ofcalcium and the absorption of iron (Ando et al., 1977; Spivey-Fox et al., 1971; Jacobs et cd., 1974). Because of their interactions with copper, the effects of cadmium and zinc on animals are likely to be influenced by the dietarypopper intake and by physiological conditions which modify the requirement of the animal for copper. The object of this study was to examine the effects of cadmium and zinc, separately and in combination, on the copper status of pregnant sheep maintained on diets contain* Present address: Boots Co. Ltd., Nottingham.
LJ.K 0013-9351/79/050001-13$02.00/O Copyright All right\
0 1979 by Academic Press. Inc. of reproduction in any form rexervrd.
2
CAMPBELL
AND
MILLS
ing a copper concentration which was inadequate by normal standards (Agricultural Research Council, 1965) but was judged to be sufficiently high to prevent the control sheep becoming grossly copper deficient within the time course of the experiment. The concentrations of cadmium and zinc employed were chosen to approximate those which can occur in industrially contaminated h&age (Mills and Dalgarno, 1972). Two experiments were conducted: The first examined the effects of cadmium and zinc on copper status during pregnancy and the second was intended to investigate whether supplementary copper would alleviate the adverse effects that were found to arise from a high zinc intake during the first study. Experiment
I: Animals,
MATERIALS AND METHODS Housing, and Management
Thirty-six Cheviot sheep, O-6 weeks pregnant, were allocated at random to six groups. The sheep were housed in groups of six in concrete-floored pens with wooden partitions. Sawdust was provided as bedding. Tap water was freely available in porcelain or polypropylene containers and feed was offered to appetite in wooden troughs. Feed was weighed in batches of 2.5 kg and added to the troughs as required. This method allowed feed consumption to be measured on a weekly basis but was unsuitable for measurement of day to day variations in feed intake. Sheep were weighed at the beginning of the experiment and at intervals thereafter. Diets
The basal diet was that of Suttle and Field (1968) supplemented to provide 2.5 mg Cu/kg. A 2 x 3 factorial design was employed in which animals were offered the basal diet supplemented with zinc or cadmium sulfate to give cadmium concentrations of 0.1 and 3.0 mg/kg and zinc concentrations of 30, 150, and 750 mg/kg diet. Sampling
Techniques
and Analytical
Methods
Blood samples were taken from the jugular vein into heparinized tubes. Liver samples were obtained by biopsy as described by Phillippo (1973). Plasma ceruloplasmin (ferroxidase I) activity was determined by the method of Houchin (1958) using Bandrowski’s base as external standard (Rice, 1962). Monoamine oxidase activity was determined by the method of Tabor et al. (1954) using benzylamine hydrochloride as substrate; a molar extinction coefficient for benzaldehyde of 12 x lo3 at 250 nm (Deardsen and Forbes, 1958) was assumed for calculations of activity. All trace element analyses were performed on a Varian Techtron AA5 atomic absorption spectrophotometer (Techtron Pty, Melbourne, Australia) after wet oxidation of tissue and diet samples. Statistical
Analysis
The spread of mating of ewes on this experiment was 40 days. To avoid comparing animals at different stages of pregnancy, the regression on time of each variable was calculated for each animal. Estimated values for each variable were then obtained for 90 and 20 days prior to parturition, and analysis of variance was carried out on these values. Data on feed consumption and efficiency of feed utilization could not be handled in this way as feed consumption of individual animals could not be measured.
ZINC
TOXICITY
IN
PREGNANT
3
SHEEP
Experiment 2: Animals, Housing, and Management Eighteen Cheviot sheep, 6 weeks pregnant, were allocated at random to three groups. Housing and management were identical to those of Experiment 1 except that feed intake was measured daily and the sheep were weighed weekly. Diets The basal diet was identical to that used in Experiment with copper and zinc (as sulfate) as follows:
1 but was supplemented
(1) 2.5 mg Cu + 30 mg B/kg (control) (2) 2.5 mg Cu + 750 mg %/kg (Group 7502) (3) 10 mg Cu + 750 mg Zn/kg (Group 7502 + Cu) Sampling Techniques and Analytical Methods These were identical to those employed in Experiment
1.
Statistical Analysis In this experiment, animals were successfully mated within a 7-day period and, being small in relation to the overall period of treatment, differences in the stage of pregnancy were ignored in analysing the data. Student’s t test was used to test the differences between means for statistical significance, except when the variance of groups differed significantly. In these cases, the Mann- Whitney U test was employed (Mann and Whitney, 1947). The distribution of nonviable lambs among treatment groups was examined using the Fisher exact probability test (Fisher, 1934) as the number of animals involved was too small to permit the use of the more usual x2 test. RESULTS
Experiment I Statistical analysis revealed no significant effects attributable to cadmium and no evidence of interactions between cadmium and zinc. The results are therefore discussed only in terms of the effects of zinc. In sheep receiving the highest dietary zinc concentration (group 7502) feed consumption and weight gain were reduced. Table 1 shows rate of gain up to parturition and feed intake and efficiency of feed utilization (EFU) up to Day 61. Figures for rate of gain apply only to pregnant animals but the figures for feed consumption and EFU include animals found to be nonpregnant because their contribution to the group mean feed intake could not be eliminated. Two sheep in the control group and one sheep in group 7502 were found to be nonpregnant and were removed at Day 61. The adverse effect of 750 mg Zn/kg diet on feed intake occurred within the first 10 days of the experiment but the method employed for measuring feed consumption did not allow the exact time course of the effect to be followed. After the initial drop in feed intake in group 7502, feed intake continued at these levels in each group for the remainder of the experiment. Weight gain in group 7502 was approximately one-third of that in the control group (P < 0.001). EFU was also severely depressed in these animals. In sheep receiving 150 mg Zn/kg diet (group 150Z) neither rate of gain nor EFU was reduced relative to the control group. At 20 days prior to parturition copper status in group 7502 was severely de.
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CAMPBELL
AND MILLS
TABLE EXPERIMENT
1: RATE OF WEIGHT
Diet (mg Zdkg) 30 150 750
1
GAIN, FEED INTAKE, AND EFFICIENCY IN PREGNANT SHEEP”
OF FEED
UTILIZATION
Rate of gain (kg/day)
Feed intake (kg/sheep/day)
EFUb
0.26 ” 0.03 (WC 0.24 a 0.02 (12) 0.09 2 0.04*** (5)”
2.1 (12) 2.0 (12) 1.6 (12)
0.11 0.11 0.03
Factorial analysis Linear zinc effect P < 0.001
-
-
0 Figures for rate of gain cover the period from the start of the experiment to parturition and refer only to pregnant sheep. Figures for feed intake and EFU cover the period up to Day 61 of the experiment and include nonpregnant sheep since their contribution to feed intake could not be estimated. After Day 61 some animals were lost due to deaths, abortions, and removal of nonpregnant sheep from the experiment. b Efficiency of feed utilization: kg weight gain&g feed consumed. c Two ewes not pregnant. d Two ewes died, one ewe not pregnant, four ewes aborted early in the experiment. All of these were excluded from the analysis as no parturition date could be assigned to them. *** Significantly different from control, P < 0.001.
