Effect of Vitamin D on the Ability of Cows to
Mobilize Blood Calcium 1
L. A. MUIR, ~ J. W. HIBBS, and H. R. CONRAD
Department of Dairy Science, Ohio Agricultural Research and Development Center, Wooster
Abstract agent, ethylenediaminetetraacetic acid (EDTA),
The intravenous infusion of ethylene- to challenge a cow's ability to mobilize calcium
diaminetetraacetic acid (EDTA) was used and maintain the normal blood calcium level
as a blood calcium challenge technique to (6, 7, 8, 11).
measure the relative ability of vitamin D The purposes of this study were to explore
fed and control cows to mobilize blood further the use of the EDTA blood calcium
ealcimn, and as an indicator of available challenge technique as an indicator of available
calcium reserves. Based on one-half re- calcium reserves and to determine if differences
covery time, six- to 10-minute infusions existed between the available calcium reserves
revealed no differences in blood cMcium of vitamin D~ fed and control dairy cows as
mobilizing ability due to vitamin D feed- reflected in their ability to maintain or re-
ing. However, cows with previous milk cover their normal blood calcium levels under
fever history had a longer one-half re- the stress of EDTA infusions.
covery time than cows with no milk fever Experimental Procedures
history. A more severe challenge, four-hour
Eighteen mature Jersey cows ranging in age
EDTA infusions, showed older cows to
from four to 11 years were selected from the
require longer for one-half recovery of
Research Center herd and used in three ex-
blood calcimn, but no differences due to
periments involving 18 EDTA infusions. I n
vitamin D feeding or level of milk produc-
Experiment 1 five cows were selected from a
tion. The linear regression coefficients
group which had been receiving supplementary
calculated from reciprocals of serum cal-
vitamin D~ for over a year at the rate of
cium plotted against infusion time, for
32,000 units/0.4536 kg grain concentrates, with
vitamin D and ~ontrol cows, were statis-
a minimum of 320,000 units/day. These cows
tically different and indicated that vita-
were paired with five control cows on the basis
rain D fed cows could withstand a similar
of body weight, milk production, and previous
drain on their blood calcium at least one-
milk fever history. Each cow was infused with
and one-half times longer than the controls.
a 10% aqueous solution of disodium EDTA.
Herein may lie the key to the effectiveness
The 10% EDTA solution was infused at the
of vitamin D feeding in milk fever pre-
rate of 0.173 ml/second into the right jugular
vein using the technique described by Smith
and Brown (11). The infusion time (six to
Vitamin D fed continuously at the rate of
ten minutes) was varied with body weight so
32,000 units s'0.4536 kg of concentrate (3) or
that the total dosage -,'as 18.5 mg of E D T A /
in massive doses, 20 to 30 million units/day,
kilogram of body weight. Blood samples were
for three to seven days prior to partlrrition
collected at 0, 10, 15, 20, 25, 30, 45, 60, 75, 90,
(2, 5) has been shown to reduce the incidence
105, 120, 130, 140, 150, 160, 170, 180, 190, and
of parturient paresis (milk fever) in dairy
200 minutes after the beginning of infusion
cows with previous milk fever history. Although
and analyzed for total serum calcium and in-
vitamin D is known to increase the intestinal
absorption of calcium (3), it is not known to organic phosphate.
I n the second experiment, eight dry cows
what extent the available calcium reserves may
were chosen which had no previous milk fever
be increased by vitamin D feeding.
