Studies on the Role of Nickel in the Ruminant

Abstract

Lambs were fed 6 to 7% of metabolic body weight per day of a basal purified diet low in nickel (65 ppb) or the basal diet plus 5 ppm nickel for a 97 day period in an attempt to demonstrate an essential role for nickel in the ovine. Weight gains for the entire period and digestibility of dry matter and of protein at 28 and 56 days were not different between the two groups. At 28 days, but not at 56 days, urinary nitrogen was less and percentage retention of absorbed nitrogen was greater in the supplemented lambs. Total serum proteins were higher at 56 days and serum alanine transaminase was higher throughout the experiment in the nickel supplemented lambs, but only significantly so at 56 days. When lambs were given an oral dose of 65Ni, the low nickel lambs tended to excrete more in the feces and retained less in the kidney, lung, and liver at 72 hours post dosing. The major excretory route of nickel was via the feces. The kidney retained the highest concentration of 65Ni of the organs examined.

Full text

Studies on the Role of Nickel in the Ruminant
JERRY W. SPEARS, E. E. HATFIELD,
RICHARD M. FORBES ANDSTEVE E. KOENIG1
Department of Animal Science, University of Illinois,
Urbana, Illinois 61801
ABSTRACT Lambs were fed 6 to 7% of metabolic body weight per
day of a basal purified diet low in nickel (65 ppb) or the basal diet plus
5 ppm nickel for a 97 day period in an attempt to demonstrate an essential
role for nickel in the ovine. Weight gains for the entire period and di
gestibility of dry matter and of protein at 28 and 56 days were not different
between the two groups. At 28 days, but not at 56 days, urinary nitrogen
was less and percentage retention of absorbed nitrogen was greater in the
supplemented lambs. Total serum proteins were higher at 56 days and
serum alanine transaminase was higher throughout the experiment in the
nickel supplemented lambs, but only significantly so at 56 days. When
lambs were given an oral dose of 63Ni, the low nickel lambs tended to
excrete more in the feces and retained less in the kidney, lung, and liver
at 72 hours post dosing. The major excretory route of nickel was via the
feces. The kidney retained the highest concentration of 63Ni of the organs
examined. J. Nutr. 108: 313-320, 1978.
INDEXING KEY WORDS nickel •lambs •nitrogen •alanine
transaminase
The essentiality of nickel has recently
been shown for the rat and chick (1-4).
Nickel deficiency in the chick has been
characterized by: 1) alterations in skin pig
mentation; 2) ultrastructural changes in
the liver including swelling of the mito
chondria in the matrix region and dilation
of the cisterns of the rough endoplasmic
reticulum; 3) decreased oxidative ability;
and 4) alterations in liver total lipids,
phospholipids and cholesterol (1-3, 5).
Deficiency symptoms in rats, raised through
successive generations, have been charac
terized by a decreased growth rate (6) and
an increased mortality toward the end of
the suckling period (1, 4) in addition to
liver changes similar to those listed above.
Limited data suggest that nickel is also
essential for swine ( 7 ). These findings were
based on a decreased growth rate, impaired
reproduction and a rough hair coat in pigs
fed a diet containing 100 ppb of nickel.
Studies dealing with nickel in the rumi
nant have been centered around the ex
cretion and tissue concentrations of this
element (8-10). Homeostatic control mech
anisms are implied in the bovine by pre
venting tissue accumulation of nickel (10,
11). Nickel is also relatively non-toxic to
ruminants (12). The fact that nickel is
under homeostatic control, and non-toxic
at normal levels, is consistent with nickel
being an essential element, but evidence to
support this hypothesis has not been pre
sented. The present study was an attempt
to demonstrate if nickel has an essential
role in the ovine.
MATERIALS AND METHODS
Twelve lambs averaging 19.2 kg were
randomly allotted by weight to either a
basal purified diet low in nickel or the
basal diet plus 5 ppm of nickel as NiCl2-
Received for publication February 2, 1977.
1Present address : Department of Animal Sciences,
University of Kentucky, Lexington, Kentucky 40506.