pressed (Table 2). Plasma copper concentration was reduced by 50% relative to the control group (P < 0.01) and similar reductions occurred in plasma ceruloplasmin and amine oxidase activity. In spite of the low copper concentration of the basal diet, plasma copper concentration, plasma ceruloplasmin, and plasma amine oxidase activity in the control group remained within normal limits throughout pregnancy, In group 15OZ, plasma copper concentration, plasma ceruloplasmin activity, and plasma amine oxidase activity were slightly increased relative to the control group. Although the increases were not in themselves statistically significant they appeared in the statisticai analyses as a significant quadratic effect of zinc (P < 0.001). The concentration of zinc in plasma was not significantly altered in group 15OZ but was greatly increased in group 7502. After 90 days on treatment plasma zinc TABLE
2
EXPERIMENT 1: COPPER CONCENTRATION, CERULOPLASMIN ACTIVITY, AND AMINE OXIDASE ACTIVITY IN THE PLASMA OF PREGNANT SHEEP 20 DAYS PRIOR TO PARTURITION
Diet (w Z&d
Number of animals
Plasma Cu (mfliter)
Plasma cerulopiasmin (U/liter)
Plasma amine oxidase (U/liter)
10 12 5
0.98 2 0.09 1.20 2 0.09 0.54 + 0.13**
10.1 2 1.4 12.7 rt 1.3 1.1 4 2.0**
55.9 ? 4.8 67.0 k 4.3 32.4 k 6.7**
P < 0.02 P < 0.001
P < 0.01 P < 0.001
P < 0.02 P <0.002
30 150 750 Factorial analysis Linear zinc effect Quadratic zinc effect
** Significantly different from control, P < 0.01.
ZlNC
TOXICITY
IN
PREGNANT
TABLE EXPERIMENT
5
SHEEP
3
1: DISTRIBUTION OF VIABLE AND NONVIABLE LAMBS INDICATED CONCENTRATIONS OF DIETARY
AMONG ZINC”
EWES RECEIVING
Diet (mg Zn/kg)
Viable
Number of lambs nonviable”
Total
30 150 750
11 19 3
3 4 17
14 23 20
THE
” x2 test: P < 0.001. h A nonviable iamb is defined as a lamb which was aborted, stillborn, or died for any reason within 7 days of birth.
concentration of this group was 6.71 + 0.83 (10) compared to 1.05 + 0.04 (12) in the control group [mean 2 SE(n)]. This difference is significant at the 0.1% level of probability. Reproductive performance was severely impaired in group 7502. Numerous abortions and perinatal deaths occurred. The distribution of abortions, stillbirths, and perinatal deaths among treatment groups is shown in Table 3. For the purpose of this table, a nonviable lamb is defined as a lamb which was aborted, stillborn, or died for any reason within 7 days of birth. The distribution is heavily biased towards group 7502 and a x2 test indicates that such a distribution would be unlikely to occur by chance (P < 0.001). The cause of death in these lambs could not be determined. Lambs born alive were weak and unable or unwilling to stand or suckle; some trembled violently or were ataxic. Deaths, preceded by convulsions, usually occurred within 48 hr, although in one case the terminal symptoms were delayed until 4 days of age. Two lambs (twins) which were bottle fed with cow’s milk survived until 5 weeks of age before they collapsed with symptoms similar to those observed in lambs which had died at birth. Of 20 lambs conceived in this group, only one survived more than 5 weeks of postnatal life. Two findings common to all of the nonviable lambs in group 7502 were very high tissue zinc concentrations and very low tissue copper concentrations (see Table 4). Lambs from group 7502 had liver copper concentrations approximately onetenth of that in control lambs (P < 0.02). In lambs from group 15OZ, there was a TABLE EXPERIMENT
1: CONCENTRATION
OF COPPER
4
AND
ZINC IN LIVER OF NONVIABLE
LAMBS”
Diet tmg Zn/kg)
Number of lambs
Liver Cu b-m&z)
Liver Zn Wk)
30 150 750
3 4 12
79.8 f 34.6 161.3 r 17.7 8.4 2 1.7’
251.9 + 20.6 915.8 -t 1843* 2044 It 359.s***
” Lambs were born to ewes maintained for approximately taining the indicated concentration of zinc. * Significantly different from control: P < 0.05. **= Significantly different from control: P < 0.001,
110 days prior to parturition on diets con-
FIG. 1. Experiment 1: Bone A was taken from a control lamb (6.5 kg) which died during birth. Death was attributed to dystocia. Bone B was t aken from a lamb born to a ewe receiving 750 mg Zn/kg diet. The lamb weighed 3.0 kg at birth and died from indeterminate cause 48 hr after birth.
ZINC
TOXICITY
IN PREGNANT
7
SHEEP
trend toward higher liver copper concentrations but the effect was not statistically significant. The zinc concentration of lamb liver was significantly increased by both levels of zinc supplementation. A dietary zinc concentration of 150 mg/kg resulted in a threefold increase in liver zinc concentration (P < 0.05) and a dietary zinc concentration of 750 mg/kg caused a lo-fold increase (P < 0.001). Nonviable lambs from the other two groups died from a variety of causes unconnected with the dietary treatment. Radiographs of nonviable lambs from group 7502 revealed lines of arrested growth in the long bones. These lines were not observed in lambs from the other two groups (Fig. 1). Severe renal damage was observed in one nonviable lamb from group 7502. Lesions included hyperemia and extravasation of blood cells in the medulla and almost complete ischemic necrosis of the cortex with necrosis of the arcuate and interlobular arteries. Experiment 2 In view of the findings from Experiment 1 that a dietary zinc concentration 750 mg/kg induced copper deficiency, a second experiment was undertaken
of to
2.5 9a x $
*.O
2
4
6
0 Day
10
12
No.
FIG. 2. Feed consumption (kg/sheep/day) in pregnant + 2.5 mg Cu/kg (0). 750 mg Zn + 2.5 mg Cu/kg (0). analysis could be performed on these data.
14
5
5
7 9 Week
11
13
ewes maintained on diets containing 30 mg Zn 750 mg Zn + 10 mg Cuikg (0). No statistical
8
CAMPBELL
AND
MILLS
determine whether provision of extra dietary copper would prevent the high lamb mortality in sheep maintained on high zinc diets during pregnancy. Feed consumption (Fig. 2) was identical in all groups during the first 7- 10 days of the experiment. Thereafter, feed intake in both the high zinc groups declined regardless of copper intake to a level about 20% below that of the control group and remained at that level for the duration of the experiment. Efficiency of feed utilization was reduced by about 50% in both of the zinc-supplemented groups. At the beginning of the experiment, copper status as judged by the concentration of copper and the activity of monoamine oxidase and ceruloplasmin in plasma, was identical in all three groups (Fig. 3). However, by Day 74 plasma copper concentration in group 7502 (750 mg Zn + 2.5 mg Cu/kg diet) was little more than half that of the control group (P < 0.05) and falling rapidly. In contrast plasma copper concentration in group 7502 + Cu (750 mg Zn + 10 mg Cu per kg diet) remained similar to that of the control group. The rise in plasma copper concentration just after the start of the experiment is difficult to explain. Since it occurred only in the zinc-supplemented groups, it appears to be caused by the zinc content of the diet. Although supplementary copper prevented the decline in the copper status of ewes receiving 750 mg zinc/kg diet it did not alleviate the effect of zinc on reproductive performance. Table 5 shows the distribution of viable and nonviable lambs among the three groups. The distribution was tested using the Fisher exact probability test because there were too few animals to allow the use of the more usual x2 test. The proportion of nonviable lambs in group 7502 and group 7502 + Cu was
FIG. 3. Plasma copper concentration, (mg/liter) in pregnant ewes maintained on diets mg Zn + 2.5 mg Co/kg (0), 750 mg Zn + 2.5 mg Cu/kg ( l ), 750 mg Zn + IO mg Cu/kg (0). the mean and standard error of six animals unless otherwise indicated. Figures adjacent indicate the number of animals if less than six. Significantly different from control (0)
containing 30 Each point is to the points “P < 0.05.