history, no previous supplementary vitamin D
Measm~ing available calcium reserves pre-
feeding, and which were six to nine months
sents a difficult problem. One approach has
pregnant. They were paired according to body
been to use infusions of a calcium chelating
weight and one group was given 320,000 units
Received for publication January 2, 1968. of vitamin D~ per day orally for at least two
Published with the approval of the Associa:te weeks prior to infusion, while the other group
Director of the Ohio Agricultural Research and served as controls. I n this experiment 97.9 mg
Development Center, Wooster, as Journal Article of EDTA/kilogram of body weight was infused
no. 98-67. during a four-hour period at the rate of 2.55
" Data. in this paper were presented to the Gra.du-
ate Faculty of The Ohio State University in partial rot/minute, the concentration being varied with
fulfillment of the requirements for the degree Mas- body weight. Blood samples were taken at 0,
ter of Science. 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
V I T A M I N D AND CALOIU1Vf 1047
6.0, 6.5, 7.0, 7.5, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0,
14.0, 24.0, 36.0, and 48.0 hours after the be-
rODO SERUM Co
ginning of infusion and analyzed for total
serum calcium and inorganic phosphate. Urine
samples were collected f r o m Cow 1502 before
infusion and at two, four, eight, ten, and 12 8.00 ~, - - GONTROL
hours after the beginning of infusion and these . . . . VIT. D
samples were also analyzed for total phosphate. ~ SAMPLED
Total serum calcium was determined by an 0
adaptation of the Watson-Rogers method (13) "~ ~ ~ SERUM P
as follows: 1 ml of serum was added to 3 ml • s"-. SS '
of 10% triehloroaeetic acid and centrifuged.
Two milliliters of 7.5 N potassium hydroxide 4.00 "*'""~'~"
and ten drops of calcein indicator were added
to 2 ml of the serum-TCA supernatant and ~ ~..f-( EOTA INFUSED)
titrated with dilute 1,2-diaminocyclohexane-N- ~oo~
tetraacetate ( D C T A ) , using as the end point
the disappearance of green fluorescence and
20 60 ,oo ,40 ,so
the appearance of a pink color, under a day- MINUTES
light lamp, against a black background. A
FIG. 1. Effect of short, six- to ten-minute EDTA
calcium carbonate standard was used. infusions on average serum Ca aa~d P of control
Serum inorganic phosphate was determined and vitamin D fed cows.
by the Briggs modification of the Bell-Doisy
colorimetric procedure (1). The control cows (Fig. 1, Table 1) received
To p r e p a r e the urine samples for analysis an average of 7.96 g of E D T A ( 1 8 . 5 - m g /
40 ml of urine were evaporated to dryness and kilogram body weight) in 7.34 minutes, which
wet ashed using a 3:1 ntric:perchloric acid chelated 0.81 g of calcium. The average serum
mixture and then dried. The remaining ash was calcium pool for the control cows, based on
diluted to 60 ml and 0.1 ml was used for de- the 9.81 rag/100 ml preinfusion calcium level
termining total phosphate, employing the san~e and 416 kg body weight, contained an estimated
colorimetric reaction used in the serum inor- 1.46 g of calcium. As in the vitamin D fed
ganic phosphate procedure (1). group, more than half the initial serum calcium
was chelated. The average serum calcium level
Results and Discussion just after infusion was 7.99 rag/100 m l ; there-
Experiment I. The vitamin D fed cows fore, 0.55 g of calcium was mobilized into the
(Fig. 1, Table 1) received an average of 8.86 blood calcium pool during the infusion. The av-
g of E D T A (18.5 rag/kilogram body weight) erage one-half recovery time f o r the control
in 8.53 minutes which, assuming that one mole-
cule of E D T A binds one calcium ion, chelated TABLE 1. Effects of eight- to ten-minute E D T A
infusions (Experiment 1).
