Effects of Different Levels of Vitamins A and E on the
Utilization of Cholecalciferol by Broiler Chickens1
A. ABURTO and W. M. BRITTON2
Department of Poultry Science, University of Georgia, Athens, Georgia 30602-2772
exposed to UV fluorescent light or no UV light, two
levels of dietary vitamin E (10 and 10,000 IU/kg) and
three levels of dietary vitamin D3 (0; 500 and 2,500 IU/
kg) in a 2 × 2 × 3 factorial arrangement. The high level of
vitamin E significantly (P < 0.05) reduced body weight,
bone ash, plasma calcium, and increased rickets but only
at 500 IU/kg of vitamin D3. Feeding 2,500 IU/kg of
vitamin D3 overcame the effects of the high level of
vitamin E, causing a significant (P < 0.05) interaction.
Ultraviolet light also prevented the detrimental effects of
the high level of vitamin E. The results of these studies
indicate that high dietary levels of vitamins A and E
negatively affected the utilization of vitamin D3 only
when D3 was present at a marginal level (500 IU/kg) in
the diet but not when it was synthesized in the bird by
exposure to UV light or supplemented at 2,500 IU/kg in
the diet.
ABSTRACT
Three experiments were conducted to
determine the effects of high dietary levels of vitamins A
and E on the utilization of cholecalciferol by broiler
chicks. In Experiment 1, chicks were fed six levels of
vitamin A (5,000, 10,000, 20,000, 40,000, 80,000, and
160,000 IU/kg). Cholecalciferol (vitamin D3) was not
added to the basal diet but all birds were exposed to
ultraviolet (UV) fluorescent light. Body weight was
decreased only at levels of vitamin A of 80,000 IU/kg or
above. In Experiment 2, birds were exposed to UV
fluorescent light or no UV light, two levels of dietary
vitamin A (1,500 and 45,000 IU/kg) and three levels of
dietary vitamin D3 (0, 500, and 2,500 IU/kg) in a 2 × 2 ×
3 factorial arrangement. The high level of vitamin A
reduced (P < 0.001) bone ash but only at a marginal level
of vitamin D3 (500 IU/kg) and when the birds were not
exposed to UV light. In Experiment 3, birds were
(Key words: vitamin A, cholecalciferol, vitamin E, ultraviolet light, broiler)
1998 Poultry Science 77:570–577
nosis A caused bone fragility. Administration of excess
vitamin A reduced the effects of hypervitaminosis D in
the rat (Clark and Bassett, 1962; Clark and Smith, 1964);
whereas extra vitamin D3 protected the rat (Vedder and
Rosenberg, 1938), dog (Frey et al., 1975), and the chick
(Taylor et al., 1968; Veltmann and Jensen, 1985; Veltmann et al., 1986, 1987) against vitamin A toxicosis.
Although most workers have agreed on the existence of
nutritional relationships among fat-soluble vitamins in
general and nutritional interactions among vitamins A,
D3, and E in particular, there has been considerable
disagreement as to whether it is due essentially to an
interaction among the three vitamins in the intestinal
tract, prior to or during absorption, or in the tissues of
the animals after absorption. Unfortunately, there are
not very sensitive methods available to evaluate the
status of vitamins A and E of animals. The measurement
of vitamins A and E in plasma and liver by HPLC
analysis has been used for several years, but the values
obtained are not easily related to dietary needs.
INTRODUCTION
High dietary levels of vitamins A and E are believed
to interact with vitamin D3. March et al. (1973) reported
that with a calcium-deficient or vitamin D-deficient diet,
bone calcification was further depressed when chicks
were given excess vitamin E. Similarly, Murphy et al.
(1981) observed reduced bone ash and plasma calcium
and phosphorus when chicks were given large doses of
vitamin E. Abawi and Sullivan (1989) found that feeding
high levels of vitamin A decreased body weight when
dietary vitamin D was low; however, increasing dietary
vitamin D reversed the effect producing a significant A
by D interaction. The administration of single high
levels of vitamins A or D3 has been shown to affect
growth and bone metabolism. Davies and Moore (1934),
and Moore and Wang (1945) showed that hypervitami-
Received for publication June 10, 1997.
Accepted for publication November 19, 1997.
1Supported by state and Hatch funds allocated to the Georgia
Agricultural Stations of the University of Georgia.
2To whom correspondence should be addressed:
[email protected]
Abbreviation Key: UV = ultraviolet; vitamin D3 = cholecalciferol.
