effects of dietary copper and copper: zinc ratio on rainbow trout salmo gairdneri
TRANSCRIPT
Aquoculture, 27 (1982) 111-119 111 Elsevier Scie.ntific Publishing Company, Amsterdam - Printed in The Netherlands
EFFECTS OF DIETARY COPPER AND COPPER: ZINC RATIO ON RAIN- BOW TROUT SALMO GAIRDNERI
DAVID KNOX, COLIN B. COWEY and JOHN W. ADRON
Institute of Marine Biochemistry, St Fittick’s Road, Tarry, Aberdeen ABl 3RA (Great Britain)
(Accepted 24 August 1981)
ABSTRACT
Knox, D., Cowey, C.B. and Adron, J.W., 1982. Effects of dietary copper and copper :zinc ratio on rainbow trout Salmo goirdneri. Aquaculture, 27: 111-119.
Purified diets with 16 and 150 mg supplemental copper/kg with dietary copper : zinc ratios of 1: :L and 1: 4 were fed for 20 weeks to rainbow trout of mean initial weight 15 g. No gross pathologies were noted in any group of fish; growth and feed conversion were the same in all groups of trout. Plasma zinc levels were positively correlated with dietary zinc intake but dietary copper level had no effect on plasma copper. Hepatic levels of copper and zinc were also related to the dietary intake of the respective minerals. The dietary copper :zinc ratio caused some small changes in the plasma and hepatic levels of a few minerals, but no evidence was found to suggest any zincrcopper antagonism in rainbow trout. The activity of the copper, zinc metahoenzyme superoxide dismutase in liver was unaffected by dietary copper or zinc intake.
INTRODUCTION
Copper and zinc are essential dietary elements for fish (La11 and Bishop, 1977) and\ optimal dietary intakes have been established for a number of species (Ogino and Yang, 1978,1979,1980; Murai et al., 1981). Little is yet known, however, about the interaction of these elements in the diet of fish, even though there are indications that Zn-Cu antagonism may occur in rain- bow trout particularly under conditions where absorption of Zn may be im- paired (Spinelli et al., 1979). Whether such an impairment may be overcome by using increased dietary concentrations of Zn and what effects such increased concentrations may have on tissue levels of other minerals has so far received only limited attention (Jeng and Sun, 1981).
The ob#ject of the present experiment was to determine the effect of high dietary Zn levels on growth, tissue mineral levels, activity of the cupro-zinc metalloenzyme superoxide dismutase (EC. 1.15.1 .l) and other enzymes in rain- bow trout. Because of the preliminary indications of Zn-Cu antagonism in rainbow trout already referred to, dietary Cu : Zn ratios of either 1: 1 or 1: 4
0044-8486~82/0000-00001$02.7 5 0 1982 Elsevier Scientific Publishing Company
112
were used. Two dietary Cu levels, one moderate (15 mg/kg) and one high (150 mg/kg) were employed giving four dietary Zn levels (15, 60,150 and 600 mg/
kg).
METHODS
Rainbow trout of mean weight approximately 15 g were obtained from Dornoch Fisheries, Dornoch, Great Britain, and were evenly distributed be- tween eight circular glassfibre tanks of diameter 1 m, depth 0.6 m and con- taining 400 1 of water (30 fish/tank). The water from the tanks was partially recirculated with a constant “bleed in” of fresh tap water from the City of Aberdeen domestic supply (75 l/h per tank). The water contained 4.1 pg Cu and 1.3 pg Zn/l. The tanks were housed in an aquarium room with an ambient temperature averaging 15°C.
