Developmental Changes in Serum Ferritin Concentration of Dairy Calves
Y. MIYATA, K. FURUGOURI, and K. SHIJIMAYA
ABSTRACT
Serum ferritin concentration of nursing
calves was measured by a two-site im-
munoradiometric assay to assess de-
velopmental changes and to evaluate
relationships of serum ferritin with
iron-related blood characteristics. Serum
ferritin concentration of calves was low at
birth and elevated slightly at 3 days of
age. From 1 wk of age onward, serum
ferritin concentration of untreated calves
and calves fed only whole milk for 4 wk
decreased and remained low throughout
the nursing period. This finding almost
coincided with hematological char-
acteristics. However, that nearly normal
hematological measures and weight gain
persisted and that they did not develop
any anemic symptom indicate that serum
ferritin concentration is more sensitive
than other hematological characteristics
to iron depletion. However, serum ferritin
concentration of calves administered 130
mg of ferrous fumarate (40 mg iron) daily
from 3 to 22 days of age or injected with
4 ml of iron-dextran intramuscularly (400
mg iron) at 3 days and 2 wk of age
increased sharply just after treatment and
persisted high for 2 to 6 wk of age.
However, there was large variation
between animals for serum ferritin
concentration. Thus, it seems likely that
serum ferritin concentration is an index
for monitoring prelatent iron deficiency
of calves.
INTRODUCTION
Nursing calves fed only whole milk often
develop anemia within a few weeks (3, 5, 20,
26). In the veal industry especially, where
milk diet is commonly fed and rapid growth
occurs, iron deficiency anemia is common (2, 4,
Received July 8, 1983.
1984 J Dairy Sci 67:1256--1263
Hokkaido National Agricultural Experiment Station
Sapporo 061-01, Japan
15, 17). Also, where iron deficient, susceptibility
of calves to some pathogenic agents increases
(13, 21). Administration of iron compounds to
newborn calves is effective for calf growth and
hematological aspects (5, 12, 25, 27, 30, 31).
Thus, economic damage from iron deficiency is
considerable. In the earlier studies, character-
istics in blood for monitoring iron nutritional
status were hemoglobin (Hb), hematocrit (Ht),
red blood ceils (RBC), serum iron (Sl), and total
iron-binding capacity (TIBC). There were close
relationships among measurements in blood and
storage iron in calves (29).
More recently, ferritin concentration in
blood serum has attracted attention as a useful
survey tool to monitor iron status, because
as ferritin in blood serum is directly propor-
tional to iron stores under normal condition
(32). In previous papers (7, 18) authors reported
a highly sensitive two-site immunoradiometric
assay measuring ferritin concentration in blood
serum of dairy cows and bulls to assess their
iron nutritional status. Also, our study on
serum ferritin of nursing piglets indicated that
circulating ferritin is an indicator for assessment
of storage iron (8). Although there is con-
siderable evidence on blood characteristics of
nursing calves, only limited data are available
on serum ferritin levels of calves.
Objectives of this study were to assess de-
velopmental changes in serum ferritin of calves
and to evaluate relationships of serum ferritin
with iron-related blood characteristics.
MATERIALS AND METHODS
Animals and Samples
Twenty male and 20 female Holstein calves
were allotted into 4 groups of 10 calves by
weight and sex. They were housed in individual
calf pens on concrete and bedded with straw
for the 13-wk experiment. Pens were cleaned
every morning. Water was available ad libitum.
The feeding program for calves is in Table 1.
Calves were full fed colostrum three times a day
for 1 wk and thereafter fed whole milk twice a
1256
 SERUM FERRITIN OF CALVES 1257

TABLE 1. Feeding program for calves.

Weeks of age
1 2 3 4 5 6 7 8 9 10 11 12 13

Groups 1, 2, and 3
Whole milk,
kg/day a 5.0 5.0 5.0 5.0 5.0 5.0 5.0 4~0 2.0
Calf starter,
kg/day .1 .2 .4 .5 .8 1.0 1.2 1.5 1.5 1.0 .5
Concentrate,
kg/day .5 1.0 1.5 2.0
Orchardgrass ad libitum
hay
Iron treatment
(oral) b

(i.m.) C C

Group 4
Whole milka
kg/day 5.0 6.0 7.0 6.0 5.0 5.0 5.0 4.0 2.0
Calf starter
kg/day .2 .4 .6 .8 1.2 1.5 1.5 1.0 .5
Concentrate
kg/day .5 1.0 1.5 2.0
Orchardgrass ad libitum
hay

aFed colostrum ad libitum.