570
UTILIZATION OF CHOLECALCIFEROL WITH HIGH VITAMIN A AND E
TABLE 1. Composition of the basal diet
Ingredients
Ground yellow corn
Soybean meal (dehulled)
Poultry fat
Dicalcium phosphate
Limestone
Iodized sodium chloride
DL-methionine
Vitamin B premix1
Mineral premix2
Calculated composition
Crude protein
ME, kcal/kg
Calcium
Phosphorus, nonphytate
Amount
(%)
55.86
35.00
5.00
1.86
1.28
0.45
0.20
0.25
0.10
22.08
3,174.00
0.98
0.47
1Vitamin B premix provided in milligrams per kilogram diet
(except as noted): riboflavin, 4.4; calcium pantothenate, 12; nicotinic
acid, 44; choline Cl, 220; vitamin B12, 9 mg; vitamin B6, 3; thiamin (as
thiamin mononitrate), 2.2; folic acid, 3; biotin, 0.3; and ethoxyquin, 125.
2Trace mineral premix provided in milligrams per kilogram diet:
MnO2, 222; ZnO, 150; FeSO4·7H2O, 200; FeCO3, 83; CuSO4·5H2O, 29;
and Ca(IO3)2, 15; Na2SeO3, 0.22.
However, bone ash has proved to be a very sensitive
measure relative to dietary need for vitamin D in broiler
chicks. In preliminary studies, we used HPLC techniques to measure vitamins A and E, and bone
parameters (bone ash and rickets) for vitamin D3 status
in broiler chicks, in an attempt to determine the
nutritional relationships among these vitamins. We
found that the nutritional antagonism occurs, at least in
large proportion, at the intestinal absorption level
(Aburto and Britton, unpublished observations). Edwards et al. (1994) reported that birds fed no vitamin D3
that were exposed to ultraviolet light from battery
fluorescence tubes required 800 to 1,600 IU/kg of
dietary vitamin D3 to provide maximum response for
16-d body weight, gain:feed ratio, bone ash, and plasma
calcium, and reduction of rickets comparable to the UV
light values. Ultraviolet light causes a photochemical
reaction in the skin, in which 7-dehydrocholesterol is
converted to previtamin D and then to vitamin D
(Holick, 1981). If the nutritional antagonism among
vitamins A, D3, and E occurs at the intestinal level prior
to absorption and vitamin D3 is supplied by exposure to
UV light then the feeding of high levels of dietary
vitamins A and E should not effect vitamin D3
metabolism. The purpose of the experiments reported
herein was to elucidate the effects of high dietary levels
of vitamins A and E on the utilization of vitamin D3 by
broiler chicks when vitamin D3 was supplied in the diet
or supplied by UV light induction.
3Arm-a-lite, Thermoplastic Processes,
4Hoffmann-La Roche Co., Nutley, NJ
Stirling, NJ 07980.
07110.
571
MATERIALS AND METHODS
Day-old male (Ross × Ross) broiler chicks were used
in all experiments. Four replicates of 10 chicks each were
fed each dietary treatment. Chicks were wing-banded
and housed in electrically heated battery brooders with
wire mesh floors. The temperature of the room was
maintained at 22 C. Feed and water were provided for
ad libitum consumption and all experiments were
conducted for 16 d. Experiments were approved by the
University of Georgia Animal Care Committee. The
basal diet, shown in Table 1, was used in all experiments. Sunlight was excluded from the room by taping
black plastic over the windows. The overhead fluorescent lights in the room were fitted with Arm-a-Lite3
sleeves, FR312W-T-12, to prevent emission of ultraviolet
light into the room. The fluorescent lights used in the
batteries were General Electric, F15T8-CW, providing
3.4% of the wattage in the ultraviolet range (260 to 400
nm). These lights were covered with plastic sleeves to
prevent exposure of the chicks to UV light. A diagram of
the configuration of the pens and lights in relation to
chicks is described by Edwards et al. (1994).
At the termination of the experiments, birds were
weighed by pen and their feed consumption recorded.
They were then killed by carbon dioxide asphyxiation
and examined for vitamin D-type rickets without
knowledge of treatment. The birds were diagnosed as
having rickets when the subepiphyseal growth-plate
band was lengthened (Long et al., 1984). The degree was
scored on a 0 to 3 basis, with 0 being no rickets and 3
very severe rickets. The left tibia was removed for bone
ash determination on a dry fat-free basis (AOAC, 1995).