0.93 g of calcium. Assuming also that 3.7%
of the body weight was serum (9) and that the Vitamin D
specific gravity of serum was 1.027, the vita- Group Control (5) ~ fed (5)"
min D fed group, with an average body weight Ilfitial serum
of 480 kg and an average initial serum cal- Ca (mg/100 nil) 9.81 ± 0.69 ~ 10.06 ± 0.24 b
cium level of 10.06 mR/100 ml, had an average Initial Ca in
blood pool (g) 1.46 _ 0.07 1.74 ± 0.22
serum calcium pool of 1.74 g. Therefore, more Infusion
than half of the initial serum calcium was time (min) 7.34 "4- 0.89 8.53 ± 0.87
chelated. The average serum calcium level just E D T A infused (g) 7.96 ± 0.83 8.86 ± 1.04
a f t e r infusion was 7.64 mR/100 ml; therefore, Ca bound by
E D T A (g) 0.81 ± 0.09 0.93 ± 0.10
it was calculated that 0.51 g of calcium had Minimum serum
been mobilized into the blood calcium pool Ca r (rag/100 ml) 7.99 ± 0.58 7.64 ± 0.75
during the infusion. The average one-half One-half recovery
recovery time (the time required for the serum time ~ (min) 32.0 + 7.40 25.4 +_ 17.3
calcium level to recover to one-half the dif- " Numbers in parentheses indicate number of
ference between the preinfusion level and the cows per group.
postinfusion level) for the vitamin D fed group b Standard deviation.
Serum Ca ten minutes after beginning infusion.
was 25.4 minutes, with a large standard devia- ,l One-half recovery time started when first blood
tion of 17.3 minutes. sample was taken after infusion.
g. DAIRY SGIEIqCE VOL 51, No. 7
1048 51VlR, t I I B B S , AND CONRAD
cows was 32.2 minutes, with a s t a n d a r d devia- 12DO~- --CONTROL
tion of 7.4 minutes.
The s e r m n p h o s p h a t e levels of both g r o u p s I000i .... VIT. D /
were lowered b y the E D T A i n f u s i o n ( F i g . 1),
the v a r i a t i o n between cows in b o t h g r o u p s was
large, a n d no consistent difference was noted
between the groups.
M u l t i p l e regression analysis was used to
. s.oolh ..............
d e t e r m i n e the effect of v i t a m i n D t r e a t m e n t ,
milk f e v e r history, age, a n d milk p r o d u c t i o n on
the one-half recovery time. The one-half re-
covery time was used instead of the complete
recovery tin]e, because serum calcium r e t u r n e d
to slightly lower t h a n p r e i n f u s i o n levels.
Cows with p r e v i o u s nfilk f e v e r h i s t o r y were
otV I F SD)
I I I I f
I0 15 20 25 30 35 40 45 510
I I I
f o u n d to have longer one-half recovery times.
This effect a p p r o a c h e d significance, with a
p r o b a b i l i t y of a p p r o x i m a t e l y 0.06. P a y n e ( 8 ) , FIO. 2. Effect of four-hour E D T A infusions on
the average serum Ca of control and vitamin D
on the other h a n d , gave equivalent doses of so- fed cows.
dium oxalate (24 r a g / k i l o g r a m of body weight
at the r a t e of 1 g / m i n u t e ) to various g r o u p s a n average of 49.4 g of E D T A (97.9 m g / k g
of cows a n d f o u n d t h a t the one-half recovery body w e i g h t ) , which chelated 5.17 g of cal-
time of cows with nfilk fever h i s t o r y did not cium. The average initial serum calcium level
differ significantly f r o m n o r n m l cows. V i t a - f o r the control g r o u p with a n average body
min D t r e a t m e n t , age, a n d milk p r o d u c t i o n w e i g h t of 504 k g was 10.64 m g / 1 0 0 m l ; there-
h a d no detectable effect on the one-half recov- fore, the average serum calcium pool f o r this
ery time with this short, six- to t e n - m i n u t e in- g r o u p was calculated to contain 1.94 g of cal-
fusion. cium. Again, a b o u t two a n d one-half times
Experiment II. Since the short, six- to ten- the total a m o u n t of calcium in the serum ealci-
minute, i n f u s i o n m e t h o d i n E x e r i m e n t I failed u m pool was chelated. Since the a v e r a g e s e r m n
to detect a significant difference in one-half ca!titan level of the control g r o u p a t the end
recovery time, it was r e a s o n e d t h a t a longer of i n f u s i o n was 4.42 rag/100 ml, a calculated
infusion, which would more severely challenge average of 4.03 g of calcium was mobilized into
the cows' ability to mobilize calcium, m i g h t be the s e r u m calcium pool d u r i n g the infusion.