The composition and mineral content of the basal diet are shown in Table I. The dry ingredients were thoroughly mixed and made to a moist paste by the addition of distilled water (300 ml containing 15 or 150 mg supplemental copper/kg dry diet (as CuS04) and sufficient ZnS04 to give dietary copper : zinc
TABLE I
Composition of the basal diet given to rainbow trout
Ingredient g/kg dry diet
Casein 550.00 Pre-cooked starch 200.00 Capelin oil 120.00 Vitamin mix’ 28.00 Mineral mix2 50.00 (Y -cellulose 46.03 Ascorbyl palmitate 0.40 BHA mix3 0.50 Cystine 5.00 Rovimix4 0.074 Measured copper level 2.5 mg/kg dry diet Measured zinc level 22.3 mg/kg dry diet
i Supplied (/kg dry diet): riboflavin 200 mg, pyridoxine hydrochloride 40 mg, nicotinic acid 800 mg, calcium pantothenate 280 mg, myo-inosito14 g, biotin 6 mg, pteroylmono- glutamic acid 15 mg, p-aminobenzoic acid 400 mg, choline chloride 8 g, ascorbic acid 2 g, DLa-tocopheryl acetate 400 mg, menaphthone 40 mg, cyanocobalamin 90 rg, thiamin hydrochloride 50 mg. “Supplied (/kg dry diet): Ca(H,PO,) H,O 34.45 g, CaCO, 2.6 g, MgCO, 4.55 g, FeSO, * 7H,O 1.5 g, KC1 2.5 g, NaCl 4 g, AI,(SO,), 16H,O 0.01 g, MnSO, * 4H,O 0.18 g, KI 0.01 g, coso, l 7H,O 0.005 g. ’ Butylated hydroxyanisole 200 g/l, propylgallate 60 g/l and citric acid 40 g/l in propylene glycol. 4 Supplied (/kg dry diet): vitamin A 37 000 units, vitamin D, 7400 units (obtained from Roche Products Ltd, London, Great Britain).
113
ratios of 1: 1 or 1: 4). The paste was then extruded through a commercial mincing m,achine from which the cutting blades had been removed. The resultant qpaghetti-like diet (3 mm diameter) was then freeze-dried and broken into small pieces for feeding. Duplicate tanks of fish were given one of the four test diets at a level of 20 g diet/kg biomass of fish per day (6 days/week). The trout were weighed every 4 weeks throughout the experimental period and the amount of food given to each tank was adjusted in accordance with the measured biomass.
At the end of the experimental period 10 fish from each tank, i.e. 20 fish per dietary treatment, were selected by random methods and samples of blood and liver were obtained. Blood packed cell volume was determined as described by Blaxhall and Daisley (1973). Haemoglobin levels were measured using a commercial assay kit with methaemoglobin as a standard (Sigma Chemical Company.: Poole, Great Britain). Plasma aspartic aminotransferase (EC. 2.6.1.2) (GOT) and alanine aminotransferase (EC.2.6.1.1) (GPT) were measured under optimal assay conditions for rainbow trout as described by D’Appolonia and Anderson (1979). Mineral analyses of plasma and liver were carried out as de- scribed by Cowey et al. (1977).
Samples of liver from each fish were homogenised in nine volumes of 0.05 M potassium phosphate buffer at pH 6.5, containing 10e4 M EDTA and TritonX 100 (10 g/l). The homogenate was centrifuged for 3 min at 12 500 X g. The supernatant obtained was used to determine the liver superoxide dismutase (SOD) activity by the method of Heikkila et al. (1976).
The statistical significance of the experimental data and correlation coeffi- cients were determined as described by Fisher (1960).
RESULTS
No gross pathologies were observed in any of the experimental groups of fish; good growth was obtained on all treatments, live weight increasing about 10 fold over the 20-week period (Table II). Mortalities were very low; the two deaths th;at occurred seeming unconnected with dietary treatment. Food con- version rate was good and feed efficiency ratios were also unaltered by dietary copper or zinc intake (Table II).
In all groups of trout the hepatosomatic index was virtually the same (Table III). However, both packed cell volume and haemoglobin level were greater in trout given diet 1 (15 mg Cu/kg diet - Cu : Zn, 1: 1) than in fish given the same dietary copper level but with a Cu: Zn ratio of 1: 4 (P < 0.05) (Table III).
The concentrations of several minerals in the plasma of trout given the experimental diets are shown in Table IV. Plasma zinc levels were positively correlated with dietary zinc intake (r 0.48, n 98, P < 0.001). Plasma copper levels remained constant although fish given diet 4 (150 mg supplemental copper/kg diet, Cu : Zn ratio 1: 4) contained significantly more plasma copper than trout given the other diets (P < 0.06).