Experimental Design
Experiment 1. This experiment was conducted to
determine the effects of feeding increasing levels of
vitamin A on the utilization of vitamin D3 by broiler chicks
and evaluate whether the effect of vitamin A occurs at the
absorption level. Six levels of vitamin A (as retinyl acetate4
5,000, 10,000, 20,000, 40,000, 80,000, and 160,000 IU/kg)
were added to the basal diet. No vitamin D3 was
supplemented to the basal diet. However, all pens of
chicks were exposed to UV light from the fluorescent
lights detailed above in the arrangement described by
Edwards et al. (1994). The other fat-soluble vitamins were
added individually (20 IU/kg of vitamin E as dl-atocopheryl acetate4 and 2 mg/kg of vitamin K as
menadione sodium bisulfite4) to the basal diet. The
sources of the vitamins used in these experiments were
commercial concentrate vitamins. Vitamins A, D3,4 and E
were spray-dried, water-dispersible products. Vitamin A
activity was 500,000 IU/g; vitamin D3 activity was 500,000
IU/g; vitamin E activity was 500 IU/g; and vitamin K had
33% menadione. These concentrated forms of the vitamins
were premixed with rice hulls where appropriate for
mixing into the feed. Analysis of variance and simple
572
ABURTO AND BRITTON
TABLE 2. Effects of dietary vitamin A on 16-d body weight, gain:feed ratio, bone ash, and the incidence and severity
of rickets in broiler chicks receiving ultraviolet light (UV) and no dietary cholecalciferol, Experiment 1
Treatments
Gain:feed
ratio1
Bone
ash1,2
(g/chick)
422
426
427
420
398
402
10
(g:g)
0.715
0.799
0.765
0.750
0.753
0.738
0.017
(%)
38.9
37.5
37.9
38.1
38.2
37.9
0.77
df
5
0.22
0.04
0.84
0.93
0.76
0.07
1
0.02
0.43
0.81
0.84
0.78
0.06
UV
(IU/kg)
5,000
10,000
20,000
40,000
80,000
160,000
Pooled SEM
(+/–)
+
+
+
+
+
+
ANOVA
Source
Vitamin A
Regression analysis
Vitamin A
Rickets
16-d
BW1
Vitamin A
Score1
0.4
0.4
0.5
0.5
0.4
0.4
0.06
Probabilities
Incidence1
24
23
28
26
24
24
2
No. 3 Score1
(%)
11
3
9
10
13
13
2
1Means
of four pens per treatment with 10 chicks per pen.
2Percentage of dry fat-free bone.
regression analysis for levels of vitamin A were computed
(SAS Institute, 1990).
Experiment 2. This experiment was conducted to
determine the effects of feeding low and high levels of
vitamin A on the absorption of vitamin D3 by broiler
chicks. Half of the 48 pens of chicks were exposed to UV
light from the fluorescent lights and the other half of the
chicks were placed in pens with sleeves covering the
fluorescent light to prevent exposure to UV light. Two
supplemental levels of vitamin A (1,500 and 45,000 IU/kg)
and three levels of vitamin D3 (0, 500, and 2,500 IU/kg)
were added to the basal diet. Vitamins E and K were
maintained constant by adding 20 IU/kg and 2 mg/kg,
respectively, in the basal diet. The experimental design
was a 2 × 2 × 3 factorial arrangement of treatments and the
data were analyzed by analysis of variance with UV light,
vitamin A, and vitamin D3 as main effects. This analysis
was performed overall and by category of UV light (with
or without) (SAS Institute, 1990).
Experiment 3 was conducted to determine the effects of
feeding low and very high levels of vitamin E on the
absorption of vitamin D3 by broiler chicks. Half of the 48
pens of chicks were exposed to UV light from the
fluorescent lights and the other half of the chicks were
placed in pens with sleeves covering the fluorescent light
to prevent exposure to UV light. Two supplemental levels
of vitamin E (10 and 10,000 IU/kg) and three levels of
vitamin D3 (0; 500; and 2,500 IU/kg) were added to the
basal diet. Vitamins A and K were maintained constant by
adding 8,000 IU/kg and 2 mg/kg, respectively, to the
basal diet. The experimental design was a 2 × 2 × 3 factorial
5Section N-31, Techincon
Autoanalyzer Methodology, (1969) Techincon Corp., Tarrytown, NY 10951.
6Section N-46, Techincon Autoanalyzer Methodology, (1969) Techincon Corp., Tarrytown, NY 10951.
7Sigma Chemical Co., St Louis, MO 63178-9916.
arrangement of treatments and the data were analyzed by
analysis of variance with UV light, vitamin E, and vitamin
D3 as main effects. This analysis was performed overall
and by category of UV light (SAS Institute, 1990).