313
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314 SPEARS, HATFIELD, FORBES AND KOENIG
TABLE 1
Composition of the basal diet
Ingredient Percent
Dried skimmilk1Solka
floe2Glucose'Starch3Corn
OilMineral
Mix4Vitamin
Mix6Antibiotic638.245.005.0047.663.000.950.100.05
1Land O'Lakes Inc., Minneapolis, Minn.
2Brown Company, Chicago, Illinois. ' A. E.
Staley Manufacturing Company, Decatur, Illinois.
4Minerals furnished per kg diet (g).
Mineral mix for basal diet
Ingredient g/Kg diet
NaClMgSO4FeSO4-7H2OCuSO4-5H20KIMnSO4-H2ONaaMoO4-2H2OCoCl2-6H,ONa2SeO3CrCl,-6H2ONaFZnOSnCl2-2H2ONaSiOu-OHjONH4VO,5.00003.50000.30000.03000.02620.05900.00250.00190.00020.00100.00220.06200.00380.50000.0005
*Expressed as units or mg/kg, retinyl acetate,
680,400 units; Cholecalciferol, 68,040 units; D,a-
tocopheryl acetate, 4,536 units ; Riboflavin, 227 mg ;
d-Pantothenic Acid, 1,247 mg; Niacin, 3,402 mg;
Choline Chloride, 34,020 mg; Vitamin Bi2, 3.6 mg.
6Aureo-10 (Chlortetracycline).
6H2O. The basal diet is shown in table 1
and contained approximately 65 ppb of
nickel. All known essential trace elements
for the rat with the exception of nickel
were provided by the mineral mix (table 1).
The lambs were maintained in wooden
metabolism crates with stainless steel
waterers. Both waterers and feeders were
coated with polyurethane to prevent pos
sible nickel contamination. Lambs were
fed twice a day according to metabolic
body weight and offered distilled water ad
libitum throughout the experimental pe
riod of 97 days. Feed allotments varied
from 6 to 1% of metabolic body weight per
day during the 97-day period. Feed not
consumed by an animal was weighed and
recorded. Each lamb was weighed at 2-
week intervals with feed intake being ad
justed accordingly at this time. Blood sam
ples were collected from the jugular vein
on days 28, 56, and 84 of the experiment
for measurement of various serum compo
nents and for hematological studies includ
ing hematocrit, hemoglobin and leukocyte
count. Five day total collections of urine
and feces were conducted at the end of 28
and 56 days for the determination of di
gestibility and nitrogen balance. Urine and
feces were collected daily with aliquots
being composited over the 5-day period.
On day 94 each lamb was given a single
oral dose of 63NiCl23 (specific activity 9.3
mCi/mg) in a gelatin capsule. Each lamb
was dosed at a rate of 40 /xCi/kg metabolic
weight. Total urine and fecal collections
were made for 72 hours post dosing at
which time the lambs were killed. Portions
of the liver were rapidly removed for mea
surement of oxygen consumption. In addi
tion to the liver, the entire kidney, spleen,
heart, testes, lung and brain were removed,
weighed and an aliquot was taken for de
termination of tissue zinc and 63Ni activity.
Serum parameters measured included
total serum protein, urea nitrogen, creati-
nine, glucose, calcium, phosphorus and
alanine transaminase activity. Total serum
protein was determined by the biuret
method (13). Blood urea nitrogen was
measured directly using 2,3-butanedione
monoxime4 with the pink complex formed
being read colorimetrically in a spectropho-
tometer at 525 nm. Creatinine in serum was
determined by a color reaction with alka
line picrate as described by London et al.
(14). Glucose was determined directly by
the method of Sudduth et al. (15) using
o-toluidine. Serum calcium was measured
by a modification of the method of Gitel-
man (16). Serum phosphorus was deter
mined using a commercial phosphorus
reagent.4 Alanine transaminase activity in
serum was determined by reacting the py-
ruvate formed from L-alanine and a-keto-
glutaric acid with 2,4-dinitrophenylhydra-
zine ( 17). Urinary and fecal nitrogen were
determined using the kjeldahl procedure
3New England Nuclear, Chicago, Illinois.