Plasma concentrations of calcium, phosphorus and potassium were unaltered
114
TABLE II
Effect of dietary copper and zinc on growth, feed efficiency ratio and mortalities in rain- bow trout
Supplemental Dietary copper level Cu:Zn (mg/kg)
Initial InitiaI Final number mean mean of fish weight weight
(g) (9)
Feed Mortalities efficiency ratio
Diet 1 15 1:l 30 15.4 157.7 1.34 1 15 1:l 30 15.0 149.5 1.37 0
Diet 2 15 1:4 30 14.9 136.0 1.29 0 15 1:4 30 15.4 164.5 1.41 0
Diet 3 150 1:l 30 15.0 151.6 1.36 1 150 1:l 30 15.6 144.0 1.35 0
Diet 4 150 1:4 30 15.7 144.7 1.29 0 150 1:4 30 15.8 156.6 1.38 0
TABLE III
Effect of dietary copper and zinc on hepatosomatic index, packed cell volume and haemoglobin levels in rainbow trout
Diet’ Hepatosomatic indexa Packed cell volume ( W) Haemoglobin (g/l)
Mean’ SE3 Mean SE Mean SE
1 1.27 0.05 46.5 2 1.22 0.07 38.7 3 1.33 0.06 41.8 4 1.37 0.04 43.3
a For details see Table II. ’ (Liver weight (g)/body weight (g)) X 100. ‘Mean and SE for 20 fish/dietary treatment.
1.0 95.5 3.0 1.1 85.7 2.6 1.2 91.0 2.3 1.3 88.3 2.7
by the dietary treatments used. In trout given 15 mg copper/kg diet and a Cu : Zn ratio of 1: 1, plasma sodium (P < 0.001) was lower and magnesium (P < 0.01) higher than in trout given the same dietary copper level but with a Cu : Zn ratio of 1: 4. No analogous changes in plasma magnesium or sodium were found in the groups given 150 mg supplemental copper/kg diet.
Liver copper levels were greater than those of the other trace elements (zinc and iron) studied (Table V). Dietary zinc level did not affect liver copper concentrations in this experiment, but increasing the dietary copper supple- ment to 150 mg/kg led to much higher (by 2-3 fold) copper levels in the liver. Liver zinc concentrations were related to dietary zinc intake (being greater in trout given diet 2 than in those given diet 1 and higher in trout given diet 4 than in those given diet 3) and apparently little affected by dietary copper level.
TA
BL
E
IV
Th
e ef
fect
of
die
tary
cop
per
and
zin
c in
tak
e on
pla
sma
min
eral
lev
els
in r
ain
bow
tr
out
Die
t’
Ca
Mg
P
Na
K
Zn
“_
_ U
,u
Fe
(mm
ol/i)
(m
mol
/l)
(mm
ol/l)
(m
mol
/l)
(mm
ol/l)
(m
mol
/l)
(rtm
oi/i
) (~
moi
/l)
Mea
n’
SE
1 M
ean
S
E
Mea
n
SE
M
ean
S
E
Mea
n
SE
M
ean
S
E
Mea
n
SE
M
ean
S
E
1 3.
65
0.06
0.
87
0.02
4.
54
0.13
16
1.00
1.
60
3.55
0.
43
0.30
0.
02
14.0
7 0.
63
3.55
0.
43
2 3.
63
0.11
0.
79
0.02
4.
49
0.14
16
7.01
1.
37
3.98
0.
30
0.36
0.
01
14.0
6 0.
58
3.98
0.
30
3 3.
61
0.11
0.
83
0.02
4.
58
0.13
16
2.81
1.
72
3.77
0.
35
0.34
0.
01
14.2
4 0.
77
3.77
0.
35
4 3.
77
0.10
0.
86
0.02
4.
53
0.10
16
2.23
1.
84
3.89
0.
23
0.43
0.
02
17.1
1 0.
96
3.89
0.
23
1 For
det
ails
see
Tab
le I
I.
lMea
n
valu
es a
nd
stan
dard
err
ors
for
20 f
ish
/tre
atm
ent.