ZINC
IN
TOXICITY
PREGNANT
TABLE EXPERIMENT
2:
DISTRIBUTION INDICATED
OF VIABLE CONCENTRATIONS
AND
5
NONVIABLE
LAMBS
OF DIETARY
30 750 750
1
2 3
AMONG
COPPER
Diet mg Zn/kg
9
SHEEP
EWES
AND
RECEIVING
THE
ZINC”
Number of lambs mg &/kg
Viable 9 0 1
2.5 2.5 10.0
” Ewes were maintained on the indicated diets for approximately probability test: Comparison, 1-2, l-3: P < 0.001.
Nonviable 2 12 13
Total 11 12 14
100 days prior to parturition.
Exact
significantly greater than would be expected to occur by chance. The concentrations of copper and zinc in the livers of these lambs are presented in Table 6. Copper concentration in the liver of lambs from group 7502 was reduced by 50% relative to the control group (P < 0.05). The depression of liver copper concentration was less severe than that resulting from 750 mg Zn/kg diet in the previous experiment. The group mean was distorted however by three lambs with liver copper concentrations similar to those of the control lambs. Most of the lambs in group 7502 had hepatic copper concentrations between 4.0 and 8.0 mg/kg. In lambs from group 7502 + Cu, liver copper concentration was not significantly different from that of the control group. Hepatic zinc concentration in lambs from the zinc-supplemented groups was approximately 10 times the control level. As in Experiment 1, lines of arrested growth were observed in the bones of nonviable lambs from both of the zinc-supplemented groups. DISCUSSION
Cadmium at a concentration of 3.0 mg/kg diet had no detectable effect on copper status or breeding performance. This is consistent with the work of Mills and Dalgamo (1972) who found that 3.5 mg Cd/kg diet had no effect on hepatic copper concentration in pregnant ewes and only depressed liver Cu of their offspring when Cd-containing solid diet rather than ewe milk constituted the primary source of food. The effect of excess dietary zinc on the copper metabolism of the pregnant ewe was more severe than was anticipated. Ott et ul. (1966a) reported major effect on copper status of sheep only when dietary zinc concentration exceeded 1000 TABLE EXPERIMENT TO EWES
2:
6
CONCENTRATION RECEIVING THE
OF COPPER AND ZINC IN THE LIVER OF NONVIABLE INDICATED CONCENTRATIONS OF DIETARY COPPER
Diet
mg Znlkg
mg Culkg
1
30 750 750
2 3
‘I 2.5 2.5 10.0
Liver Cu
Liver
27.0 2 5.9 13.0 t 2.9” 18.3 rt 3.9
262.2 5 151.7 3265 k 5g7”*’ 2456 k 381*“”
” Figures in parentheses = number of animals. * Significantly different from group 1: P < 0.05. *** Significantly different from group 1: P < 0.01.
LAMBS BORN AND ZINC
Zn
-
(3)” (10) (8)
10
CAMPBELL
AND
MILLS
mg/kg. These workers however were using nonpregnant sheep and the copper content of their diet, although not stated, may have been greater than that used in this study judging from the ingredients used. Copper deficiency in our ewes was reflected by an equally severe copper deficiency in the fetus and neonate. Copper deficiency, in both ewe and lamb, was prevented by copper supplementation but in spite of the substantially higher copper content of the diet of their dams, liver copper in lambs from group 7502 + Cu was no different from that of the control group. The high zinc concentration of the diet therefore prevented the increase in liver copper concentration which might have been expected in the livers of lambs whose dams were receiving four times the dietary copper concentration of the control sheep. Many of the lambs had liver copper concentrations around 4 or 5 mg/kg which is within the range in which swayback might ,be expected to occur (Mills and Williams, 1962). However, examination of sections from the spinal cord and red nucleus (Mills and Fell, 1960) of nonviable lambs revealed no demyelination and no degeneration of motor neurones. The significant quadratic effect of zinc on copper-dependent parameters in ewes suggests that a moderate level of zinc supplementation is in some way beneficial to the copper status of the sheep. Thus plasma copper concentration, plasma ceruloplasmin activity, and plasma amine oxidase activity were all higher in group 15OZ than in the control group. Subsequent work with the offspring of these sheep maintained on the same diet (manuscript in preparation) has shown a similar pattern of copper status. Lambs receiving 150 mg Zn/kg diet tended to accumulate hepatic copper more readily than control lambs. Under these conditions however, the total concentration of copper in the liver or plasma may not be a true reflection of the functional copper status of the animal. The large increases in tissue zinc concentration observed in this study were not in complete accord with the findings of other workers. In this study plasma zinc concentration rose progressively from the time the sheep were first exposed to the high zinc diets and at parturition some animals had plasma zinc concentrations of up to 12 mg/liter. Ott et al. (1966~) found that diets containing up to 1000 mg Zn/kg caused plasma zinc concentrations to double and large increases in plasma zinc concentration occurred only at dietary zinc concentrations in excess of 1000 mg/kg. The sheep used by Ott et al. were however of a different breed to those used in this experiment, were of a different age, weight, and sex, and were not pregnant. Any of these factors may modify the change in plasma zinc concentration produced by excess dietary zinc. High plasma zinc concentrations also developed in our lambs. Two lambs born in group 7502 when sampled within 24 hr of birth were found to have plasma zinc concentrations of 5 mg/liter. The effect of this level of zinc in plasma on the health of the lambs is not known. Adverse effects of excess dietary zinc on feed intake and EFU have been reported-although at much higher dietary concentrations than those employed in these experiments. Ott et al. (1966a, b, c) reported reduced feed consumption and EFU in sheep receiving dietary zinc concentrations of 1000 mg/kg or more. They attributed the reduced feed intake to the unpalatability of diets containing high concentrations of zinc salts and the decline in EFU to a decline in microbial activity in the rumen. In the study reported here, the food intake of sheep receiving high zinc rations remained normal for 7 to 10 days before declining. It seems
ZINC
TOXICITY
IN
PREGNANT
SHEEP
II
likely, therefore, that the low feed intake was due not to the taste of the diet but rather to some systemic effect of zinc. Rumen metabolism was not investigated but microscopic examination of rumen contents indicated that rumen microflora was identical in all the sheep regardless of their zinc intake. The effect of zinc on EFU was much greater in Experiment 1 than in Experiment 2. No explanation can be offered for this finding. The most important finding in this study was the effect of high dietary zinc concentrations on the viability of lambs. This effect has not been previously reported in sheep. Several hypothesis can be advanced to explain the low viability of the lambs. First, the very high concentrations of zinc in the tissues may have been toxic to the fetus. In particular, the kidneys may have been severely affected. It is known that in suckling lambs exposed to high dietary zinc concentrations, the kidneys accumulate large amounts of zinc which leads to severe renal damage (Davies et ~11.. 1977). The very limited evidence available from this study suggests that the kidneys of the fetus may be similarly susceptible to damage by high concentrations of zinc. The lamb from which the single kidney sample was obtained was dying with symptoms identical to those observed in other nonviable lambs when it was killed, specifically for histological study. The extensive renal damage observed in this lamb must have contributed to its poor condition and in the light of the findings of Davies et al. (1977) it seems possible that the debilitation and death of other nonviable lambs was due in part to renal damage caused by high concentrations of zinc in the kidney. Unfortunately, no other samples could be obtained for microscopic examination and no definite conclusion can be reached. A second possibility is that the depressed feed intake of the ewes may have caused the high mortality rate among lambs. Inadequate nutrition of ewes during late pregnancy leads to low birthweight and increased perinatal mortality in lambs (Thomson and Thomson, 1949; Hight and Jury, 1970). Thomson and Thomson found that when the dry matter intake of pregnant ewes was restricted to about 50% of normal, perinatal lamb mortality approached 70%. The reduction in feed intake induced by excess dietary zinc in the present study was much less than that imposed by Thomson and Thomson and it is therefore unlikely that reduced feed intake was responsible for the reduced viability of lambs. However, the weight changes in pregnant ewes reported by Thomson and Thomson were similar to those observed in the experiments described here. The very low rate of weight gain in pregnant ewes receiving 750 mg Zn/kg was presumably due therefore to the combined effect of low feed intake and the reduced EFU resulting from this treatment. This resulted in a degree of malnutrition comparable with that observed by Thomson and Thomson which may have contributed to the reduced viability of lambs. Discontinuous growth of lambs in utero is indicated by the transverse bands in the bones. It has been suggested that these “lines of arrested growth” may be produced by disturbance of fetal nutrition (Harris, 1923, 1931). Similar lines can be produced in the bones of growing rats by subjecting them to periods of severe malnutrition (Park and Richter, 1953) and it is tempting, therefore, to interpret the lines in lamb bones as evidence of fetal malnutrition. Toxoplasmosis has also been suggested as a cause of lines of arrested growth in lambs (H. M. ROSS, North of Scotland College of Agriculture, unpublished observations) but toxoplasmosis was not detected in any of these animals.
