25-Hydroxyvitamin D in Plasma of Cattle M. H I D I R O G L O U 1, J. G. PROULX 2, and D. R O U B O S 2 Animal Research Institute Research Branch Agriculture Canada Ottawa, Ontario K1A OC6
pound in the skin of cattle could be limited because of insufficient ultraviolet radiation. In addition, grass silage, which on many farms is the sole fodder used in the northern area of Ontario, is low in vitamin D (1). We evaluated the effects of season and supplementation with vitamin D on plasma 25-hydroxyvitamin D (25-OH-D) in cattle. This metabolite, which is the major form of vitamin D in blood, can be assayed in small quantities of plasma and provides the most readily determinable index of the vitamin D status of the animal. One experiment was at the Kapuskasing Experimental Farm (latitude 49 ° 28 t elevation 218 m) where the sun is less than 20 ° from the horizon for 6 to 7 mo of the year and where most of the ultraviolet irradiation required is removed from the sun's rays (2, 12).
ABSTRACT
Blood samples from 12 Shorthorn heifers, fed a grass silage ration and housed indoors or outdoors, were assayed for 25-hydroxyvitamin D during 9 mo. There was more in plasma during the summer from the outdoor group, reflecting a greater exposure to ultraviolet light and dermal synthesis of vitamin D, the precursor of 25-hydroxyvitamin D. In another experiment, assays were in four groups of six heifers each; 1) control, 2) single oral dose of 1,000,000 IU vitamin Da, 3) injected intramuscularly with 1,000,000 IU of vitamin D3, and 4) free access to a mineral mixture containing 32,000 IU vitamin D3/kg. In all groups, 25-hydroxyvitamin D was higher in plasma in summer than in winter. All heifers given vitamin D had more 25hydroxyvitamin D in plasma during winter than controls. Animals injected with vitamin D had more 25-hydroxyvitamin D in plasma during part of the winter than those on the other treatments with vitamin D.
EXPERIMENTAL Experiment 1
INTRODUCTION
Cattle can obtain vitamin D from two sources, direct ultraviolet irradiation of the skin and by eating irradiated plants. In an animal's skin 7-dehydrocholesterol is converted to vitamin D3 whereas vitamin D2 is produced by ultraviolet irradiation of forage ergosterol. In Canada, especially in its northern latitudes during winter months, the photochemical conversion of provitamin D to its active c o m -
Received February 5, 1979. 1Contribution No. 824, Animal Research Institute, Ottawa, Ontario K1A OC6. 2Kapuskasing Experimental Farm, Kapuskasing, Ontario P5N 2X9. 1979 J Dairy Sci 62:1076-1080
PROCEDURE
In November, 1977, a group of twelve 18-mo-old Shorthorn heifers which had been together all summer, was divided into two equal groups balanced by weight: 1) wintered outdoors in an open yard and 2) wintered indoors. Group 2 remained inside until the experiment ended in July, 1978. Both groups were fed ad libitum a grass-legume silage containing 25 to 30% dry matter. All animals had free access to water and to a mineral mixture containing 65% cobalt iodized salt a n d 35% dicalciumphosphate. Experiment 2
A group of 24 Shorthorn heifers, 8 mo old, was divided at the end of December, 1977, into groups of six each balanced by weight. All animals were winter-fed grass-legume silage and housed inside until the end of May, 1978. The treatments were 1) control, 2) a single oral dose of vitamin D3 (1,000,000 IU) in oil solution, 3) a single intramuscular dose of
1076
25-HYDROXYVITAMIN D 1N PLASMA vitamin D3 (1,000,000 IU) in oil solution administered in the gluteus medius muscle, 4) free access to the same mineral mixture as in Experiment 1 but enriched with 32,000 IU vitamin D3/kg of the mineral mixture. The vitamin D3 oral and im doses (groups 2 and 3) were given at the beginning of the experiment, after the first blood samples were taken. Groups 1, 2, and 3 also had access to the same mineral mixture but without vitamin D3. All animals were turned onto a common pasture on June 1, 1978, and the experiment ended July 18, 1978. Analyses