bOral administration of 130 nag of ferrous fumarate (40 mg iron) daily.
Injected lron-dextran (400 mg iron) intramuscularly.

day. Groups 1, 2, and 3 were on similar feeding
programs. They were fed calf starter after 1 wk
of age. Relative to the other groups, group 4
received additional whole milk and no starter
until 4 wk of age. All calves were fed orchard-
grass hay for ad libitum consumption after 1
wk of age. They were weaned at 9 wk of age
and fed concentrate after weaning. Group 1
received an oral dose of 130 mg of ferrous
fumarate (40 mg iron) daily from 3 to 22 days
of age. Group 2 received 4 ml of iron-dextran
intramuscularly (400 mg iron) at 3 days and 2
wk of age. This iron level was based upon dietary
requirement of calves of 40 mg/day per head
reported by Matron et at. (17), Roy et al. (28),
and Hansard (10). Iron content of the rations
was 283 ppm for calf starter, 179 ppm for
concentrate, and 190 ppm for orchardgrass hay
dry matter.
Observation and sampling of calves were at
birth, 3 days, 1, 2, 3, 4, 6, 8, and 13 wk of age.
Blood samples were collected by jugular punc-
ture into nonheparinized and heparinized
tubes. The RBC were counted by TO A microcell-
counter (Model CC-108, TOA tyo Denshi,
Kobe) and Hb, Ht, and plasma total protein
(TP) were determined as in (7). Serum was
extracted from whole blood and frozen at
- 3 0 ° C for later analysis of SI, TIBC, and serum
ferritin. The SI and TIBC were determined as in
(7). However, SI and TIBC of calves ad-
ministered iron-dextran intramuscularly was
not measured because of difficulty of separation
of Fe from iron-dextran complex.
Two-Site Immunoradiometric Assay
for Bovine Serum Ferritin
Serum ferritin concentration was determined
by a two-site immunoradiometric assay as in
(7). Bovine ferritin was isolated from bovine
spleen by the method of Penders et al. (24) and
purified by high speed liquid chromatography

Journal of Dairy Science Vol. 67, No. 6, 1984

1258 MIYATA ET AL.

(HLC-803A, T o y o Soda, T o k y o ) . The antisera
against purified bovine ferritin was o b t a i n e d
f r o m rabbit i m m u n i z e d by repeat subcutaneous
injections of purified ferritin m i x e d with
F r e u n d ' s c o m p l e t e adjuvant. G a m m a globulin
fraction was salted out with Na2 SO4, f o l l o w e d
by affinity c h r o m a t o g r a p h y on a ferritin-coupled
CNBr-activated Sepharose 4B column. A part of
purified anti-bovine spleen ferritin a n t i b o d y
was coupled on silicone rubber strings (3 m m in
diameter, 4 m m long). The o t h e r part of
purified a n t i b o d y was labeled with radio-
active 125 I. Iodination of purified anti-ferritin
a n t i b o d y was according to the m e t h o d of Niitsu
et al. (23). Ferritin standard solutions and two-
or threefold diluted serum samples were sand-
wiched between two antibodies, and radio-
activity of antibody-antigen-antibody linkage of
silicone rods was c o u n t e d by an auto g a m m a
scintillation c o u n t e r (LKB Wallac, Turku). All
samples were analyzed at least in triplicate. A
standard curve for this assay showed a positive
relation with ferritin c o n c e n t r a t i o n over a range
of .1 to 500 ng/ml.
RESULTS A N D DISCUSSION
Changes in b o d y weight of calves are shown in
Table 2. Daily gains of calves in groups 1, 2, 3,
and 4 were .84, .75, .82, .78 kg. No difference
was significant among daily gains of four groups
(P< .05).
Hematological changes of calves are shown in
Figures 1 and 2. The RBC, Hb, and Ht of
u n t r e a t e d calves and calves fed only whole milk
declined from birth to 1 wk of age. F r o m 1 wk
of age onward, RBC gradually increased,
whereas Hb and Ht remained low. However,
calves did n o t show any anemic s y m p t o m . The
Hb and Ht increased gradually from 6 wk of
age. These results are similar to (2, 4, 15, 17).
Thus, it seems likely that the neonatal anemia
of calves does n o t develop severely in a short
period, as c o m p a r e d with that of piglets (6, 8).
Under the present condition, severe iron
deficiency may develop when calves are reared
on exclusive milk diet for a tong time.
However, hematological m e a s u r e m e n t s of
calves administrated iron orally or intra-
muscularly increased sharply just after t r e a t m e n t
and reached m a x i m u m at 2 to 4 w k of age. The
RBC, Hb, and Ht were higher (P<.01) for
treated calves than for u n t r e a t e d calves during
this period. According to ferrokinetic studies
on calves (11, 14, 22), m a x i m u m u p t a k e by red
blood cell of radioactive S9Fe has been 12 to 16
days after iron injection. Our findings almost
coincided with the ferrokinetic data. However,
no difference was significant between oral and
injected groups for RBC, Hb, and Ht. Mean
corpuscular v o l u m e (MCV) declined slightly
from birth to 8 wk of age. The MCV was higher
for iron treated calves than for u n t r e a t e d calves
between 1 wk and 6 wk of age. Mean corpuscular
h e m o g l o b i n (MCH) declined gradually from
birth to 6 wk of age. The MCH was not affected
so sharply as MCV by iron treatment. Mean