1Hycel Inc., Houston, Texas.
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NICKEL IN THE RUMINANT 315
(18). For the hematological work, hemo
globin was determined using a hemoglobin
reagent.4 Blood was stained with Wright's
stain for leukocyte counting.
Oxygen uptake was determined on a
portion of liver which had been removed,
immediately homogenized (10% W/V) in
Krebs-Ringer buffer and then stored in ice
until analysis was made, using an oxygen
electrode.5 Glucose was used as the sub
strate.
Tissue samples were prepared for liquid
scintillation counting8 and zinc analysis 7
by digesting an approximately 10 g sample
in concentrated nitric acid followed by
oxidation with hydrogen peroxide. Analysis
of variance was conducted on all data ( 19).
RESULTS
No deficiency symptoms were observed
in the lambs fed the low nickel diet in this
study. Average daily gain and gain:feed
ratio for the 97-day period did not differ
between treatments (table 2). Protein and
dry matter digestibility were also not af
fected by nickel (table 2). The amount of
gain produced per unit of feed was slightly
higher in the lambs receiving supplemental
nickel (table 2). However the difference
was not significant.
Nitrogen excretion during the two 5-day
collection periods is shown in table 3. Since
lambs were fed according to metabolic
weight, results are expressed on a meta
bolic body weight basis when appropriate.
Urinary nitrogen excretion was significantly
higher in the low nickel lambs during the
first collection period. Fecal nitrogen was
not affected by dietary nickel. No treat
ment effect was apparent with respect to
nitrogen excretion in the second collection
period.
Serum parameters measured during the
experiment are presented in table 4. Serum
urea-nitrogen was not affected by dietary
nickel at any time period. Total serum pro
teins were consistently lower throughout
the experiment in the lambs fed the low
nickel diet with the difference being signifi
cant at 56 days. Serum creatinine levels
were not affected by dietary nickel. Serum
calcium and phosphorus were not affected
by nickel. Alanine transaminase activity in
the serum was substantially higher in the
nickel supplemented lambs at all dates.
TABLE 2
Average daily gain (ADG), feed efficiency and nutrient
digestibility in lambs fed low nickel or
nickel supplemented diets
Low Ni 5 ppm Ni
ADG(£)Gain
:feedDry
matter dig.1(%)28
days56
daysProtein
dig.'(%)28
days56
days137.7
±12.1»0.18±
0.0390.5
±0.591.7
±0.577.9
±1.978.9
±2.1139.4
±15.80.20
±0.0389.2
±1.490.2
±0.575.7
±2.579.6
±0.9
1Results of a 5 day collection period at each date.
±SEM(n - 6). 'Mean
Because of animal variability, differences
were significant only at 56 days. Serum glu
cose levels followed the same trend as ala-
nine transaminase with the low nickel
lambs tending to have slightly lower levels
at 28, 56, and 84 days.
Hematological data are shown in table 5.
Hemoglobin concentrations, hematocrits or
white blood cell counts were not signifi
cantly different between treatments at any
of the times studied.
The rate of oxidation of glucose by liver
homogenate preparations tended to be
slightly greater for the nickel supplemented
lambs (3.78 ±0.5 versus 4.54 ±0.6). Be
cause of the variation within a treatment
the differences were not significant.
The effect of dietary nickel on organ
weights is shown in table 6. The only organ
significantly affected by nickel was the
lung with the low nickel lambs having
smaller lungs.
The 63Ni dose given orally tended to be
excreted to a greater extent and retained
to a lesser extent in the tissues of the low
nickel lambs as compared to the Iambs re
ceiving 5 ppm of nickel (table 7). At 72
hours post dosing the low nickel lambs had
excreted 74.4% of the total 63Ni via the
feces while the lambs receiving supple
mented nickel had excreted only 64.7% via
this route, the decrease was not significant.