Blood was sampled approximately every 3 wk from the jugular vein of all heifers and centrifuged immediately. Plasma then was removed and stored at - 2 0 C. 25-Hydroxyvitamin D was determined in triplicate in the plasma by the method of Hollis et al. (9). Vitamin D content of silage samples was assayed by the method of Cohen and Lapointe (5). Comparison of means of groups was by one-way analysis of variance. If the F-test showed unequal variance, then Cochran's approximation to the Behrens-Fisher solution was used (4). Comparison of paired samples was by the paired t-test. All the statistical methods were as described by Snedecor and Cochran (19). RESULTS AND DISCUSSION Experiment 1
Figure 1 shows 25-OH-D in plasma according
1077
to the dates of sampling. Between November and mid-May 25-OH-D in plasma averaged 19 ng/ml for both groups. This shows that the vitamin D status in the winter months was low for both groups due probably to the scant solar irradiation (Table 1). In the northern latitude during the winter months, wave lengths o f less than 306 nm are removed completely because of the greater angle of the sun to the earth's atmosphere (3). Between May 31 and July 31 the mean 25-OH-D in plasma was 49 ng/ml for the outdoor group compared to 21 ng/ml plasma for the indoor group. The data indicate an increase of 25-OH-D in plasma for the outdoor animals at the end of May, apparently due to the increased exposure to ultraviolet light (Table 1). Estimated wave lengths at this time are between 290 and 313 nm and are optimal in June and July (3). The highly significant difference in 25-OH-D of plasma between winter and summer periods in the outdoor group indicates that endogenous synthesis was by far the chief source of vitamin D in plasma. It is unlikely that the low dietary vitamin D2, which was less than 5 ng/kg dry matter in the silage, had any determinant effect on the vitamin D status of the cattle. The effect of summer sunlight on 25-OH-D in serum in man is documented (20), and Pettifor (16) attributed a seasonal decline in man to a fall in environmental temperature. In this experiment during winter, the difference in 25-OH-D in plasma was not significant (P>.05) between cattle wintered outdoors and indoors because during this period there was little or no solar anti-
TABLE 1. Temperature, sunshine, and ultraviolet radiation during the experimental period. 1978
1977
Temperature (C) Mean maximum Mean minimum Sunshine (daily average) (h)
Nov
Dec
Jan
Feb
March
Apr
May
June
July
--1.8 1.7 --5.2 1.9
--14.8 --11 --18.5 1.7
--19.7 14.0 --25.3 3.1
--16.7 --10.2 23.1 4.3
11.2 3.1 --19.3 5.5
--1.9 4.1 --7.9 7.0
10.0 16.1 3.8 6.5
12.0 18.1 5.9 6
16.8 22.8 10.7 7.9
Ultraviolet radiation a (W/s cm-2 mo -1 ) for: Latitude
Jan
Feb
March
Apr
May
June
July
Aug
Sept
Oct
Nov
Dec
Year
50°N
.09
.03
1.2
2.8
4.0
4.5
4.3
3.1
1.8
.7
.2
.06
23.1
aAt 307.5 nm (10A°) by P. Bener, cited by Cutchis (6). Journal of Dairy Science Vol. 62, No. 7, 1979
1078
HIDIROGLOU ET AL. In s u m m e r , 25-OH-D in this e x p e r i m e n t was similar to 25-OH-D o f 48 n g / m l and 45 n g / m l in N o r t h A m e r i c a by K o s h y (11) and Hollis (10) in plasma o f p a s t u r e d dairy cows. In Australia, G e i m e i n e r (7) r e p o r t e d 35 n g / m l 25-OH-D in c o w s ' plasma. In Scotland, vitamin D in b l o o d in sheep was low during t h e w i n t e r (18) w h i c h c o r r e s p o n d s to o u r findings.
MEAN1 SE
ttt
!t NOV
t
t
t
Experiment 2 OEC
JAN
~EB
MaN
*p~
M*V
JUNE
JUrY
Figure 1. 25-Hydroxyvitamin D 3 in plasma of outdoor and indoor wintered beef cattle (ng/ml plasma).
rachitic activity (136 m W / m i n per cm 2 ultraviolet radiation) (3). Also the t h i c k hair coat m a y have d e c r e a s e d f u r t h e r the effectiveness o f t h e U V rays. Tisdall and Brown (22) s h o w e d t h a t vitamin D activity increased m a r k e d l y in rats e x p o s e d to sunlight. Their e x p e r i m e n t s w e r e in T o r o n t o ( ~ 4 4 ° N ) and s h o w e d t h a t c h o l e c a l c i f e r o l p r o d u c t i o n in n o n p i g m e n t e d e p i d e r m i s u n d e r clear sky was 10 to 20 I U c m -2 in D e c e m b e r t o J a n u a r y , 159 t o 250 IU cm -~ in April, and over 400 IU cm -2 during the s u m m e r .