TA
BL
E
V
Eff
ect
of d
ieta
ry c
oppe
r an
d zi
nc
on l
iver
min
eral
lev
els
(mm
ol/k
g w
et t
issu
e) i
n r
ain
bow
tr
out
Die
t’
Ca
Mg
P
Na
K
cu
Mea
n”
SE
2 M
ean
S
E
Mea
n
SE
M
ean
S
E
Mea
n
SE
M
ean
S
E
Zn
F
e
Mea
n
SE
M
ean
S
E
1 1.
46
0.12
6.
53
0.23
70
.46
3.67
29
.20
1.31
68
.72
2.65
1.
01
0.09
0.
28
0.01
0.
56
0.05
2
1.63
0.
15
7.27
0.
20
72.4
9 3.
25
34.6
3 2.
06
79.7
1 2.
40
0.95
0.
09
0.34
0.
01
0.50
0.
05
3 1.
74
0.09
7.
12
0.17
74
.10
3.18
31
.27
1.01
73
.73
2.17
2.
35
0.15
0.
32
0.01
0.
52
0.05
4
1.27
0.
11
7.68
0.
19
74.9
7 3.
58
34.3
7 0.
70
78.7
4 1.
56
2.51
0.
20
0.37
0.
01
0.55
0.
05
1 For
det
ails
see
Tab
le I
I.
2Mea
n v
alu
es a
nd
stan
dard
err
ors
for
20 f
ish
/tre
atm
ent.
116
TABLE VI
The effect of dietary copper and zinc intake on the activity of plasma aspartic amino- transferase (GOT), plasma aianine aminotransferase (GPT) and liver superoxide dismutase (SOD) in rainbow trout
Diet’ Plasma GOT (U/l) Plasma GPT (U/l) Liver SOD (units/g wet tissue)3
Mean” SE Mean SE Mean SE
1 182.4 9.3 6.2 0.6 661.5 18.1 2 197.7 10.8 6.4 0.7 680.9 16.4 3 189.9 9.7 5.5 0.7 658.8 14.2 4 172.1 9.9 6.6 1.3 640.7 7.9
! For details see Table II. *Mean values and standard errors for 20 fish/treatment. 3 One unit of SOD activity was defined as the amount of enzyme needed to obtain 50% inhibition of the substrate auto-oxidation.
In the fish given diets 1 and 2 containing 15 mg supplemental copper/kg, liver calcium and phosphorus were unaffected by the dietary Cu : Zn ratio; increased hepatic levels of magnesium (P < 0.02), sodium (P < 0.05) and potassium (P < 0.01) were, however, present in trout given diet 2 with a Cu : Zn ratio of 1: 4 (Table IV). In those trout given diets 3 and 4 containing 150 mg supplemental copper/kg, liver levels of magnesium (P < 0.05) and sodium (P < 0.02) increased and hepatic calcium decreased (P < 0.01) in diet 4 with a Cu : Zn ratio of 1: 4. Liver concentrations of phosphorus and potassium were unaffected by increases in dietary zinc.
Measurements of superoxide dismutase activity (SOD) in liver showed that neither dietary copper nor zinc intake influenced the activity of this enzyme (Table VI). Similarly no changes in plasma aspartate aminotransferase (GOT) and.alanine aminotransferase (GPT) activities were found.
DISCUSSION
The absence of any gross pathologies or significant reduction in growth over the 20 week feeding period contrasts with the findings of Murai et al. (1981). These authors found that in channel catfish Ictaluruspunctutus, diets contain- ing more than 17.5 mg copper/kg depressed growth and feed conversion. Similarly a reduction of growth rate in common carp Cyprinus carpio was found when diets containing 294 or more mg zinc/kg diet were given (Jeng and Sun, 1981). Although packed cell volume and haemoglobin levels were significantly reduced in those trout given diet 2 (cf. diet l), the values obtained for all four dietary treatments fell within the range given by McCarthy et al. (1973,1975).