Urinary excretion of 63Ni was low in both
groups. Lambs receiving supplemental
6Y S I Model 53 Biological Oxygen Monitor.
' Packard Trl-Carb Liquid Scintillation Spectrom
eter, Model 574.
7Perkln-Elmer-306 Atomic Absorption Spectropho-
tometer.
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316 SPEARS, HATFIELD, FORBES AND KOENIG
TABLE 3
Nitrogen excretion in lambs fed a low nickel or nickel supplemented diet for 28 or 56 days1
28 days 56 days
Low Ni 5 ppm Ni Low Ni 5 ppm Ni
mg/day/kg bodyu>/3/4Urinary
nitrogen2
Fecal nitrogen2
Nitrogen retained2
Absorbed nitrogen
Retained794.8±38.3J.»
304.3±24.9
286.7±66.625.7
±5.7"652.6±30.7"
337.9±34.9
410.2±39.4<?,38.8±3.3-<680.3±37.5
280.6±27.3
365.3±19.735.1
±2.3695.7±10.7
268.9±15.4
384.9±34.935.5
±2.4
'Five day collection period at each date. 2mg/day/kg BW3'4. 3 Mean±SEM (n =6). "•*Sig
nificantly different (P < 0.05) within a time period. "•''Significantly different (P < 0.10) within a time
period.
nickel tended to retain a greater concentra
tion of 63Ni in the kidney, lung and liver
at 72 hours. The kidney showed the great
est amount of 63Ni per kg fresh tissue of
the organs examined and was the only
organ to retain a substantial amount of the
isotope.
The effect of nickel on the zinc content
of various tissues is shown in table 8. As
can be seen from the standard error of the
means, animal variability was great and no
significant differences were found between
nickel levels in any of the tissues studied.
DISCUSSION
Gain was not affected by nickel in the
present study. However, the efficiency of
converting feed to gain tended to be
slightly higher for the lambs receiving 5
ppm of nickel. Growth responses to nickel
have been reported in swine (7) and rats
(6) but nickel deprived chicks (3, 5, 19)
and quail (21) have usually gained simi-
larily to controls.
No significant differences in dry matter
or protein digestibility were found in this
study. O'Dell et al. (12) reported that
digestibility was not affected with dietary
levels of up to 1,000 ppm of nickel, so
apparently the rumen ecosystem can adapt
to high levels of nickel. Recent work indi
cated that rumen bacterial urease required
nickel (22). Therefore, nickel appeared to
have a role in the rumen independent of
any role that it may have in the mam
malian system.
The significantly higher urinary nitrogen
excretion noted in the low nickel lambs
during the first collection period suggested
an involvement of nickel in protein metab
olism. Total serum proteins tended to be
slightly lower throughout the experiment
in the lambs receiving the low nickel diet
although differences were only significant
at 56 days. However, these data are con
founded by the fact that no difference in
nitrogen excretion was observed between
the low and adequate nickel groups in the
TABLE 4
Serum parameters in lambs fed a low nickel or nickel supplemented diet
28 days 56 days 84 days
Low Ni 5 ppm Ni Low Ni 5 ppm Ni Low Ni 5 ppm Ni
Urea Nitrogen (mg/100ml)Total
Serum Protein (g/100ml)Creatinine
(mg/100ml)Ca
(mg/100ml)P
(mg/100ml)Glucose
(mg/100ml)Alanine
Transaminase (units/1)15.2
±1.2'5.7
±0.12.0±
0.213.3
±0.49.3
±0.580.2
±2.459.3±12.613.7
±1.25.9
±0.12.0
±0.112.6
±0.38.9
±0.381.0
±3.696.4
±23.915.2±1.05.4±0.1°1.3
±0.211.