Table 2 s h o w s the results o f vitamin D s u p p l e m e n t a t i o n o n 25-OH-D in plasma. Until mid-May all cattle s u p p l e m e n t e d with vitamin D had higher 25-OH-D t h a n t h e c o n t r o l (P
pound in the skin of cattle could be limited because of insufficient ultraviolet radiation. In addition, grass silage, which on many farms is the sole fodder used in the northern area of Ontario, is low in vitamin D (1). We evaluated the effects of season and supplementation with vitamin D on plasma 25-hydroxyvitamin D (25-OH-D) in cattle. This metabolite, which is the major form of vitamin D in blood, can be assayed in small quantities of plasma and provides the most readily determinable index of the vitamin D status of the animal. One experiment was at the Kapuskasing Experimental Farm (latitude 49 ° 28 t elevation 218 m) where the sun is less than 20 ° from the horizon for 6 to 7 mo of the year and where most of the ultraviolet irradiation required is removed from the sun's rays (2, 12).
ABSTRACT
Blood samples from 12 Shorthorn heifers, fed a grass silage ration and housed indoors or outdoors, were assayed for 25-hydroxyvitamin D during 9 mo. There was more in plasma during the summer from the outdoor group, reflecting a greater exposure to ultraviolet light and dermal synthesis of vitamin D, the precursor of 25-hydroxyvitamin D. In another experiment, assays were in four groups of six heifers each; 1) control, 2) single oral dose of 1,000,000 IU vitamin Da, 3) injected intramuscularly with 1,000,000 IU of vitamin D3, and 4) free access to a mineral mixture containing 32,000 IU vitamin D3/kg. In all groups, 25-hydroxyvitamin D was higher in plasma in summer than in winter. All heifers given vitamin D had more 25hydroxyvitamin D in plasma during winter than controls. Animals injected with vitamin D had more 25-hydroxyvitamin D in plasma during part of the winter than those on the other treatments with vitamin D.
EXPERIMENTAL Experiment 1
INTRODUCTION
Cattle can obtain vitamin D from two sources, direct ultraviolet irradiation of the skin and by eating irradiated plants. In an animal's skin 7-dehydrocholesterol is converted to vitamin D3 whereas vitamin D2 is produced by ultraviolet irradiation of forage ergosterol. In Canada, especially in its northern latitudes during winter months, the photochemical conversion of provitamin D to its active c o m -
Received February 5, 1979. 1Contribution No. 824, Animal Research Institute, Ottawa, Ontario K1A OC6. 2Kapuskasing Experimental Farm, Kapuskasing, Ontario P5N 2X9. 1979 J Dairy Sci 62:1076-1080
PROCEDURE
In November, 1977, a group of twelve 18-mo-old Shorthorn heifers which had been together all summer, was divided into two equal groups balanced by weight: 1) wintered outdoors in an open yard and 2) wintered indoors. Group 2 remained inside until the experiment ended in July, 1978. Both groups were fed ad libitum a grass-legume silage containing 25 to 30% dry matter. All animals had free access to water and to a mineral mixture containing 65% cobalt iodized salt a n d 35% dicalciumphosphate. Experiment 2
A group of 24 Shorthorn heifers, 8 mo old, was divided at the end of December, 1977, into groups of six each balanced by weight. All animals were winter-fed grass-legume silage and housed inside until the end of May, 1978. The treatments were 1) control, 2) a single oral dose of vitamin D3 (1,000,000 IU) in oil solution, 3) a single intramuscular dose of
1076
25-HYDROXYVITAMIN D 1N PLASMA vitamin D3 (1,000,000 IU) in oil solution administered in the gluteus medius muscle, 4) free access to the same mineral mixture as in Experiment 1 but enriched with 32,000 IU vitamin D3/kg of the mineral mixture. The vitamin D3 oral and im doses (groups 2 and 3) were given at the beginning of the experiment, after the first blood samples were taken. Groups 1, 2, and 3 also had access to the same mineral mixture but without vitamin D3. All animals were turned onto a common pasture on June 1, 1978, and the experiment ended July 18, 1978. Analyses