a more sensitive measure of available calcium re- Therefore, the average control mobilized 0.1 g
serves. I n E x p e r i m e n t I I the p e r cent E D T A TABLI~ 2. Effects of four-hour EDTA infusions
in the solution infused was v a r i e d with body (Experiment 2).
weight a n d the i n f u s i o n time was s t a n d a r d i z e d
at f o u r hours. Group Control (4)" fed (4) ~
The v i t a m i n D fed cows (Fig. 2, Table 2)
received a n average of 49.0 g of E D T A (97.9 Initial serum
Ca (rag/100 ml) 10.64 __ 0.45 b 11.32 ~ 0.58 b
n / g / k i l o g r a m body w e i g h t ) , which chelated Initial Ca in
5.14 g of calcium. Since the average initial blood pool (g) 1.94 ± 0.28 2.06 _+ 0.14
s e r m a calcium level f o r this g r o u p was 11.32 EDTA
rag/100 nfl a n d the average b o d y w e i g h t was infused (g) 49.4 _+5.30 49.0 ~ 1.70
Ca bound by
501 kg, tt:e average s e r m n calcium pool f o r the E D T A (g) 5.17 +_ 0.55 5.14 +_ 0.18
v i t a m i n D ted g r o u p was calculated to be 2.06 Minimum serum Ca c
g. On this basis, a p p r o x i m a t e l y two a n d one- (mg/100 m]) 4.42 _+ 0.19 5.80 ~+ 0.98
h a l f times the a m o u n t of calcimn i n i t i a l l y pres- One-half recovery
time '1 (hr) 22.4 _ 7.40 19.0 ~+ 14.40
ent in the s e r u m calcium pool was chelated. Immediately
The average serum calcium level of this g r o u p available Ca
at the end of i n f u s i o n was 5.80 rag/100 m l ; reserves (g) 10.27 -- 1.26 14.30 e ~ 4.41 ~
therefore, a n a v e r a g e of 4.13 g of calcium was Numbers iu parentheses indicate number of
mobilized into the serum calcium pool d u r i n g cows per group.
the infusion. The average one-half recovery b Standard deviation.
time was 19 hours, with a s t a n d a r d deviation ¢ Serum Ca at end of four hours' infusion.
'~ One-half recovery time starts at end of infu-
of 14.4 hours. sion.
The control cows (Fig. 2, Table 2) received Average of three cows.
J. DAIRY SCIENCE VOL. 51, NO. 7
V I T A M I N I) AND CALCIUM 1049
less calcimn during the infusion period than 1502 did not return to the one-hMf preinfusion
the average vitamin D fed cow. The average one- calcium level during the 48-hour sampling pe-
hMf recovery time for the control cows was riod. Age significantly affected the one-half
22.4 hours, with a standard deviation of 7.4 recovery time. The older cows required more
hours. time to return to their one-half preinfusion
The serum phosphate levels of both groups calciunl level than did the younger cows. I t
were lowered by the EDTA infusion (Fig. 3), is of interest that it was the oldest cow in
as was also noted in Experiment 2. I n this ex- the experiment, Cow 1502, that did not return
periment the serum phosphate levels fell more to her one-half preinfusion serum calcium level
uniformly and to lower values than was found during the experimental period. The effect of
in Experinmnt I ; however, no differences were age on reducing ability to mobilize caleimn is
observed between groups. also indicated by the fact that milk fever usual-
Urine samples collected from Cow 1502 ly occurs in cows that have had at least two
showed that the urinary phosphate level in- previous parturitions (4). Vitamin D treat-
creased between the second and fourth hours ment and the level of milk production had no
of infusion from 1.81 to 9.75 rag/100 ml of significant effect on the one-half recovery time.