12
CAMPBELL
AND
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Although the mechanisms involved are not clear, it is evident that the pregnant sheep is vulnerable to concentrations of dietary zinc which have little effect on the nonpregnant animal. Whereas Ott el al. (1966a, c) found signs of toxicity at dose rates of around 50 mg/kg body weight per day, our sheep suffered almost complete reproductive failure at a dose rate which declined from 20 mg/kg body weight per day at the start of the experiment to around 10 mg/kg/day during the last 10 weeks of pregnancy. Similar findings have been reported in rats. Campbell and Mills (1972) reported that a diet containing 300 mg Zn/kg had no major effect on the health of male rats, while Kumar (1976) found a significant increase in fetal resorption in pregnant rats receiving a diet containing 150 mg Zn/kg. This corresponds to a zinc intake of approximately 10 mg/kg body weight per day. Thus a zinc intake of around 10 mg/kg body weight per day, which is considerably less than that previously thought to be harmful, can have a serious effect on the course of pregnancy in two species. Until more is known about the effects of zinc on reproduction, zinc supplementation of livestock should be undertaken with this limitation in mind, Furthermore, it should be noted that herbage with concentrations of zinc up to and exceeding 750 mg/kg has been found in the vicinity of some industrial complexes (Goodman and Roberts, 1971; Lagerwerffet al., 1972; Mills and Dalgarno, 1972). Although the forms in which this zinc exists in or on the herbage have rarely been identified and its availability to animals may vary, this study seems to emphasize that pregnant animals may be at risk from such situations. ACKNOWLEDGMENTS We are grateful to Rio Tinto Zinc Services Limited, Bond Street, Bristol for a research grant in aid of this work. We are also grateful to Dr. B. F. Fell and Mr. R. Boyne for preparation and examination of histological samples, to Mr. G. Wenham for radiography and surgical procedures, and to Mr. S. Mann for examination of rumen contents. Thanks are also due to Mr. A. C. Dalgarno for his advice and assistance, to Mrs. L. Miller for technical assistance, and to Mr. G. Calder for management of the animals.
REFERENCES Agricultural Research Council (1965). The Nutritional Requirements of Farm Livestock. No. 2 Ruminants: Technical Reviews. H. M. Stationery Office. Ando, M., Sayoto, Y., Tonomura, M., and Osawa, T. (1977). Studies on excretion and uptake of Ca by rats after continuous oral administration of cadmium. Toxicol. Appl. Pharmaco/. 39, 321-328. Campbell, J. K., and Mills, C. F. (1974). Effects of dietary cadmium and zinc on rats maintained on diets low in copper. Proc. Nurr. Sot. 33, 15A-17A. Cox, D. H., Schlicker, S. A., and Chu, R. G. (1969). Excess dietary zinc for the maternal rat and zinc, iron, copper, calcium and magnesium content and enzyme activity in maternal and fetal tissues. J. Nurr. 98, 459-466. Davies, N. T., Soliman, H. S., Corrigall, W., and Flett, A. (1977). The susceptibility of suckling lambs to zinc toxicity. Brit. J. Nurr. 38, 153- 156. Dearsden, J. C., and Forbes, W. F. (1958). Light absorption studies. Part XII. Ultraviolet absorption spectra of benzaldehydes. Canad. J. Chem. 36, 1362-1370. Fisher, R. A. (1934). “Statistical Methods for Research Workers,” 5th ed. Oliver and Boyd, Edinburgh. Goodman, G. T., and Roberts, T. M. (1971). Plants and soils as indicators of metals in the air. Narure (London)
231,
287-292.
Grant-Frost, D. R., and Underwood, copper. Ausr. J. Exp. Biol. Med.
E. J. (1958). Zinc toxicity in the rat and its interrelation Sci.
36, 339-346.
with
ZINC TOXICITY
IN PREGNANT
SHEEP
13
Harris, H. A. (1924). The growth of the long bones. Proc. ~fthp Anaf. SW. of Ct. Brituin und Irdmtl. p. 94. Harris, H. A. (1931). Lines of arrested growth in the long bones in childhood: The correlation of histological and radiographic appearances in clinical and experimental conditions. bit. J. Rudid. N.S. 6, 622-640. Hight, G. K.. and Jury, K. E. (1970). Hill country sheep production. II. Lamb mortality and birth weights in Romney and Border Leicester x Romney flocks. N.Z. J. Agric,. Rrs. 13, 735-752. Houchin, 0. B. (1958). Rapid calorimetric method for the quantitative determination of copper oxidase activity (caeruloplasmin). C/in. Chrm. 4, 519-523. Jacobs, R. M., Spivey-Fox, M. R.. Fry, B. E.. Jr., and Harland. B. F. ( 1974). The effect of a two day exposure to dietary cadmium on the concentration of elements in duodenal tissue of Japanese Quail. In “Trace Element Metabolism in Animals-2” (W. G. Hoekstra, J. W. Suttie, H. E. Ganther, and W. Mertz, Eds.), pp. 684-686. University Park Press, Baltimore. Ketcheson, M. R., Barron, G. P., and Cox. D. H. (1%9). Relationship of maternal dietary zinc during gestation and lactation to development and zinc, iron and copper content of the postnatal rat. J. Nutr. 98, 303-311. Kinnamon, K. E. (1963). Some independent and combined effects of copper, molybdenum and zinc on the placental transfer of zinc-65 in the rat. J. Nurr. 81, 312-320. Kumar, S. (1976). Effect of zinc supplementation on rats during pregnancy. Nutr. Rep. Int. 13,33-36. Lagerwerff, J. V., Brower, D. L., and Biersdorf, G. T. (1972). Accumulation of cadmium, copper, lead and zinc in soil and vegetation in the proximity of a smelter. In “Trace Substances in Environmental Health” (D. D. Hemphill, Ed.), Vcl. VI, pp. 71-78. University of Missouri. Missouri. Mann, H. B., and Whitney, D. R. (1947). On a test of whether one of two random variables is stochastically larger than the other. Ann. Math. Stcltist. 18, 50-60. Mills, C. F., and Dalgarno, A. C. (1972). Copper and zinc status of ewes and lambs receiving increased dietary concentrations of cadmium. Nature (London) 239, 171- 173. Mills. C. F., and Fell, B. F. (1960). Demyelination in Iambs born of ewes maintained on high intakes of sulphate and molybdate. Ntrture (London) 185, 20-22. Mills, C. F.. and Williams, R. B. (1962). Copper concentration and cytochrome oxidase and ribonucIease activities in the brain of copper deficient lambs. Biwhem. J. 85, 629-632. Ott, E. A., Smith, W. H., Harrington, R. B., and Beeson, W. M. (1%6a). Zinc toxicity in ruminants. I. Effect of high levels of dietary zinc on gains. feed consumption and feed efficiency of lambs. ./. Anim. Sci. 25, 414-418. Ott. E. A., Smith, W. H., Harringron, R. B., and Beeson, W. M. (1966b) Zinc toxicity in ruminants. II. Effect of high levels of dietary zinc on gains, feed consumption and feed efficiency of beef cattle. J. Anim. Sci. 25, 419-423. Ott. E. A.. Smith, W. H.. Harrington, R. B., Stob, M., Parker. H. E., and Beeson, W. M. (1966~). Zinc toxicity in ruminants. III. Physiological changes in tissues and alterations in rumen metabolism in Iambs. J. Anim. Sci. 25, 424-431. Park, E. A., and Richter, C. P. (1953). Transverse lines in bone: The mechanism of their development. &r/l.