TABLE 2. Changes in body weight of calves.

No. of Weeks of age

Groups animals 0 4 8 13 Daily gain

X, SE ,X SE X SE X SE X SE
Group 1a 10 46.7 1.8 kg 58.6 2.2 kg 90.3 3.2 kg 123.2 3.4 kg .84 .04 kg
Group 2b 10 44.4 2.3 57.1 1.5 86.0 2.0 113.6 2.6 .75 .02
Group 3c 10 45.1 .9 57.0 1.7 83.4 3.4 119.5 3.1 .82 .04
Group 4d 10 48.5 1.8 60.8 1.4 86.3 1.7 119.2 2.9 .78 .03

aFed calf starter after 1 wk of age, weaned at 9 wk of age, and received an oral dose of 130 mg of ferrous
fumarate (40 mg iron) daily from 3 to 22 days of age.
bFed calf starter after 1 wk of age, weaned at 9 wk of age, and received 4 ml of iron-dextran intramuscularly
(400 mg iron) at 3 days and 2 wk of age, respectively.
CFed calf starter after 1 wk of age, weaned at 9 wk of age.
dFed calf starter after 4 wk of age, weaned at 9 wk of age.

Journal of Dairy Science Vol. 67, No. 6, 1984

 SERUM FERRITIN OF CALVES 1259

o Group1 £oral iron~

• Group 2 , i. m iron>
o - - - - - Group 3 (untreated)
o _ _ _ G r o u p 4 *whole m i l k f o r 4 weeks)

000

~t
0 900
X
V

0 800
m
n~
700 I t I i I I I I t

4O
o~
V

-~¢. "-,~_ _ _ - , ~ . ~ . . . . ..o~ ~" ~" -....

30 i I I I I I I i

12
"(3

v
O~
_13 fO
T 9 I I I "-- L I I
03 I 2 3 4 6 8 13
! I I., ql
DAYS
oral iron WEEKS

[ i.m iron f
Figure 1. Changes in red blood cells (RBC), hemoglobin concentration (Hb), and hematocrit (Ht) of dairy
calves.

corpuscular hemoglobin c o n c e n t r a t i o n (MCHC) chronic changes during the nursing period.

was relatively low at birth and elevated at 3 Changes in TP, SI, and TIBC are shown in Fig-
days of age, and thereafter it remained almost ure 3. The TP and serum transferrin concentra-
u n c h a n g e d t h r o u g h o u t the experiment. Eryth- tion increased rapidly from birth to 1 wk of age
rocyte of calves show microcytic and n o r m o - and thereafter plateaued. Although intense and

Journal of Dairy Science Vol. 67, No. 6, 1984

1260 MIYATA ET AL.

immediate change of TP occurs as a result of orally from 2 to 4 wk of age. The SI was

intact absorption from the small intestine,
elevation of serum transferrin is derived from
endogenous sources (16). Developmental
changes in TIBC are similar to those reported
by Martinsson et al. (16). The TIBC was slightly
higher in calves fed only whole milk than in
untreated calves and calves administered iron
relatively low at birth and 3 days of age. The SI
of untreated calves and calves fed only whole
milk increased gradually from 1 wk of age and
was relatively constant after 4 wk of age.
However, SI of calves administered iron orally
increased temporarily with a peak at 1 wk of
age (P<.01). This finding is consistent with that

o Group1 ~oral ironJ

_- Group2 * i m iron>
(>----- Group3 ' u n t r e a t e d ,
50-
~___ Group4,v#lulerrnilkfor4weeks'

45-

>
0
40-

35 I I I L I _1 I

~ 14--
O_

(.~ 12

""~0-- ~ _ ~ -- ~ l - - ~

I0 1 I J I I I J

30

25 I I I I I I
03 2 3 4 6 8 13
L w I

DAYS
orat iron WEEKS
I ~-

1' i m l r o r , f

Figure 2. Changes in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCI-I), and mean
corpuscular hemoglobin concentration (MCHC) of dairy calves.