Hepatic levels of copper and zinc were related to the dietary intake of these
117
minerals. In all treatments liver copper concentrations exceeded those of zinc despite the fact that diets 2 and 4 contained much more zinc than copper. This may be due to a high turnover rate of zinc in rainbow trout, although it is known that copper tends to accumulate in the liver where it can be indicative of the onset of pathologies. Ogino and Yang (1978) in experiments designed to determine the dietary zinc requirement of fingerling rainbow trout (1.5.-3.4 g live weight) also observed higher concentrations of copper than zinc in the liver. These authors also found that increasing the dietary zinc level from 1 to 30 mg zinc/kg diet had no effect upon hepatic zinc levels, but increased the zinc concentrations in vertebrae three times and more than doubled the level in the intestine. Variable effects of increasing dietary zinc intake have been reported in other species. Ogino and Yang (1979) found greatly increased levels of zinc in the hepatopancreas, intestine and kidney of carp Cyprinus carpb when dietary zinc was increased from 1 to 100 mg/kg diet. Jeng and Sun (1981), on the other hand, found that diets containing up to 1972 mg zinc/kg fed to common carp for 8 weeks had no significant effect on hepatopancreatic zinc levels, However, zinc did accumulate first in the digestive tract, then in the skeletal tissue. When the skeletal tissues were “saturated” with zinc, the excess amount was then stored in skin and muscle. Thus in rainbow trout, zinc may be accumulated in other tissues except liver, e.g. vertebrae and intestine.
Murai et al. (1981) found that increasing dietary copper from 1.5 to 33.5 mg/kg diet elevated liver copper from 0.08 to 0.15 mmol/kg dry weight liver in channel catfish. The latter concentration was found in fish that were suffer- ing a marked depression of growth and feed conversion rate. Comparison of these values with those found in trout from the present experiment given a diet containing approximately the same dietary Cu level (diet 1) indicates much higher liver copper levels in trout (3.39 mmol copper/kg dry liver cf. 0.15 mmol copper/kg dry liver). This suggests that trout are markedly less sensitive to dietary copper level than are channel catfish.
It is known (Bremner et al., 1976) that supplementing the diet of lambs with 420 mg zinc/kg diet lowers hepatic copper concentrations with a concomitant reduction in the extent of liver damage caused by copper toxi- cosis. By contrast, supplementing rat diets with 560 mg zinc/kg (Kang et al., 1977) or 1000 mg zinc/kg diet (Chvapil et al., 1974) produced no alterations in liver copper levels. Similarly, in the present experiment, at the dietary copper levels used, increasing the Cu : Zn ratio had no effect on hepatic copper levels. There were no signs of any zinc- copper antagonism, and alteration of tissue levels of other elements as a consequence of high zinc in- takes was slight. Consequently under conditions where a reduction in bio- availability of dietary zinc is suspected, as for example when soybean proteins are inclu’ded in the diets (Spinelli et al., 1979), then elevated levels of supple- mentary zinc may, in the case of rainbow trout, be used to prevent such an occurrence.
The activities of the two plasma aminotransferases were very similar in all
118
four groups of trout. Suttle and Mills (1966) showed that in pigs the activity of serum GOT was greatly increased when diets containing 460 mg copper/kg diet were fed. Supplementing these diets with 500 mg zinc/kg diet produced normal serum GOT activities. It is possible that in the present experiment the normal GOT and GPT levels found were produced because the levels of zinc in the diets were sufficient to prevent any copper toxicity. ’
In all groups of fish the hepatic SOD activity was the same. The assay sys- tem used measured not only the copper-zinc enzyme, but also the manganese- containing superoxide dismutase known to be present in trout tissues (Knox et al., 1981). The values obtained for the SOD activity were no different from values previously found in livers of trout fed a commercial diet and using the same assay system and conditions (Knox, unpublished results). The absence of any changes in enzyme activity further supports the idea that neither copper nor zinc metabolism had been affected by the dietary mineral intake. Evidence has been found to show that in rainbow trout the activity of hepatic SOD (both copper-zinc and manganese enzymes) was reduced when liver levels of their respective mineral components were reduced (Knox et al., 1981).
Overall, the data suggests that rainbow trout (in the size range 15 g live weight upward) can tolerate relatively high dietary levels of copper and zinc (analysis of diet 4 showed mineral levels of 178 mg copper and 683 mg zinc/ kg dry diet) at water concentrations of 1.3 yg Zn and 4.1 pg Cu/l. Although the copper : zinc ratio in the diet caused some small changes in certain plasma and liver mineral levels in the present experiment, neither dietary copper nor zinc levels nor copper: zinc ratio had any effect on growth or health of rain- bow trout.