Johns
Hopkins
Hosp.
93, 234-248.
Phihippo, M. (1973). A technique for liver biopsy in sheep. J. Agric. Sci. Cumb. 80, 313-314. Rice, E. W. (1962). Standardization of caeruloplasmin activity in terms of international enzyme units. Oxidation of “Bandrowski‘s Base” fromp-phenylenediamine by caeruloplasmin. Alrtr/. Bi<,c,hrrn. 3, 452-456. Spivey-Fox, M. R., Fry, B. E., Jr., Harland, B. F., Schertel, M. E., and Weeks, C. E. (1971). Effect of ascorbic acid on cadmium toxicity in the young Coturnk. J. Nutr. 101, 1294- 1305. Suttle, N. F.. and Field, A. C. (I%8). Effect of intake of copper, molybdenum and sulphate on copper metabolism in sheep. I. Clinical condition and distribution ofcopper in blood ofthe pregnant ewe../. Camp. Pathol. 78, 351-362. Tabor. C. W.. Tabor, H., and Rosenthal, S. M. (1954). Purification of amine oxidase from beef plasma. J. Bid. Chem.208, 645-661. Thomson, A. M., and Thomson, W. (1949). Lambing in relation to the diet of the pregnant ewe. Brir. J. Nutr.
2. 290-305.
RESEARCH
The
20,
l-13
Toxicity J.K.
(197%
of Zinc to Pregnant CAMPBELL*
AND C.F.
Sheep
MILLS
Received November 7, 1978 The effects on pregnant sheep of cadmium and excess dietary zinc, separately and in combination, were investigated, Zinc induced severe copper deficiency in the ewes and caused a high incidence of abortions and stillbirths. Feed consumption, weight gain. and efficiency of feed utilization were all reduced in sheep receiving 750 mg Znkg diet. Supplementary copper prevented the development of copper deficiency but failed to prevent the adverse effect of high zinc on weight gain, feed consumption, effkiency of feed utilization, and lamb viability. The possible mechanisms involved in these effects are discussed. No adverse effect on reproductive performance resulted from the inclusion of 3 mg Cd/kg diet.
INTRODUCTION
Zinc is a relatively nontoxic element, but toxicity has been reported to arise in sheep and cattIe when dietary concentrations exceed 900- 1000 mg/kg (Ott ef ai., 1%6a, b). These experiments by Ott and his collaborators varied in duration from a few days up to 12 weeks and the dietary zinc concentrations employed ranged from 500 to 4000 mg/kg dry matter (D.M.). In sheep, toxic symptoms appeared at a zinc intake of approximately 50 mg Zn/kg body weight per day. Higher dose rates, up to 240 mg/kg body weight per day, caused acute toxicity. Although detailed studies have not been carried out on the mechanisms underlying zinc intoxication, there is evidence that at high zinc intakes antagonistic effects on copper, iron, and calcium metabolism can arise. High levels of dietary zinc have been shown to induce copper deficiency in rats (Grant-Frost and Underwood, 1958; Campbell and Mills, 1974) and to interfere with the metabolism of calcium (Cox ef al., 1969) and iron (Grant-Frost and Underwood, 1958). Zinc concentrations of up to 5000 mg/kg in the diet of pregnant rats induced hypocuprosis in the fetuses and caused a high incidence of stillbirths and fetal resorptions (Ketcheson et af., 1969). In contrast, Kinnamon (1963) reported no resorption in rats receiving similar levels of zinc. Little is known of the toxicity of zinc to human subjects but it has been reported (Kumar, 1976) that of four women dosed orally with zinc (300 mg Zn/d) during pregnancy, three gave birth prematurely and the child of a fourth was stillborn. Cadmium is also a metabolic antagonist of copper and has been shown to inhibit the accumulation of copper by the developing fetus (Mills and Dalgarno, 1972). Cadmium also had adverse effects upon the absorption ofcalcium and the absorption of iron (Ando et al., 1977; Spivey-Fox et al., 1971; Jacobs et cd., 1974). Because of their interactions with copper, the effects of cadmium and zinc on animals are likely to be influenced by the dietarypopper intake and by physiological conditions which modify the requirement of the animal for copper. The object of this study was to examine the effects of cadmium and zinc, separately and in combination, on the copper status of pregnant sheep maintained on diets contain* Present address: Boots Co. Ltd., Nottingham.
LJ.K 0013-9351/79/050001-13$02.00/O Copyright All right\
0 1979 by Academic Press. Inc. of reproduction in any form rexervrd.
2
CAMPBELL
AND
MILLS
ing a copper concentration which was inadequate by normal standards (Agricultural Research Council, 1965) but was judged to be sufficiently high to prevent the control sheep becoming grossly copper deficient within the time course of the experiment. The concentrations of cadmium and zinc employed were chosen to approximate those which can occur in industrially contaminated h&age (Mills and Dalgarno, 1972). Two experiments were conducted: The first examined the effects of cadmium and zinc on copper status during pregnancy and the second was intended to investigate whether supplementary copper would alleviate the adverse effects that were found to arise from a high zinc intake during the first study. Experiment
I: Animals,
MATERIALS AND METHODS Housing, and Management
Thirty-six Cheviot sheep, O-6 weeks pregnant, were allocated at random to six groups. The sheep were housed in groups of six in concrete-floored pens with wooden partitions. Sawdust was provided as bedding. Tap water was freely available in porcelain or polypropylene containers and feed was offered to appetite in wooden troughs. Feed was weighed in batches of 2.5 kg and added to the troughs as required. This method allowed feed consumption to be measured on a weekly basis but was unsuitable for measurement of day to day variations in feed intake. Sheep were weighed at the beginning of the experiment and at intervals thereafter. Diets
The basal diet was that of Suttle and Field (1968) supplemented to provide 2.5 mg Cu/kg. A 2 x 3 factorial design was employed in which animals were offered the basal diet supplemented with zinc or cadmium sulfate to give cadmium concentrations of 0.1 and 3.0 mg/kg and zinc concentrations of 30, 150, and 750 mg/kg diet. Sampling
Techniques
and Analytical
Methods
Blood samples were taken from the jugular vein into heparinized tubes. Liver samples were obtained by biopsy as described by Phillippo (1973). Plasma ceruloplasmin (ferroxidase I) activity was determined by the method of Houchin (1958) using Bandrowski’s base as external standard (Rice, 1962). Monoamine oxidase activity was determined by the method of Tabor et al. (1954) using benzylamine hydrochloride as substrate; a molar extinction coefficient for benzaldehyde of 12 x lo3 at 250 nm (Deardsen and Forbes, 1958) was assumed for calculations of activity. All trace element analyses were performed on a Varian Techtron AA5 atomic absorption spectrophotometer (Techtron Pty, Melbourne, Australia) after wet oxidation of tissue and diet samples. Statistical
Analysis
The spread of mating of ewes on this experiment was 40 days. To avoid comparing animals at different stages of pregnancy, the regression on time of each variable was calculated for each animal. Estimated values for each variable were then obtained for 90 and 20 days prior to parturition, and analysis of variance was carried out on these values. Data on feed consumption and efficiency of feed utilization could not be handled in this way as feed consumption of individual animals could not be measured.