Blood was sampled approximately every 3 wk from the jugular vein of all heifers and centrifuged immediately. Plasma then was removed and stored at - 2 0 C. 25-Hydroxyvitamin D was determined in triplicate in the plasma by the method of Hollis et al. (9). Vitamin D content of silage samples was assayed by the method of Cohen and Lapointe (5). Comparison of means of groups was by one-way analysis of variance. If the F-test showed unequal variance, then Cochran's approximation to the Behrens-Fisher solution was used (4). Comparison of paired samples was by the paired t-test. All the statistical methods were as described by Snedecor and Cochran (19). RESULTS AND DISCUSSION Experiment 1
Figure 1 shows 25-OH-D in plasma according
1077
to the dates of sampling. Between November and mid-May 25-OH-D in plasma averaged 19 ng/ml for both groups. This shows that the vitamin D status in the winter months was low for both groups due probably to the scant solar irradiation (Table 1). In the northern latitude during the winter months, wave lengths o f less than 306 nm are removed completely because of the greater angle of the sun to the earth's atmosphere (3). Between May 31 and July 31 the mean 25-OH-D in plasma was 49 ng/ml for the outdoor group compared to 21 ng/ml plasma for the indoor group. The data indicate an increase of 25-OH-D in plasma for the outdoor animals at the end of May, apparently due to the increased exposure to ultraviolet light (Table 1). Estimated wave lengths at this time are between 290 and 313 nm and are optimal in June and July (3). The highly significant difference in 25-OH-D of plasma between winter and summer periods in the outdoor group indicates that endogenous synthesis was by far the chief source of vitamin D in plasma. It is unlikely that the low dietary vitamin D2, which was less than 5 ng/kg dry matter in the silage, had any determinant effect on the vitamin D status of the cattle. The effect of summer sunlight on 25-OH-D in serum in man is documented (20), and Pettifor (16) attributed a seasonal decline in man to a fall in environmental temperature. In this experiment during winter, the difference in 25-OH-D in plasma was not significant (P>.05) between cattle wintered outdoors and indoors because during this period there was little or no solar anti-
TABLE 1. Temperature, sunshine, and ultraviolet radiation during the experimental period. 1978
1977
Temperature (C) Mean maximum Mean minimum Sunshine (daily average) (h)
Nov
Dec
Jan
Feb
March
Apr
May
June
July
--1.8 1.7 --5.2 1.9
--14.8 --11 --18.5 1.7
--19.7 14.0 --25.3 3.1
--16.7 --10.2 23.1 4.3
11.2 3.1 --19.3 5.5
--1.9 4.1 --7.9 7.0
10.0 16.1 3.8 6.5
12.0 18.1 5.9 6
16.8 22.8 10.7 7.9
Ultraviolet radiation a (W/s cm-2 mo -1 ) for: Latitude
Jan
Feb
March
Apr
May
June
July
Aug
Sept
Oct
Nov
Dec
Year
50°N
.09
.03
1.2
2.8
4.0
4.5
4.3
3.1
1.8
.7
.2
.06
23.1
aAt 307.5 nm (10A°) by P. Bener, cited by Cutchis (6). Journal of Dairy Science Vol. 62, No. 7, 1979
1078
HIDIROGLOU ET AL. In s u m m e r , 25-OH-D in this e x p e r i m e n t was similar to 25-OH-D o f 48 n g / m l and 45 n g / m l in N o r t h A m e r i c a by K o s h y (11) and Hollis (10) in plasma o f p a s t u r e d dairy cows. In Australia, G e i m e i n e r (7) r e p o r t e d 35 n g / m l 25-OH-D in c o w s ' plasma. In Scotland, vitamin D in b l o o d in sheep was low during t h e w i n t e r (18) w h i c h c o r r e s p o n d s to o u r findings.
MEAN1 SE
ttt
!t NOV
t
t
t
Experiment 2 OEC
JAN
~EB
MaN
*p~
M*V
JUNE
JUrY
Figure 1. 25-Hydroxyvitamin D 3 in plasma of outdoor and indoor wintered beef cattle (ng/ml plasma).
rachitic activity (136 m W / m i n per cm 2 ultraviolet radiation) (3). Also the t h i c k hair coat m a y have d e c r e a s e d f u r t h e r the effectiveness o f t h e U V rays. Tisdall and Brown (22) s h o w e d t h a t vitamin D activity increased m a r k e d l y in rats e x p o s e d to sunlight. Their e x p e r i m e n t s w e r e in T o r o n t o ( ~ 4 4 ° N ) and s h o w e d t h a t c h o l e c a l c i f e r o l p r o d u c t i o n in n o n p i g m e n t e d e p i d e r m i s u n d e r clear sky was 10 to 20 I U c m -2 in D e c e m b e r t o J a n u a r y , 159 t o 250 IU cm -~ in April, and over 400 IU cm -2 during the s u m m e r .
Table 2 s h o w s the results o f vitamin D s u p p l e m e n t a t i o n o n 25-OH-D in plasma. Until mid-May all cattle s u p p l e m e n t e d with vitamin D had higher 25-OH-D t h a n t h e c o n t r o l (P