urine. The urinary phosphate level remained Payne (7) infused approximately 13.3 g of
high through the eighth hour after infusion EDTA per hour for four hours and measured
and then began to fluctuate. Increased urinary the plasma calcium level for two to three hours
phosphate dm'ing EDTA infusion has been following completion of infusion. By assmning
noted also by others (7, 8, 11, 12). Spencer that the recovery of the normal serum calcium
(12) suggested that this increase in urinary level was a linear process, Payne estimated
phosphate may indicate an increase in para- calcium mobilization rate (the rate at which
thormone secretion which, in turn, causes in- a cow can mobilize her available calcium re-
creased phosphate excretion. I n view of the serves) and immediately available calcium re-
recent work of Sherwood et al. (10), who have serves (the amount of calcium immediately
shown an increase in circulating parathormone available for transfer to the blood). However,
during EDTA infusion, Spencer% theory seems the data obtained in this experiment indicated
plausible. Further support was found for this that there were two recovery phases (Fig. 2) :
explanation when we added EDTA to blood a rapid nonlinear initial recovery phase dur-
serum in vitro and found no effect on the phos- ing the first four hours after the end of infu-
phate level, suggesting that the lowered phos- sion, followed by a linear secondary phase.
phate in vivo due to EDTA infusion resulted We reasoned that the immediately available
from a physiological process. calcium reserves could be more accurately esti-
Multiple regression analysis was used to mated by obtaining the Y intercept value (a)
determine the effect of vitamin D, age, and from the linear regression equation of the se-
the level of milk production on one-half re- rum ealcimn values of the secondary recovery
covery time. None of the cows in this experi- phase on the time after the beginning of EDTA
ment had a history of milk fever. Only seven infusion and using this value in the following
of the eight cows infused could be included formula :
in the nmltiple regression analysis because Cow Immediately available calcium reserves (g)
= Cak/[(Cat -- a ) / C m ] where Ca~ ---- amount
CONTROL of Ca chelated by the EDTA infused; Ca~ =-
.... VIT. D initial serum Ca level; and a ---- the Y intercept
6.00! ,~ S A M P L E D
value of the linear regression equation of the
J secondary recovery phase calcium values on the
time after the beginning of EDTA infusion.
Applying this formula to the data obtained
in this experiment (Table 2), the control cows
had an average immediately available calcium
2.00 reserve of 10.27 g, while three of the vitamin
(EDTA INFUSED) D fed cows had an average of 14.30 g. The
fourth vitamin D fed cow was omitted because
for some reason she did not reach the secondary
5 Io 15 20 25 35 0 50
phase during the experimental period but re-
HOURS mained at a reduced serum calcium level.
FIG. 3. Effect of four-hour infusions on the av-
erage serum P of control and vitanlin D fed cows. The 4.03-g difference between the immediate-
5. DAII~Y SCIENCE VOI:, 51, NO. 7
1050 ~u~R, I-IIBBS, AND CONRAD
ly available calcium reserves of the two g r o u p s f o r d e d by vitamin D feeding t h r o u g h increased
was not statistically significant ( P = 0.2) with intestinal a b s o r p t i o n of calcium (2, 3, 5) can
this n u m b e r of cattle; however, it may prove be a t t r i b u t e d to the additional time p r o v i d e d
to be a real difference when tested more criti- f o r a d j u s t m e n t of the calcium r e g u l a t o r y mech-
cally in f u t u r e e x p e r i m e n t s with large Utah- anism u n d e r the stress of b e g i n n i n g lactation.
The sermn calcium levels of the cows in both References
g r o u p s declined in a nonlinear m a n n e r during (1) Briggs, A. P. 1922. A modification of the
the E D T A infusion. However, when the re- Bell-Doisy phosphorus determination. J.
ciprocals of the serum calcium values were Biol. Chem., 53: 13.
plotted against infusion time, s t r a i g h t lines (2) Hibbs, J. W., and H. R. Conrad. 1960.
Studies of milk fever in dairy cows. VI.
with different slopes were obtained ( F i g u r e 4).