ZINC
TOXICITY
IN
PREGNANT
3
SHEEP
Experiment 2: Animals, Housing, and Management Eighteen Cheviot sheep, 6 weeks pregnant, were allocated at random to three groups. Housing and management were identical to those of Experiment 1 except that feed intake was measured daily and the sheep were weighed weekly. Diets The basal diet was identical to that used in Experiment with copper and zinc (as sulfate) as follows:
1 but was supplemented
(1) 2.5 mg Cu + 30 mg B/kg (control) (2) 2.5 mg Cu + 750 mg %/kg (Group 7502) (3) 10 mg Cu + 750 mg Zn/kg (Group 7502 + Cu) Sampling Techniques and Analytical Methods These were identical to those employed in Experiment
1.
Statistical Analysis In this experiment, animals were successfully mated within a 7-day period and, being small in relation to the overall period of treatment, differences in the stage of pregnancy were ignored in analysing the data. Student’s t test was used to test the differences between means for statistical significance, except when the variance of groups differed significantly. In these cases, the Mann- Whitney U test was employed (Mann and Whitney, 1947). The distribution of nonviable lambs among treatment groups was examined using the Fisher exact probability test (Fisher, 1934) as the number of animals involved was too small to permit the use of the more usual x2 test. RESULTS
Experiment I Statistical analysis revealed no significant effects attributable to cadmium and no evidence of interactions between cadmium and zinc. The results are therefore discussed only in terms of the effects of zinc. In sheep receiving the highest dietary zinc concentration (group 7502) feed consumption and weight gain were reduced. Table 1 shows rate of gain up to parturition and feed intake and efficiency of feed utilization (EFU) up to Day 61. Figures for rate of gain apply only to pregnant animals but the figures for feed consumption and EFU include animals found to be nonpregnant because their contribution to the group mean feed intake could not be eliminated. Two sheep in the control group and one sheep in group 7502 were found to be nonpregnant and were removed at Day 61. The adverse effect of 750 mg Zn/kg diet on feed intake occurred within the first 10 days of the experiment but the method employed for measuring feed consumption did not allow the exact time course of the effect to be followed. After the initial drop in feed intake in group 7502, feed intake continued at these levels in each group for the remainder of the experiment. Weight gain in group 7502 was approximately one-third of that in the control group (P < 0.001). EFU was also severely depressed in these animals. In sheep receiving 150 mg Zn/kg diet (group 150Z) neither rate of gain nor EFU was reduced relative to the control group. At 20 days prior to parturition copper status in group 7502 was severely de.
4
CAMPBELL
AND MILLS
TABLE EXPERIMENT
1: RATE OF WEIGHT
Diet (mg Zdkg) 30 150 750
1
GAIN, FEED INTAKE, AND EFFICIENCY IN PREGNANT SHEEP”
OF FEED
UTILIZATION
Rate of gain (kg/day)
Feed intake (kg/sheep/day)
EFUb
0.26 ” 0.03 (WC 0.24 a 0.02 (12) 0.09 2 0.04*** (5)”
2.1 (12) 2.0 (12) 1.6 (12)
0.11 0.11 0.03
Factorial analysis Linear zinc effect P < 0.001
-
-
0 Figures for rate of gain cover the period from the start of the experiment to parturition and refer only to pregnant sheep. Figures for feed intake and EFU cover the period up to Day 61 of the experiment and include nonpregnant sheep since their contribution to feed intake could not be estimated. After Day 61 some animals were lost due to deaths, abortions, and removal of nonpregnant sheep from the experiment. b Efficiency of feed utilization: kg weight gain&g feed consumed. c Two ewes not pregnant. d Two ewes died, one ewe not pregnant, four ewes aborted early in the experiment. All of these were excluded from the analysis as no parturition date could be assigned to them. *** Significantly different from control, P < 0.001.
pressed (Table 2). Plasma copper concentration was reduced by 50% relative to the control group (P < 0.01) and similar reductions occurred in plasma ceruloplasmin and amine oxidase activity. In spite of the low copper concentration of the basal diet, plasma copper concentration, plasma ceruloplasmin, and plasma amine oxidase activity in the control group remained within normal limits throughout pregnancy, In group 15OZ, plasma copper concentration, plasma ceruloplasmin activity, and plasma amine oxidase activity were slightly increased relative to the control group. Although the increases were not in themselves statistically significant they appeared in the statisticai analyses as a significant quadratic effect of zinc (P < 0.001). The concentration of zinc in plasma was not significantly altered in group 15OZ but was greatly increased in group 7502. After 90 days on treatment plasma zinc TABLE
2
EXPERIMENT 1: COPPER CONCENTRATION, CERULOPLASMIN ACTIVITY, AND AMINE OXIDASE ACTIVITY IN THE PLASMA OF PREGNANT SHEEP 20 DAYS PRIOR TO PARTURITION
Diet (w Z&d
Number of animals
Plasma Cu (mfliter)
Plasma cerulopiasmin (U/liter)
Plasma amine oxidase (U/liter)
10 12 5
0.98 2 0.09 1.20 2 0.09 0.54 + 0.13**
10.1 2 1.4 12.7 rt 1.3 1.1 4 2.0**
55.9 ? 4.8 67.0 k 4.3 32.4 k 6.7**
P < 0.02 P < 0.001
P < 0.01 P < 0.001
P < 0.02 P <0.002
30 150 750 Factorial analysis Linear zinc effect Quadratic zinc effect
** Significantly different from control, P < 0.01.
ZlNC
TOXICITY
IN
PREGNANT
TABLE EXPERIMENT
5
SHEEP
3
1: DISTRIBUTION OF VIABLE AND NONVIABLE LAMBS INDICATED CONCENTRATIONS OF DIETARY
AMONG ZINC”
EWES RECEIVING
Diet (mg Zn/kg)
Viable
Number of lambs nonviable”
Total
30 150 750
11 19 3
3 4 17
14 23 20
THE
” x2 test: P < 0.001. h A nonviable iamb is defined as a lamb which was aborted, stillborn, or died for any reason within 7 days of birth.
concentration of this group was 6.71 + 0.83 (10) compared to 1.05 + 0.04 (12) in the control group [mean 2 SE(n)]. This difference is significant at the 0.1% level of probability. Reproductive performance was severely impaired in group 7502. Numerous abortions and perinatal deaths occurred. The distribution of abortions, stillbirths, and perinatal deaths among treatment groups is shown in Table 3. For the purpose of this table, a nonviable lamb is defined as a lamb which was aborted, stillborn, or died for any reason within 7 days of birth. The distribution is heavily biased towards group 7502 and a x2 test indicates that such a distribution would be unlikely to occur by chance (P < 0.001). The cause of death in these lambs could not be determined. Lambs born alive were weak and unable or unwilling to stand or suckle; some trembled violently or were ataxic. Deaths, preceded by convulsions, usually occurred within 48 hr, although in one case the terminal symptoms were delayed until 4 days of age. Two lambs (twins) which were bottle fed with cow’s milk survived until 5 weeks of age before they collapsed with symptoms similar to those observed in lambs which had died at birth. Of 20 lambs conceived in this group, only one survived more than 5 weeks of postnatal life. Two findings common to all of the nonviable lambs in group 7502 were very high tissue zinc concentrations and very low tissue copper concentrations (see Table 4). Lambs from group 7502 had liver copper concentrations approximately onetenth of that in control lambs (P < 0.02). In lambs from group 15OZ, there was a TABLE EXPERIMENT
1: CONCENTRATION
OF COPPER
4
AND
ZINC IN LIVER OF NONVIABLE
LAMBS”
Diet tmg Zn/kg)
Number of lambs
Liver Cu b-m&z)
Liver Zn Wk)
30 150 750
3 4 12
79.8 f 34.6 161.3 r 17.7 8.4 2 1.7’
251.9 + 20.6 915.8 -t 1843* 2044 It 359.s***
” Lambs were born to ewes maintained for approximately taining the indicated concentration of zinc. * Significantly different from control: P < 0.05. **= Significantly different from control: P < 0.001,
110 days prior to parturition on diets con-
FIG. 1. Experiment 1: Bone A was taken from a control lamb (6.5 kg) which died during birth. Death was attributed to dystocia. Bone B was t aken from a lamb born to a ewe receiving 750 mg Zn/kg diet. The lamb weighed 3.0 kg at birth and died from indeterminate cause 48 hr after birth.