Journal of Dairy Science Vol. 67, No. 6, 1984

 SERUM FERRITIN OF CALVES 1261

o Group1 (oral iron)

• Group 2 (i. m. iron~
:f ~ o - - - - Group 3 ~unt reated~
7 ~ e----- Group4 ~whole m i Ik for 4 weeks)

~'~ I I 1 I I I 1 I I

500 - / ~ " ~ --- ~ . ~

~ 400 "- ""

0 300
m
V-
200-

I00

0 J J J J I I I I l
03 I 2 3 4 6 8 13
'D/~Y~' ora'l iron
i , WEEKS
[ i.m. iron 1L
Figure 3. Changes in plasma total protein (TP), serum iron (SI), and total iron-binding capacity (TIBC)
of dairy calves.

of Getty et al. (9), but after 2 wk of age, SI of
treated calves was not different from that of
untreated calves.
Developmental changes in serum ferritin
concentration in blood serum of dairy calves
from birth to 13 wk of age are presented in Fig-
ure 4. Serum ferritin concentration of calves was
low at birth and elevated sligb ely at 3 days of age.
This is similar to tfiat of nursing piglets (8.19).
From 1 wk of age onward, ferritin concentration
in blood serum of untreated calves decreased
and remained below 10 ng/ml throughout the
experiment. Similarly, serum ferritin con-
centrations of calves fed only whole milk was
less after 1 wk of age compared with untreated
animals, and serum ferritin almost disappeared
from circulation from 2 to 4 wk of age. Ferritin
concentration in blood serum of calves ad-
ministered iron orally or intramuscularly
increased rapidly in a short period and persisted
high for 2 to 6 wk of age. However, there was a
large variation between animals in serum
ferritin concentration. Thereafter, it declined
linearly to 13 wk of age. No difference in serum
ferritin concentration was observed between
treated and untreated animals at 13 wk of age.
This indicates that active iron absorption occurs
in intestine of neonatal calves and rapid growth
accelerates in iron depletion of storage sites
following low ferritin concentration in blood
serum. This finding also indicates that most
iron administered orally or injected is utilized

Journal of Dairy Science Vol. 67, No. 6, 1984

1262 MIYATA ET AL.
E 120
~0
g IO0
V--
8O / ~----Group3,untreated'
g
Z
o 11
0i 3 J I 2 3 4
DAYS
oral iron
P m
tim iron t
Figure 4. Changes in serum ferritin concentration of dairy calves. Mean -+ standard error.
b y red b l o o d cells with o n l y a small a m o u n t
t a k e n u p b y storage sites, b e c a u s e e r y t h r o p o i e s i s
of nursing calves is active (1, 11, 14, 22). T h u s ,
it seems t h a t calves r e s p o n d well to oral ad-
m i n i s t r a t i o n in t h e n e o n a t a l period. A l t h o u g h
u n t r e a t e d calves a n d calves fed o n l y w h o l e m i l k
h a d low s e r u m f e r r i t i n t h r o u g h o u t t h e n u r s i n g
period, n e a r l y n o r m a l range of h e m a t o l o g i c a l
m e a s u r e s a n d w e i g h t gain persisted. A c c o r d i n g l y ,
s e r u m f e r r i t i n c o n c e n t r a t i o n is m o r e sensitive
t h a n o t h e r h e m a t o l o g i c a l c h a r a c t e r i s t i c s t o iron
d e p l e t i o n . T h u s , it seems likely t h a t s e r u m
f e r r i t i n c o n c e n t r a t i o n is an i n d e x for m o n i t o r i n g
prelatertt iron d e f i c i e n c y of calves.
ACKNOWLEDGMENTS
T h e a u t h o r s expressed t h e i r sincere t h a n k s
to Y. Niitsu a n d Y. K o h g o , D e p a r t m e n t o f
I n t e r n a l Medicine, S a p p o r o Medical College,
S a p p o r o , for t h e i r advice o n this s t u d y a n d R.
M a t s u m u r a for t e c h n i c a l assistance.
Journal of Dairy Science Vol. 67, No. 6, 1984
o G r o u p 1 ~oral iron~
= Group2~im. irom
---- -- " r 4 weeks~
I I
6 8 13
__
WEEKS
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Journal of Dairy Science Vol. 67, No. 6, 1984