Plasma and Tissue Analysis
At termination of Experiments 2 and 3, blood samples
were obtained from two birds per pen by cardiac
puncture, and the plasma was analyzed for total Ca5 and
dialyzable P.6 Plasma vitamin A and E concentrations
were also determined from the same plasma samples by
HPLC analysis. Vitamins A and E were extracted from the
plasma using the method of Jansson et al. (1981). The
plasma was extracted with ethanol and hexane, the top
layer removed, dried, and resuspended in ethanol for
HPLC injection. The HPLC method was the method
described by Hatam and Kayden (1979), except that a
spectrophotometric detector was used at 292 nm, which
allowed detection of vitamin A and E in the same analysis.
All-trans-retinol and a-tocopherol were used as standards.7
Liver samples from the same two birds used to obtain
plasma in Experiment 2 were extracted by the procedure
of Buttriss and Diplock (1984). Vitamin A was extracted
with hexane following saponification. The HPLC analysis
was conducted as described above.
RESULTS
Experiment 1
Regression analysis showed a significant (P < 0.02)
decrease in body weight at vitamin A levels exceeding
40,000 IU/kg (Table 2). Gain:feed ratio was significantly
decreased (P < 0.04) by the same levels of vitamin A;
however, neither bone ash nor rickets were affected by
573
UTILIZATION OF CHOLECALCIFEROL WITH HIGH VITAMIN A AND E
TABLE 3. Effects of ultraviolet light (UV) exposure, and different levels of vitamin A and cholecalciferol (D3) on 16-d body
weight, gain:feed ratio, bone ash, incidence and severity of rickets, plasma calcium, plasma phosphorus,
plasma vitamin A, and liver vitamin A in broiler chicks, Experiment 2
Treatments
UV
A
Rickets
16-d
BW1
D3
(+/–)
(IU/kg)
+
1,500
0
+
1,500
500
+
1,500
2,500
+
45,000
0
+
45,000
500
+
45,000
2,500
–
1,500
0
–
1,500
500
–
1,500
2,500
–
45,000
0
–
45,000
500
–
45,000
2,500
Pooled
SEM
Main effect means
UV
+
–
A
1,500
45,000
D3
0
500
2,500
ANOVA
Source
UV
A
D3
UV × A
UV × D3
A × D3
UV × A × D3
UV (+)
A
D3
A × D3
UV (–)
A
D3
A × D3
Gain:
feed1
(g/chick) (g:g)
355
0.739
376
0.753
363
0.722
348
0.703
334
0.712
354
0.714
271
0.738
327
0.734
329
0.715
272
0.734
309
0.737
334
0.671
df
1
1
2
1
2
2
2
Bone
ash1,2
Plasma concentrations
#3
Score1
Score1
Inc1
(%)
38.7
39.5
40.2
38.1
39.2
39.6
24.5
33.2
38.3
23.6
30.5
37.4
0.3
0.2
0.2
0.3
0.1
0.1
3.0
2.3
1.1
3.0
2.8
1.3
10
13
8
13
5
8
100
82
43
100
95
45
8
3
3
3
0
3
100
72
30
100
88
40
(%)
Ca3
P3
(mg/100 mL)
7.7
5.1
7.3
5.3
6.4
5.4
8.0
5.2
8.2
5.2
6.7
6.3
5.8
6.6
7.7
5.0
8.2
5.1
6.2
5.6
7.5
4.8
8.1
5.0
A3
Liver
A4
(mg/mL)
0.19
0.16
0.16
0.32
0.26
0.22
0.73
0.57
0.49
1.12
0.33
0.24
(mg/g)
2.6
2.5
2.2
71.6
67.7
58.7
2.8
2.8
2.5
85.4
84.8
71.7
12
0.027
0.4
0.1
5
5
0.5
0.4
0.13
5.1
355a
307b
0.724
0.722
39.2a
31.2b
0.2b
2.3a
9b
77a
3b
72a
7.6
7.2
5.4
5.3
0.22b
0.58a
34.2b
41.7a
337x
325x
0.733
0.712
35.7x
34.7y
1.2x
1.3x
43x
44x
36x
39x
7.2
7.6
5.4
5.3
0.38x
0.41x
2.6y
73.3x
311f
336e
345e
0.729
0.733
0.706
31.2g
35.6f
38.8e
1.6e
1.3f
0.7g
56e
49f
26g
53e
41f
19g
6.9
7.7
7.6
5.6
5.1
5.4
0.59e
0.33f
0.28g
40.6e
39.5e
33.8e
<0.001
<0.001
<0.001
0.05
<0.001
0.35
0.12
<0.001
0.53
<0.001
0.10
<0.001
0.66
0.20
<0.001
0.68
<0.001
0.26
<0.001
0.93
0.16
<0.001 0.89
0.08
0.19
<0.001 0.31
0.25
0.68
0.007 0.59
0.17
0.98
0.88
0.54
Probabilities
<0.001
0.30
0.35
0.15
<0.001
0.09
0.06
0.16
<0.001
<0.001
0.24
0.90
0.49
0.56
0.87
0.60
0.12
0.10
0.005
0.29
0.50
<0.001
0.69
0.005
0.39
0.03
0.14
0.22
0.02
<0.001
0.14
0.02
0.96
0.18
0.96
1
2
2
0.07
0.84
0.32
0.23
0.86
0.81
0.05
<0.001
0.82
0.27
0.44
0.63
0.56
0.70
0.46
0.18
0.30
0.59
0.08
0.31
0.67
0.39
0.10
0.32
0.002
0.17
0.62
<0.001
0.48
0.53
1
2
2
0.64
<0.001
0.53
0.52
0.23
0.65
0.002
<0.001
0.14
0.14
<0.001
0.41
0.24
<0.001
0.43
0.10
<0.001
0.43
0.97
<0.001
0.79
0.17
0.01
0.46
0.82
0.02
0.18
<0.001
0.25
0.29
a,b;e–g;x,yMeans
within a variable with no common superscript differ significantly (P < 0.05).