ZINC
TOXICITY
IN PREGNANT
7
SHEEP
trend toward higher liver copper concentrations but the effect was not statistically significant. The zinc concentration of lamb liver was significantly increased by both levels of zinc supplementation. A dietary zinc concentration of 150 mg/kg resulted in a threefold increase in liver zinc concentration (P < 0.05) and a dietary zinc concentration of 750 mg/kg caused a lo-fold increase (P < 0.001). Nonviable lambs from the other two groups died from a variety of causes unconnected with the dietary treatment. Radiographs of nonviable lambs from group 7502 revealed lines of arrested growth in the long bones. These lines were not observed in lambs from the other two groups (Fig. 1). Severe renal damage was observed in one nonviable lamb from group 7502. Lesions included hyperemia and extravasation of blood cells in the medulla and almost complete ischemic necrosis of the cortex with necrosis of the arcuate and interlobular arteries. Experiment 2 In view of the findings from Experiment 1 that a dietary zinc concentration 750 mg/kg induced copper deficiency, a second experiment was undertaken
of to
2.5 9a x $
*.O
2
4
6
0 Day
10
12
No.
FIG. 2. Feed consumption (kg/sheep/day) in pregnant + 2.5 mg Cu/kg (0). 750 mg Zn + 2.5 mg Cu/kg (0). analysis could be performed on these data.
14
5
5
7 9 Week
11
13
ewes maintained on diets containing 30 mg Zn 750 mg Zn + 10 mg Cuikg (0). No statistical
8
CAMPBELL
AND
MILLS
determine whether provision of extra dietary copper would prevent the high lamb mortality in sheep maintained on high zinc diets during pregnancy. Feed consumption (Fig. 2) was identical in all groups during the first 7- 10 days of the experiment. Thereafter, feed intake in both the high zinc groups declined regardless of copper intake to a level about 20% below that of the control group and remained at that level for the duration of the experiment. Efficiency of feed utilization was reduced by about 50% in both of the zinc-supplemented groups. At the beginning of the experiment, copper status as judged by the concentration of copper and the activity of monoamine oxidase and ceruloplasmin in plasma, was identical in all three groups (Fig. 3). However, by Day 74 plasma copper concentration in group 7502 (750 mg Zn + 2.5 mg Cu/kg diet) was little more than half that of the control group (P < 0.05) and falling rapidly. In contrast plasma copper concentration in group 7502 + Cu (750 mg Zn + 10 mg Cu per kg diet) remained similar to that of the control group. The rise in plasma copper concentration just after the start of the experiment is difficult to explain. Since it occurred only in the zinc-supplemented groups, it appears to be caused by the zinc content of the diet. Although supplementary copper prevented the decline in the copper status of ewes receiving 750 mg zinc/kg diet it did not alleviate the effect of zinc on reproductive performance. Table 5 shows the distribution of viable and nonviable lambs among the three groups. The distribution was tested using the Fisher exact probability test because there were too few animals to allow the use of the more usual x2 test. The proportion of nonviable lambs in group 7502 and group 7502 + Cu was
FIG. 3. Plasma copper concentration, (mg/liter) in pregnant ewes maintained on diets mg Zn + 2.5 mg Co/kg (0), 750 mg Zn + 2.5 mg Cu/kg ( l ), 750 mg Zn + IO mg Cu/kg (0). the mean and standard error of six animals unless otherwise indicated. Figures adjacent indicate the number of animals if less than six. Significantly different from control (0)
containing 30 Each point is to the points “P < 0.05.
ZINC
IN
TOXICITY
PREGNANT
TABLE EXPERIMENT
2:
DISTRIBUTION INDICATED
OF VIABLE CONCENTRATIONS
AND
5
NONVIABLE
LAMBS
OF DIETARY
30 750 750
1
2 3
AMONG
COPPER
Diet mg Zn/kg
9
SHEEP
EWES
AND
RECEIVING
THE
ZINC”
Number of lambs mg &/kg
Viable 9 0 1
2.5 2.5 10.0
” Ewes were maintained on the indicated diets for approximately probability test: Comparison, 1-2, l-3: P < 0.001.
Nonviable 2 12 13
Total 11 12 14
100 days prior to parturition.
Exact
significantly greater than would be expected to occur by chance. The concentrations of copper and zinc in the livers of these lambs are presented in Table 6. Copper concentration in the liver of lambs from group 7502 was reduced by 50% relative to the control group (P < 0.05). The depression of liver copper concentration was less severe than that resulting from 750 mg Zn/kg diet in the previous experiment. The group mean was distorted however by three lambs with liver copper concentrations similar to those of the control lambs. Most of the lambs in group 7502 had hepatic copper concentrations between 4.0 and 8.0 mg/kg. In lambs from group 7502 + Cu, liver copper concentration was not significantly different from that of the control group. Hepatic zinc concentration in lambs from the zinc-supplemented groups was approximately 10 times the control level. As in Experiment 1, lines of arrested growth were observed in the bones of nonviable lambs from both of the zinc-supplemented groups. DISCUSSION
Cadmium at a concentration of 3.0 mg/kg diet had no detectable effect on copper status or breeding performance. This is consistent with the work of Mills and Dalgamo (1972) who found that 3.5 mg Cd/kg diet had no effect on hepatic copper concentration in pregnant ewes and only depressed liver Cu of their offspring when Cd-containing solid diet rather than ewe milk constituted the primary source of food. The effect of excess dietary zinc on the copper metabolism of the pregnant ewe was more severe than was anticipated. Ott et ul. (1966a) reported major effect on copper status of sheep only when dietary zinc concentration exceeded 1000 TABLE EXPERIMENT TO EWES
2:
6
CONCENTRATION RECEIVING THE
OF COPPER AND ZINC IN THE LIVER OF NONVIABLE INDICATED CONCENTRATIONS OF DIETARY COPPER
Diet
mg Znlkg
mg Culkg
1
30 750 750
2 3
‘I 2.5 2.5 10.0
Liver Cu
Liver
27.0 2 5.9 13.0 t 2.9” 18.3 rt 3.9
262.2 5 151.7 3265 k 5g7”*’ 2456 k 381*“”
” Figures in parentheses = number of animals. * Significantly different from group 1: P < 0.05. *** Significantly different from group 1: P < 0.01.