8±0.47.0
±0.487.8
±5.844.5
±6.2«15.7±
1.15.7
±0.1»1.1
±0.212.3±
0.27.6
±0.590.2
±5.2103.8
±27.5*15.0±1.25.7
±0.11.3
±0.112.8±0.2e7.2
±0.675.7
±5.132.8
±4.8«15.5±1.85.9
±0.11.3
±0.213.4
±0.2*7.6
±0.279.6
±2.544.4
±3.7i
i Mean±8EU (n - 6).
period. «.'Significantly different (P < 0.05) within a time period. «.<Different (P <0.10) within a time
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NICKEL IN THE RUMINANT 317
TABLE 5
Hematological data in lambs fed a low nickel or nickel supplemented diet
28 days 56 days 84 days
Low Ni 5 ppm Ni Low Ni 5 ppm Ni Low Ni 5 ppm Ni
Hematocrit
Hemoglobin (g/100 ml)
Leukocyte count (X10S)34.7±0.9'
33.7±0.5
11.4±0.3 11.3±0.4
8.1±0.8 9.3±0.932.2±0.4
31.3±1.1
11.0±0.3 10.8±0.5
8.9±0.9 7.7±0.534.7±1.2
33.6±1.5
11.2±0.4 11.1±0.4
7.5±0.6 6.8±0.4
'Mean±SEM(n
second collection period conducted at 56
days. Based on evidence that cellular nickel
tended to be localized in the nucleic acid
fraction (23, 24) and also that nickel may
contribute to the stability of RNA (25),
DNA (26) or ribosomes (27, 28), it has
been suggested that nickel might function
in nucleic acid and/or protein metabolism.
Increased activities of RNA polymerase
and alkaline ribonuclease in the liver of
nickel deficient rats have been reported
(29).
Alanine transaminase activity was higher
in the serum of the lambs receiving sup
plemental nickel at 56 and 84 days. The
ruminant is dependent upon gluconeogene-
sis for its major supply of glucose and ala-
nine serves as a major gluconeogenic sub
strate for the ruminant (30, 31). However,
it can not be determined from this study
if the increased alanine transaminase activ
ity actually influenced gluconeogenesis.
Whether the increased activity of this en
zyme was due to a direct activation of the
enzyme by nickel or via a hormonal inter
action is not known. Intraperitoneal injec
tions of nickel have been shown to increase
TABLE 6
Organ weights of lambs fed a low nickel or nickel
supplemented diet
OrganLiver
Kidney
Spleen
Heart
Testis
Lung
BrainLow
Ni 5 ppmNi%
of BodyWeight1.92±0.12>
1.87±0.13
0.21±0.01 0.21±0.01
0.13±0.01 0.16±0.02
0.40±0.02 0.44±0.04
0.42±0.06 0.44±0.09
0.73±0.09» 1.11±0.126
0.23±0.01 0.23±0.03
urinary excretion of corticoids in the guinea
pig (32) and corticoids in turn at high
concentration can induce hepatic alanine
transaminase (33).
Serum calcium and phosphorus were not
significantly affected by dietary nickel in
this study. However, there is a slight
tendency for serum calcium and phos
phorus to be slightly higher in the nickel
supplemented Iambs at 56 and 84 days,
but not at 28 days (table 4). In isolated
tissues, nickel can replace calcium at cer
tain steps in the excitation process of nerve
cells (34) and also in the excitatory con-
tractory process of muscle cells (35). De
creased bone concentrations of calcium
have been reported in swine receiving low
nickel diets (7). Dormer et al. (36) have
recently reported that nickel may act by
inhibiting stimulus-secretion coupling of
certain secretory systems and suggested
TABLE 7
"Ni distribution and excretion in lambs fed a low
nickel or nickel suplemenled diet
Distribution (organ)
Kidney
Lung
Spleen
Heart
Testi«
Liver
BrainLow
Ni5 ppmNi%
dose/ kg freshtissue0.1282±0.0362'-°
0.3223±0.0552»
0.0095±0.0014« 0.0204±0.0028''
0.0094±0.0013 0.0208±0.0133
0.0073±0.0020 0.0078±0.0006
0.0049±0.0009 0.0104±0.0036
0.0070±0.0002« 0.0136±0.0033/
0.0068±0.0010 0.0069±0.0006
Low Ni 5 ppm Ni
% dose
Excretion (route)
Feces
Urine 74.4 ±3.3«
0.8 ±0.2 64.7 ±3.3>
2.0 ±1.0
1Mean±SEM(re = 6 for low Ni and 5 for 5 ppm
Ni). «•»Significantly different (P < 0.05).