of four pens per treatment with 10 chicks per pen.
2Percentage of dry fat-free bone.
3Means of 8 samples from two chicks per pen per treatment.
4Means of 8 livers from two chicks per pen per treatment.
1Means
increasing the dietary level of vitamin A, indicating that
the UV light was meeting most of the vitamin D3 need of
the chicks and that vitamin A influence on growth was
caused by an effect on something other than vitamin D3.
Experiment 2
Groups of birds exposed to radiation from unfiltered
fluorescent light had higher body weight and bone ash
and a lower incidence of rickets than birds exposed to
filtered fluorescent light (Table 3). Plasma and liver
vitamin A concentrations were lower in birds exposed to
unfiltered fluorescent light than those exposed to filtered
fluorescent light. Gain:feed ratio, plasma calcium, and
plasma phosphorus were not influenced by UV light.
In chicks fed 0 IU/kg of vitamin D with or without UV
light there was no change in body weights when 45,000
IU/kg of vitamin A was consumed. The same was true for
chicks fed 2,500 IU/kg of vitamin D3, but body weight was
decreased by 45,000 IU/kg of vitamin A (P < 0.08) in the
chicks fed the marginal level of vitamin D3 (500 IU/kg).
The 45,000 IU/kg level of vitamin A significantly (P <
574
ABURTO AND BRITTON
0.001) reduced bone ash, but only at the 500 IU/kg level of
vitamin D3 (500 IU/kg) when the birds were not exposed
to UV light. Vitamin D-type rickets, plasma calcium,
plasma phosphorus, and plasma vitamin A concentration
were not significantly influenced by dietary vitamin A.
Liver vitamin A significantly increased (P < 0.001) when
45,000 IU/kg of vitamin A was fed.
When dietary vitamin D3 was changed from 0 to 500
IU/kg, a significant (P < 0.01) increase in body weight,
bone ash, and plasma vitamin A concentration, and
decreases in rickets score, rickets incidence and number 3
scores were seen. When dietary vitamin D3 was increased
to 2,500 IU/kg, a further increase in bone ash and a
decrease in rickets were seen. Plasma vitamin A concentration was significant decreased (P < 0.02) by dietary
vitamin D3 only when the birds were not exposed to UV
light. The response to vitamin D3 for plasma calcium
approached significance (P < 0.09) and no effects were
observed on gain:feed, plasma phosphorus, and liver
vitamin A.
Interactions. A significant interaction (P < 0.05)
between UV light and vitamin A was observed for bone
ash and liver vitamin A concentration. Bone ash was
decreased by high levels of vitamin A but only when birds
were not exposed to UV light. Liver vitamin A was
reduced by exposure to UV light but it increased by
increasing vitamin A in the diet. When birds were not
exposed to UV light, body weight, bone ash, and plasma
calcium all decreased, with a corresponding increase in
rickets. The addition of dietary vitamin D3 prevented
these effects (P < 0.05).
Experiment 3
Chicks exposed to UV light had significantly higher (P
< 0.01) body weight, bone ash, and plasma calcium, and
reduced rickets, plasma and liver vitamin A concentrations compared to chicks receiving no UV light (Table 4).
High dietary vitamin E significantly (P < 0.05) reduced
body weight, bone ash, and plasma calcium, and increased rickets. However, the effect of high dietary
vitamin E on body weight, bone ash, and rickets was more
severe at the marginal level of vitamin D3 (500 IU/kg) and
when the birds were exposed to filtered fluorescent light.