LAMBS BORN AND ZINC
Zn
-
(3)” (10) (8)
10
CAMPBELL
AND
MILLS
mg/kg. These workers however were using nonpregnant sheep and the copper content of their diet, although not stated, may have been greater than that used in this study judging from the ingredients used. Copper deficiency in our ewes was reflected by an equally severe copper deficiency in the fetus and neonate. Copper deficiency, in both ewe and lamb, was prevented by copper supplementation but in spite of the substantially higher copper content of the diet of their dams, liver copper in lambs from group 7502 + Cu was no different from that of the control group. The high zinc concentration of the diet therefore prevented the increase in liver copper concentration which might have been expected in the livers of lambs whose dams were receiving four times the dietary copper concentration of the control sheep. Many of the lambs had liver copper concentrations around 4 or 5 mg/kg which is within the range in which swayback might ,be expected to occur (Mills and Williams, 1962). However, examination of sections from the spinal cord and red nucleus (Mills and Fell, 1960) of nonviable lambs revealed no demyelination and no degeneration of motor neurones. The significant quadratic effect of zinc on copper-dependent parameters in ewes suggests that a moderate level of zinc supplementation is in some way beneficial to the copper status of the sheep. Thus plasma copper concentration, plasma ceruloplasmin activity, and plasma amine oxidase activity were all higher in group 15OZ than in the control group. Subsequent work with the offspring of these sheep maintained on the same diet (manuscript in preparation) has shown a similar pattern of copper status. Lambs receiving 150 mg Zn/kg diet tended to accumulate hepatic copper more readily than control lambs. Under these conditions however, the total concentration of copper in the liver or plasma may not be a true reflection of the functional copper status of the animal. The large increases in tissue zinc concentration observed in this study were not in complete accord with the findings of other workers. In this study plasma zinc concentration rose progressively from the time the sheep were first exposed to the high zinc diets and at parturition some animals had plasma zinc concentrations of up to 12 mg/liter. Ott et al. (1966~) found that diets containing up to 1000 mg Zn/kg caused plasma zinc concentrations to double and large increases in plasma zinc concentration occurred only at dietary zinc concentrations in excess of 1000 mg/kg. The sheep used by Ott et al. were however of a different breed to those used in this experiment, were of a different age, weight, and sex, and were not pregnant. Any of these factors may modify the change in plasma zinc concentration produced by excess dietary zinc. High plasma zinc concentrations also developed in our lambs. Two lambs born in group 7502 when sampled within 24 hr of birth were found to have plasma zinc concentrations of 5 mg/liter. The effect of this level of zinc in plasma on the health of the lambs is not known. Adverse effects of excess dietary zinc on feed intake and EFU have been reported-although at much higher dietary concentrations than those employed in these experiments. Ott et al. (1966a, b, c) reported reduced feed consumption and EFU in sheep receiving dietary zinc concentrations of 1000 mg/kg or more. They attributed the reduced feed intake to the unpalatability of diets containing high concentrations of zinc salts and the decline in EFU to a decline in microbial activity in the rumen. In the study reported here, the food intake of sheep receiving high zinc rations remained normal for 7 to 10 days before declining. It seems
ZINC
TOXICITY
IN
PREGNANT
SHEEP
II
likely, therefore, that the low feed intake was due not to the taste of the diet but rather to some systemic effect of zinc. Rumen metabolism was not investigated but microscopic examination of rumen contents indicated that rumen microflora was identical in all the sheep regardless of their zinc intake. The effect of zinc on EFU was much greater in Experiment 1 than in Experiment 2. No explanation can be offered for this finding. The most important finding in this study was the effect of high dietary zinc concentrations on the viability of lambs. This effect has not been previously reported in sheep. Several hypothesis can be advanced to explain the low viability of the lambs. First, the very high concentrations of zinc in the tissues may have been toxic to the fetus. In particular, the kidneys may have been severely affected. It is known that in suckling lambs exposed to high dietary zinc concentrations, the kidneys accumulate large amounts of zinc which leads to severe renal damage (Davies et ~11.. 1977). The very limited evidence available from this study suggests that the kidneys of the fetus may be similarly susceptible to damage by high concentrations of zinc. The lamb from which the single kidney sample was obtained was dying with symptoms identical to those observed in other nonviable lambs when it was killed, specifically for histological study. The extensive renal damage observed in this lamb must have contributed to its poor condition and in the light of the findings of Davies et al. (1977) it seems possible that the debilitation and death of other nonviable lambs was due in part to renal damage caused by high concentrations of zinc in the kidney. Unfortunately, no other samples could be obtained for microscopic examination and no definite conclusion can be reached. A second possibility is that the depressed feed intake of the ewes may have caused the high mortality rate among lambs. Inadequate nutrition of ewes during late pregnancy leads to low birthweight and increased perinatal mortality in lambs (Thomson and Thomson, 1949; Hight and Jury, 1970). Thomson and Thomson found that when the dry matter intake of pregnant ewes was restricted to about 50% of normal, perinatal lamb mortality approached 70%. The reduction in feed intake induced by excess dietary zinc in the present study was much less than that imposed by Thomson and Thomson and it is therefore unlikely that reduced feed intake was responsible for the reduced viability of lambs. However, the weight changes in pregnant ewes reported by Thomson and Thomson were similar to those observed in the experiments described here. The very low rate of weight gain in pregnant ewes receiving 750 mg Zn/kg was presumably due therefore to the combined effect of low feed intake and the reduced EFU resulting from this treatment. This resulted in a degree of malnutrition comparable with that observed by Thomson and Thomson which may have contributed to the reduced viability of lambs. Discontinuous growth of lambs in utero is indicated by the transverse bands in the bones. It has been suggested that these “lines of arrested growth” may be produced by disturbance of fetal nutrition (Harris, 1923, 1931). Similar lines can be produced in the bones of growing rats by subjecting them to periods of severe malnutrition (Park and Richter, 1953) and it is tempting, therefore, to interpret the lines in lamb bones as evidence of fetal malnutrition. Toxoplasmosis has also been suggested as a cause of lines of arrested growth in lambs (H. M. ROSS, North of Scotland College of Agriculture, unpublished observations) but toxoplasmosis was not detected in any of these animals.
12
CAMPBELL
AND
MILLS
Although the mechanisms involved are not clear, it is evident that the pregnant sheep is vulnerable to concentrations of dietary zinc which have little effect on the nonpregnant animal. Whereas Ott el al. (1966a, c) found signs of toxicity at dose rates of around 50 mg/kg body weight per day, our sheep suffered almost complete reproductive failure at a dose rate which declined from 20 mg/kg body weight per day at the start of the experiment to around 10 mg/kg/day during the last 10 weeks of pregnancy. Similar findings have been reported in rats. Campbell and Mills (1972) reported that a diet containing 300 mg Zn/kg had no major effect on the health of male rats, while Kumar (1976) found a significant increase in fetal resorption in pregnant rats receiving a diet containing 150 mg Zn/kg. This corresponds to a zinc intake of approximately 10 mg/kg body weight per day. Thus a zinc intake of around 10 mg/kg body weight per day, which is considerably less than that previously thought to be harmful, can have a serious effect on the course of pregnancy in two species. Until more is known about the effects of zinc on reproduction, zinc supplementation of livestock should be undertaken with this limitation in mind, Furthermore, it should be noted that herbage with concentrations of zinc up to and exceeding 750 mg/kg has been found in the vicinity of some industrial complexes (Goodman and Roberts, 1971; Lagerwerffet al., 1972; Mills and Dalgarno, 1972). Although the forms in which this zinc exists in or on the herbage have rarely been identified and its availability to animals may vary, this study seems to emphasize that pregnant animals may be at risk from such situations. ACKNOWLEDGMENTS We are grateful to Rio Tinto Zinc Services Limited, Bond Street, Bristol for a research grant in aid of this work. We are also grateful to Dr. B. F. Fell and Mr. R. Boyne for preparation and examination of histological samples, to Mr. G. Wenham for radiography and surgical procedures, and to Mr. S. Mann for examination of rumen contents. Thanks are also due to Mr. A. C. Dalgarno for his advice and assistance, to Mrs. L. Miller for technical assistance, and to Mr. G. Calder for management of the animals.
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