Effect of three prepartal dosage levels of
The regression equations for these two lines Vitamin D on milk fever incidence. J.
were Y = .09489 + .03239 X f o r the control Dairy Sci., 43: 1124.
g r o u p and I7 = .08819 + .02126 X f o r the (3) Hibbs, J. W., and i~. R. Conrad. 1966.
vitamin D fed group. The difference between Re-evaluation of nutrient allowances for
regression coefficients was tested using the "t" high-producing cows. I L CMeium, phos-
test and f o u n d to be highly significant. phorus, and Vitamin D. J. Dairy Sci., 49:
W h e n the regression coefficient of the con- 243.
trol g r o u p was divided by the regression co- (4) Hibbs, J. W., W. E. Krauss, C. F. Monroe,
and T. S. Sutton. 1946. Studies on milk
efficient of the vitamin D fed group, a value
fever in dairy cows. II. The effect of vita-
of ] .5 was obtained. This value might be called min D on some of the blood changes in
a relative serum calcium maintenance constant. normal and milk fever cows at parturition.
I t indicates t h a t u n d e r a stress which reduces J. Dairy Sci., 29: 617.
blood ealcimn such as E D T A infusion or the (5) Hibbs, J. W., and W. D. Pounden. 1955.
initiation of milk secretion, the vitamin D fed Studies on milk fever in dairy cows. IV.
cow will be able to remain above the critical Prevention by short-time, prepartum feeding
blood calcium level one and a h a l f times of massive doses of Vitamin D. J. Dairy
longer than the control cow. Sei., 38:65.
(6) Jackson, H. D., A. R. Pappenhagen, and
Milk fever develops w h e n s e r m n ealcimn
G. D. Goetach. 1957. Effects of EDTA
falls to the critical level before the blood calci- and parathyroid extract on blood calcium
um r e g u l a t i n g mechanism is able to a d j u s t to levels of dairy cows. J. Anim. Sci., 16: 1023.
the increased eMcium d e m a n d s f o r lactation. On (7) Payne, J. M. 1964. The response of cows
this basis the protection a g a i n s t milk f e v e r af- to experimentally induced hypocalcaemia.
II. Chronic experimental hypocalcaemia.
I--CONTROL (~'=, 09489 +. 03239X) Vet. Record, 7.6: 77.
~.'2401---'VIT.| O (~=.08819 +.02126X) (8) Pay~lc, J. M., D. F. Sansom, and R. Mans-
ton. 1963. The responses of cows to ex-
0 perimentally induced hypocalcaemia. I.
0 Acute experimental hypocalcaemia. Vet.
• 200 Record, 75: 588.
(9) Reynolds, M. 1953. Measurement of bovine
a plasma and blood volume during pregnancy
(D and lactation. Amer. J. Physiol., 175: 118.
.160 o ~ss (10) Sherwood, L. M., J. T. Potts, A. D. Care,
G. P. Mayer, and G. D. Aurbach. 1966.
co Evaluation of radioimmunoassay of factors
controlling the secretion of parathyroid
.120 hormone. Natu~'e, 209: 52.
(11) Smith, V. R., and W. K. Brown. 1963. Re-
sponse of some blood constituents to infu-
sions of disodium ethylenediaminetetraaee-
tare in intact cattle. J. Dairy Sci., 46: 223.
(E .080 (12) Spencer, H. 1960. Studies of the effects
1 J I I I I t I I
of chelating agents in man. Ann. N. Y.
I 2 3 4 Acad. Sei., 88: 281.
(13) Watson, D., and J. Rogers. 1963. Serum
INFUSION TIME (HRS.)
calcium determination with 1,2-diaminocyclo-
Fro. 4. Effect of four-hour EDTA infusions on
the reciprocals of the serum Ca in control and hexane-N-tetra-aeetate. Clin. Chim. Acta,
vitamin D fed cows. 8 : 168.
J. DAI~Y ScIENc~ VOL. 51, NO. 7