No effect of high level of vitamin E was observed in any of
the groups when vitamin D3 was not supplemented to the
basal diet. The response to increasing dietary vitamin D3
was significant (P < 0.05) for all the criteria measured
except plasma phosphorus. Chicks fed 500 IU of D3/kg
had maximum vitamin E levels in the plasma and plasma
vitamin E declined slightly when 2,500 IU of D3/kg was
fed.
Interactions. A significant interaction (P < 0.05)
between UV light and vitamin E was observed for plasma
vitamin E concentration, and the same interaction approached significance (P < 0.06 and P < 0.09) for the
severity of rickets (number 3 scores) and plasma calcium,
respectively. The high level of dietary vitamin E greatly
increased plasma vitamin E when birds were not exposed
to UV light. The interaction between UV light and vitamin
D3 was highly significant (P < 0.001) for body weight, bone
ash, rickets score, rickets incidence, number 3 scores, and
plasma calcium, and approached significance (P < 0.07) for
gain:feed ratio. In the absence of UV light, body weight,
bone ash, and plasma calcium increased and the values for
the rickets variables decreased when vitamin D3 was
added to the diet; however, this response to D3 was much
smaller when UV light was present. The interaction of
vitamins E by D3 was significant (P < 0.05) for bone ash,
rickets score, and severity of rickets (number 3 scores).
High dietary vitamin E decreased bone ash and increased
rickets that was prevented by vitamin D3. The three-way
interaction among UV light, vitamin E, and vitamin D3
was significant (P < 0.05) for bone ash, rickets score, rickets
incidence, number 3 scores, and plasma calcium. The high
dietary vitamin E reduced bone ash and plasma calcium
and increased rickets in the absence of UV light and
presence of 500 IU/kg vitamin D3, but these changes were
corrected by the addition of 2,500 IU/kg vitamin D3.
DISCUSSION
The results of the experiments described above
indicate that the nutritional antagonism among vitamins
A, D3, and E after absorption from the intestinal tract is
of minor importance. Based on the criteria measured,
high dietary levels of vitamins A and E did not interfere
with the metabolism of vitamin D3 when it was
synthesized in the skin from UV light exposure. When
the birds were exposed to filtered fluorescent light (no
UV), high dietary levels of vitamins A and E significantly affected the utilization of dietary vitamin D3,
which reduced body weight, bone ash, and plasma
calcium and increased rickets; however, these changes
occurred only at marginal dietary vitamin D3 (500 IU/
kg). In Experiment 1, feeding vitamin A at 80,000 IU/kg
of diet caused a small reduction in body weight. This
weight reduction would appear to be caused by
something other than a vitamin A effect on vitamin D3,
as bone ash and the incidence and severity of rickets
were not affected. No vitamin D3 was supplemented in
the diet of these birds, but all groups were exposed to
direct fluorescent light that provided about 3.4% of the
wattage in the UV range (260 to 400 nm) (Edwards et al.,
1994). In experiments from our laboratory (unpublished
observations), body weight was reduced when vitamin
A was fed at 40,000 IU/kg of diet. In these experiments,
a marginal vitamin D3 (500 IU/kg) level was added to
the diet and all birds were exposed to filtered fluorescent light (no UV). These results indicated that 500 IU/
kg of diet of vitamin D3 appeared not to be enough to
produce maximum tibia bone ash and to control rickets
when there was no UV light and that increasing dietary
vitamin A decreased bone ash and increased rickets. In
Experiment 1, it appeared that the UV light alone was
575
UTILIZATION OF CHOLECALCIFEROL WITH HIGH VITAMIN A AND E
TABLE 4. Effects of ultraviolet light (UV) exposure, and different levels of vitamin E and cholecalciferol (D3) on 16-d body
weight, gain:feed ratio, bone ash, incidence and severity of rickets, plasma calcium, plasma phosphorus,
