207
Biochimica et Biophysica Acta, 475 (1977) 207--216
© Elsevier/North-Holland Biomedical Press
BBA 98861 ANTIBODY SYNTHESIS STIMULATION BY VITAMIN A IN CHICKENS
Z.K. LEUTSKAYA a and D. FAIS b a Laboratory of Helmintology of the U.S.S.R. Academy of Sciences and b Laboratory of Bioorganic Chemistry of Moscow State University, Moscow (U.S.S.R.)
(Received July 26th, 1976) (Revised manuscript received October 25th, 1976) Summary The effect of vitamin A on the immune response of chickens on primary and secondary immunization has been studied. It is demonstrated that the antibody content in chickens depends on the dose of vitamin A in the diet. In chickens fed a high dose of vitamin A in the diet the antibody content in the serum is 2 to 5 times as high as in chickens which have not been given vitamin A. An additional dose of vitamin A administered perorally any day following reimmunization increases the content of antibodies; the effect is most pronounced when vitamin A is given on the third day after reimmunization. In the period of maximal synthesis of antibodies (during the fourth day after reimmunization) labelled retinol is incorporated into the spleen of reimmunized chickens 13 times as actively as in the spleen of control chickens. In the in vitro experiments with antibody synthesis b y spleen cells it has been demonstrated that addition of retinyl palmitate to the incubation medium enhances antibody synthesis during incubation. Addition of retinyl palmitate to the spleen cells of vitamin A-deficient chickens restores the synthesis up to the level observed in the control cells. The suggestion is discussed that the effect of vitamin A on the immune process is realized at the level of immunoglobulin synthesis.
Introduction Vitamin A was recognized as being anti-infectious as early as in 1928 [1], b u t what underlied this definition was for a long time unknown. One of the authors of this paper demonstrated in 1964 that vitamin A deficiency in the diet decreases the antibody level in blood serum of chickens [2]. Then the same author [3,4] demonstrated the existence of a correlation b e t w e e n the level of vitamin A in chick liver and the quantity of antibodies synthesized. Recently results were published b y several authors [5--8] demonstrating that
208 the effect of vitamin A in the immunization process is due to its role as an adjuvant. Analysis of our previous data and the results of the recent investigations show that the effect of vitamin A on the immune response lies not so much in its adjuvanticity, but rather in its participation in antibody synthesis. Materials and Methods
Animals and diets. Use was made of White Leghorn chicks, 2 months old (average b o d y weight 400 g) at the beginning of immunization and 5 m o n t h s old (average b o d y weight 1100 g) at the m o m e n t of reimmunization. All the chicks were given the usual food which allowed normal growth in chickenraising farms until the 5th immunization injection, after which the chicks were kept on the synthetic vitamin A and carotine-deficient diet [9]. The chickens were divided into experimental groups as described in Results. Immunization o f animals. Human serum albumin or salt extraction of Ascaridia galli nematodes were used as the antigen. The chickens were immunized with 10 intraperitoneal injections of 1 ml of the antigen in physiological saline solution. The antigen was administered every two days with a 5--6 day interval after the fifth injection. Human serum albumin was administered in increasing concentrations (1-2.5 mg/ml) [10]. The concentration of the antigen from Ascaridia was 2.8 mg protein per ml of the extract [11]. Reimmunization of chickens was performed two m o n t h s after the first immunization by a single intraperitoneal injection of the respective antigen. Antibody assay. Isolation of antibodies was carried out by the immunochemical m e t h o d of Heidelberger [12] and McDuffie and Kabat [13] as modified by Gurvich [14]. The protein content was determined by the method of Lowry
[15]. Incorporation o f [~4C]retinol. Each chicken was given intraperitoneal 11 pCi retinol [14C] carbinol (6.4 Ci/mol, Radiochemical Centre, Amersham) dissolved in 1 ml of physiological saline solution containing 0.1% Tween-40 and 20% ethanol. Vitamin A from spleen tissue was extracted as described by Glover et al. [16]. The reagents were purified and prepared for extraction according to the method of Roels and Mahadevan [17]. Final chlorophorm extracts of vitamin A were applied to membrane filters and radioactivity of vitamin A was measured in a toluene scintillator using a Nuclear Chicago Mark I counter. Incubation o f spleen cells. Chickens were immunized and reimmunized as described above. On the fourth day after reimmunization the chickens were bled. All further operations were carried out in cold as described [18]. Spleens were excized, placed into cold physiological saline solution, freed from connective tissue, and the pulp was pipetted into cell suspension. The cell suspension was filtered through 4 gauze layers, washed twice with physiological saline solution and divided into 500 mg samples. The samples were incubated in 4 ml of Eagle medium [19] in an atmosphere of 5% CO2 and 95% 02 for 3 h at 37°C under shaking. Retinyl palmitate was added at a concentration of 5 pg per sample in 0.02 ml of 1% ethyl alcohol. To control samples equal amounts of ethyl alcohol were added. The samples were fixed with n-butanol and treated as described by Peters and Anfinsen [10] for antibody assay.