REFERENCES
Blaxhail, P.C. and Daisley, K,W., 1973. Routine haematological methods for use with fish blood. J. Fish Biol., 5: 771-781.
Bremner, I., Young, B.W. and Mills, C.F., 1976. Protective effect of zinc supplementation against copper toxicosis in sheep. Br. J. Nutr., 36: 551-561.
Chvapil, M., Peng, Y.M., Aronson, A.L. and Zukoski, C., 1974. Effect of zinc on lipid peroxidation and metal content in some tissues of rats. J. Nutr., 104: 434-443.
Cowey, C.B., Knox, D., Adron, J.W., George, S. and Pirie, B., 1977. The production of renal calcinosis by magnesium deficiency in rainbow trout (Salmo gairdneri). Br. J. Nutr., 38: 127-135.
D’Apollonia, S. and Anderson, P.D., 1980. Optimal assay conditions for serum and liver glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and sorbitol dehydrogenase from the rainbow trout Salmo gairdneri. Can. J. Fish. Aquat. Sci., 39: 163-169.
Fisher, R.A., 1950. Statistical Methods for Research Workers. Oliver and Boyd, Edinburgh, 354 pp.
Heikkila, R.E., Cabat, F.S. and Cohen, G., 1976. In vivo inhibition of superoxide dis- mutase in mice by diethyldithiocarbamate. J. Biol. Chem., 251: 2182-2185.
Jeng, S.S. and Sun, L.T., 1981. Effects of dietary zinc levels on zinc concentrations in tissues of common carp. J. Nutr., 111: 134-140.
119
Kang, H.K., Harvey, P-W., Valentine, J.L. and Swenseid, M.E., 1977. Zinc, iron, copper and mag.nesium concentrations in tissues of rats fed various amounts of zinc. Clin. Chem., 23: 1834-1837.
Knox, D., Cowey, C.B. and Adron, J.W., 1981. The effect of low dietary manganese intake on rainbow trout (Salmo gairdneri). Br. J. Nutr., 46: 495-501.
Lall, S.P. and Bishop, F.J., 1977. Studies on mineral and protein utilization by Atlantic salmon (Salmo salar) grown in seawater. Canadian Fish. Mar. Serv. Tech. Rep. NO. 688.
McCarthy, ‘D.H., Stevenson, J.P. and Roberts, M.S., 1973. Some blood parameters of the rainbow trout (Salmo gairdneri Richardson). I. The Kamloops variety. J. Fish Biol., 5: l-8.
McCarthy, D.H., Stevenson, JP. and Roberts, M.S., 1975. Some blood parameters of the rainbow trout (Safmo gairdneri Richardson). II. The Shasta variety. J. Fish Biol., 7: 215-219.
Murai, T., Andrews, J.W. and Smith, R.G., 1981. Effects of dietary copper on channel cat- fish. Aquaculture, 22: 353-357.
Ogino, C. and Yang, G-Y,, 1978. Requirement of rainbow trout for dietary zinc. Bull. Jap. Sot. Sci. Fish., 44: 1015-1018.
Ogino, C. and Yang, G-Y., 1979. Requirement of carp for dietary zinc. Bull. Jap. Sot. Sci. Fish., 4!i: 967-969.
Ogino, C. and Yang, G-Y., 1980. Requirements of carp and rainbow trout for dietary manganese and copper. Bull. Jap. Sot. Sci. Fish., 46: 455-458.
Spinelli, J., Mahnken, C. and Steinberg, M., 1979. Alternate sources of proteins for fish meal in salmonid diets. In: J.E. Halver and K. Tiews (Editors), Finfiih Nutrition and Fishfeed Technology. Heenemann, Berlin, 2: 131-142.
Suttle, N.F. and Mills, C.F., 1966. Studies of the toxicity of copper to pigs 1. Effects of oral zinc and iron salts on the development of copper toxicosis. Br. J. Nutr., 20: 135-148