and plasma vitamin E in broiler chicks, Experiment 3
Treatments
UV
E
D3
(+/–)
(IU/kg)
+
10
0
+
10
500
+
10
2,500
+
10,000
0
+
10,000
500
+
10,000
2,500
–
10
0
–
10
500
–
10
2,500
–
10,000
0
–
10,000
500
–
10,000
2,500
Pooled
SEM
Main effect means
UV
+
–
E
10
10,000
D3
0
500
2,500
ANOVA
Source
UV
E
D3
UV × E
UV × D3
E × D3
L × E × D3
UV (+)
E
D3
E × D3
UV (–)
E
D3
E × D3
16-d
BW1
Gain:feed1
Bone
ash1,2
(g/chick)
427
449
429
409
391
387
305
417
432
305
379
410
(g:g)
0.788
0.767
0.744
0.707
0.696
0.737
0.754
0.780
0.705
0.764
0.765
0.726
13
Rickets
Score1
Inc1
(%)
36.5
38.9
39.0
35.3
38.1
38.3
24.5
36.0
38.6
23.6
31.9
38.0
0.3
0.1
0.0
0.8
0.2
0.1
2.8
0.4
0.0
2.9
1.9
0.1
13
5
0
38
8
6
100
25
0
100
60
3
0.019
0.5
0.2
415a
375b
0.740a
0.749a
37.7a
32.1b
0.3b
1.4a
11b
48a
410x
380y
0.756x
0.732y
35.6x
34.2y
0.6y
1.0x
361f
409e
414e
0.753e
0.752e
0.728f
30.0g
36.2f
38.5e
1.7e
0.6f
0.04g
Plasma concentrations
#3
Score1
(%)
5.7
5
5
0
18
5
0
100
18
0
100
53
3
3.7
Ca3
P3
(mg/100 mL)
8.6
4.5
8.9
4.9
9.5
5.1
8.6
3.8
9.2
4.6
9.2
4.3
6.1
4.6
9.1
4.1
8.9
4.9
5.2
4.2
8.0
4.2
9.2
4.3
E3
(mg/mL)
1.0
3.3
2.7
14.0
19.3
19.0
1.2
4.1
4.0
21.9
27.0
23.4
0.5
0.7
2.1
5b
45a
9.0a
7.8b
4.5
4.4
9.9b
13.6a
24y
36x
21y
30x
8.5x
8.2x
4.7
4.2
2.7y
20.8x
63e
24f
2g
56e
20f
1g
7.1g
8.8f
9.2e
4.3
4.4
4.7
9.5f
13.4e
12.3e
<0.001
0.04
<0.001
0.09
<0.001
0.49
0.03
0.46
0.03
0.26
0.48
0.26
0.50
0.98
0.004
<0.001
0.04
0.02
0.87
0.70
0.72
df
1
1
2
1
2
2
2
<0.001
<0.001
<0.001
0.21
<0.001
0.11
0.99
0.43
0.04
0.01
0.13
0.07
0.16
0.50
<0.001
<0.001
<0.001
0.12
<0.001
0.05
0.03
<0.001
<0.001
<0.001
0.14
<0.001
0.009
<0.001
1
2
2
0.004
0.70
0.41
0.005
0.75
0.17
0.09
<0.001
0.91
0.10
0.04
0.44
0.10
0.02
0.32
0.29
0.08
0.32
0.80
0.02
0.46
0.04
0.16
0.72
<0.001
0.10
0.62
1
2
2
0.03
<0.001
0.24
0.71
0.02
0.62
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.02
<0.001
0.06
0.33
0.43
0.69
<0.001
0.28
0.77
Probabilities
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.80
0.06
<0.001
<0.001
0.21
0.01
0.004
<0.001
a,b;e–g;x,yMeans
within a variable with no common superscript differ significantly (P 0.05).
of four pens per treatment with 10 chicks per pen.
2Percentage of dry fat-free bone.
3Means of 8 samples from two chicks per pen per treatment.
1Means
not enough to produce maximum bone ash or to control
rickets, but these parameters were not affected by
dietary vitamin A. There is a possibility that under the
conditions of our studies, the birds exposed to fluorescent light were unable to synthesize enough vitamin D3
by photolysis of UV light. In contrast, Edwards et al.
(1994) reported that the exposure to UV light seemed to
give maximum response for all of the criteria that they
measured, including 16-d body weight, gain:feed ratio,
bone ash, plasma calcium, and the incidence of rickets,
as compared to values observed for birds receiving 800
or 1,600 IU/kg of vitamin D3.
Birds exposed to UV light in Experiment 2 showed a
small positive response in body weight, increased bone
ash, and lower incidence of rickets when 500 IU/kg of
vitamin D3 was added to the diet, suggesting that UV
light alone was probably not enough to support
maximum response of these criteria. This positive
response to dietary vitamin D3 was produced only when
vitamin A was added at 1,500 IU/kg of diet, because
vitamin A (45,000 IU/kg) interfered with dietary
vitamin D3 (500 IU/kg) in birds exposed to UV light.
This effect was more obvious when the birds were not
exposed to UV light and all their vitamin D3 came from
576
ABURTO AND BRITTON
the diet. Another observation that supports the inhibition of absorption of vitamin D3 by high dietary levels of
vitamin A was when the birds were not exposed to UV
light and were not supplemented with vitamin D3 in the
diet. These groups did not show any effect of high
dietary vitamin A on body weight (271 vs 272 g), bone
ash (24.5 vs 23.6%), incidence and severity of rickets (100
vs 100%), and plasma calcium (5.8 vs 6.2 mg/100 mL).