1Mrun l MEM(n = 6 for low Ni and 5 for 5 ppm Ni).
«.'Significantly different (P < 0.05). «.'Significantly dif
ferent (P < 0.01). «./Different(P < 0.10).
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318 SPEARS, HATFIELD, FORBES AND KOENIG
TABLE 8
Effect of feeding a low nickel or nickel supplemented
diet on the Zinc content of lamb tissues
Low Ni 5 ppm Ni
lÃ-Q/gfresh tissue
Kidney
Lung
Spleen
Heart
Testis
Liver
Brain38.4Õ4.01
21.2Ü.4
34.4±4.7
17.6±0.6
12.5±0.6
29.2±4.2
12.5±0.137.9±8.9
24.4±1.5
33.8±7.7
17.4±0.2
13.6±1.3
31.0±1.2
12.4±0.5
1MeanisEM (n = 6 for low Ni and 5 for 5
ppm Ni).
that this inhibition might be the result of
an antagonism between nickel and calcium.
Nickel deficiency has frequently resulted
in a decreased hematocrit in the rat and
chick (1, 3, 4). In this study, hematocrits,
hemoglobin concentrations or leukocyte
counts were not affected by dietary nickel
(table 5).
Consistent with previous chick studies
(1-3) was the tendency for a decreased
oxygen consumption in liver homogenate
preparations from the low nickel lambs.
Nielsen et al. (3) have suggested that the
decreased oxygen uptake is a reflection of
the ultrastructural changes that occur in
the liver of the deficient chick. More re
cently, Kirchgessner and Schnegg (37) re
ported that glucose 6-phosphate dehydro-
genase and malate dehydrogenase were de
creased greatly in the liver of rats deficient
in nickel.
It is clear from the present study that
the feces is the major excretory route for
nickel in the ovine (table 7). The lambs
receiving 5 ppm of nickel apparently ab
sorbed and retained a greater amount of
the tracer 63Nidose than did the low nickel
lambs, especially in the kidney, lung and
liver (table 7). This finding is in contra
diction with previous reports (20, 38) in
which nickel deficient chicks were found
to retain a greater amount of 63Nithan the
control chicks in most organs. It is known
that nickel is poorly absorbed (39) but
the mechanism involved has not been
studied. The organ distribution of 63Ni in
the present study is in general agreement
with previous studies (38, 40-42).
Lungs from the lambs supplemented
with 5 ppm of nickel were significantly
larger when expressed as a percentage of
body weight. O'Dell et al. (10) found the
lung to contain the highest concentration
of nickel when non-toxic levels of nickel
were fed to male calves. Wase et al. (41)
reported that the lung had the greatest re
tention of 63Niwhen compared to the other
organs studied and suggested a high com
plex-formation constant for Ni with lung
protein. In the present study, the lung re
tained the second highest concentration of
63Niat 72 hours post dosing.
Anke et al. (7) recently reported that
swine fed low nickel diets had lower zinc
concentrations in certain tissues. Nickel,
when added to swine diets low in zinc, has
resulted in increased serum as well as liver
and bone zinc.8 In the present study, zinc
concentrations did not differ significantly
between treatments in any of the tissues
studied. Although no deficiency symptoms
were noted in the present study, certain
metabolic parameters did appear to be in
fluenced by feeding the low nickel diet. In
the present study, the environment was
not controlled and the nickel content of the
basal diet (65 ppb) was considerably
higher than that reported in diets (—'15
ppb) previously used to develop a nickel
deficiency in rats (4, 6) or chicks (3).
This may help explain why a more dra
matic deficiency was not observed.
LITERATURE CITED
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s Chung. A. S., Hoekstra, W. G. & Grummer, R. H.
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NICKEL IN THE RUMINANT 319
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