In Experiment 3, adding vitamin D3 (500 IU/kg) to
the diet of birds exposed to UV light gave a slight
positive response in body weight, an increase in bone
ash, and a small decline in rickets, showing again that
UV light without supplementary vitamin D3 was
probably not adequate. This positive effect of dietary
vitamin D3 to increase growth and bone ash was
observed at the low level of vitamin E (10 IU/kg) when
500 IU/kg of vitamin D3 was fed; however, at the very
high dietary level of vitamin E (10,000 IU/kg), 500 IU/
kg of dietary vitamin D3 overcame the bone problems
but did not return growth to normal even at 2,500 IU/
kg of diet, which suggests that high dietary vitamin E
may affect something other than vitamin D3, causing the
growth depression. Birds exposed to filtered fluorescent
light (no UV light) were severely affected by the higher
level of vitamin E (10,000 IU/kg) when the level of
vitamin D3 was supplemented at the marginal level (500
IU/kg). The effect was seen in body weight (417 vs 379
g), bone ash (36.0 vs 31.9%), rickets incidence (25 vs
60%), rickets number 3 scores (18 vs 53%), and plasma
calcium (9.1 vs 8.0 mg/100 mL). As seen with high
vitamin A (45,000 IU/kg) in Experiment 2, birds without
UV light and without dietary vitamin D3 showed no
effect of high dietary vitamin E on body weight (305 vs
305 g), bone ash (24.5 vs 23.6%), and incidence and
severity of rickets (100 vs 100%). These results again
suggest that the most important quantitative effect of
high dietary levels of vitamins A and E on the
utilization of vitamin D3 occur at the intestinal level
prior to or during the absorption process.
The results described above are in agreement with the
findings of other investigators that reported that extra
vitamin D3 protected the rat (Vedder and Rosenberg,
1938), dog (Frey et al., 1975), cattle (Payne and Manston,
1967), and poultry (Taylor et al., 1968; Veltmann and
Jensen, 1985; Veltmann et al., 1986, 1987) against vitamin
A toxicosis. March et al. (1973) and Murphy et al. (1981)
reported that excess vitamin E can be toxic for chicks.
They could not completely prevent the toxicity with
vitamin D3, but the highest level they fed was 500 IU/kg
of diet. Furthermore, when birds were exposed to UV
light, we did not find any effect of feeding high dietary
levels of vitamins A and E, suggesting that internally
produced vitamin D3 is not affected by high dietary
levels of these vitamins. This approach, using UV light
to produce vitamin D3 compared to dietary vitamin D3,
has not been used before in the experimental designs of
experiments looking for nutritional antagonisms of
vitamins A and E on vitamin D3.
Although the absorption of vitamin D3 was not
measured in these experiments, the evidence points to
absorption as the major cause for the adverse effect of
high levels of vitamins A and E on vitamin D3;
suggestive of an antagonism of vitamin A and E on
Vitamin D3. In the marginal vitamin D3 diet (500 IU/
kg), 12.5 mg/kg was added to the diet and even with the
high vitamin D3 diet (2,500 IU/kg) only 62.5 mg/kg was
added to the diet. In contrast, vitamin A was added at
450 mg/kg (1,500 IU/kg) or 13.5 mg/kg (45,000 IU/kg)
and vitamin E was added at 10 mg/kg (10 IU/kg) or
10,000 mg/kg (10,000 IU/kg). If the vitamins share any
common mechanism of absorption, then by mass action
it is apparent that vitamin E and vitamin A would be
favored over vitamin D3. For example, if 500 IU/kg of
dietary vitamin D3 is expressed on a molar basis
compared to 45,000 IU/kg of vitamin A or 10,000 IU/kg
of vitamin E, the molar concentrations are 0.0325
micromolar for vitamin D3, 0.0471 millimolar for
vitamin A and 21.1506 millimolar for vitamin E (dl-atocopherol). Although there are large differences in
molar concentrations of vitamin A and vitamin E from
vitamin D3, increasing the molar concentration of
vitamin D3 to 0.1625 micromolar (2,500 IU/kg of diet)
overcame the problems caused by the high levels of
vitamin A and vitamin E. It would appear that studies
to work out the proper molar ratios among these
vitamins are needed.
ACKNOWLEDGMENT
We wish to thank the Hoffmann-LaRoche Company
for supplying the vitamins for these experiments.
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