209
Results
Effect of retinyl palmitate on antibody synthesis upon primary immunization At the beginning o f immunization all the chickens were given their usual food. After the fifth injection of the antigen the chickens were divided into three groups in the following way. The first group was given a synthetic diet devoid of vitamin A (vitamin A-deficient group). The second and the third group received an equal amount of the same diet devoid of vitamin A b u t containing retinyl palmitate, the second group 1000 I.U. daily (control group) and the third group 4000 I.U. daily (surplus vitamin A group). This methodological scheme was adopted to avoid metabolic disturbances that occur in the chickens if they are kept on the vitamin A-deficient diet for a long period. 6--8 chickens from each of the three groups were bled on the 7th, 14th and 21st postimmunization day to test the sera antibody content. Fig. 1 shows the results of antibody assay in the blood serum of the chickens immunized by the antigen from Ascaridia. The highest antibody content in the sera of the chickens from all three groups was registered on the 14th postimmunization day; this is in accord with the literature data for intraperitoneal antigen injections. As seen in Fig. 1, the chickens of the vitamin A-deficient group, as compared to those of the control group, display a lower antibody content in the serum. This difference is more pronounced on the seventh day. In the chickens of the surplus vitamin A group there is a higher antibody yield not only as compared to the vitamin A-deficient group (5 times as high on the 7th day, 1.9 times as high on the 14th day and 2.7 times on the 21st day), b u t also with respect to the control chickens (1.4--2 times higher). Similar results were obtained with the chickens immunized with human serum albumin (not shown in the figure).
A~
t66
m!
IO0-B :E
_~
7-TH DAY
2O0
uJ u'J
J46
z
o ~100 207
IO0-B 10
I 7
I t4
_~
2t-TH DAY
t 78 21 DAYSAFTER IMMUNIZATION
Fig. 1. E f f e c t o f v i t a m i n A o n t h e a n t i b o d y c o n t e n t i n c h i c k e n s e r u m o n p r i m a r y i m m u n i z a t i o n . Ascaridia e x t r a c t w a s u s e d as t h e a n t i g e n . A, v i t a m i n A - d e f i c i e n t g r o u p ; B , c o n t r o l g r o u p ; C, s u r p l u s v i t a m i n A . E a c h p o i n t c o r r e s p o n d s to t h e r e s u l t s o b t a i n e d f o r 6 - - 8 c h i c k e n s . V e r t i c a l lines s h o w S . D .
210
Effect of retinyl palmitate on antibody synthesis upon reimmunization T he n e x t series of exper i m ent s was c o n d u c t e d with chickens immunized and reimmunized with h u m a n serum albumin. It is k n o w n that, as a result of reimmunization, the synthesis occurs in a much m ore intensive fashion than after t h e primary immunization, and t he a n t i b o d y c o n t e n t in the serum reaches the m a x i m u m sooner. In contrast to the previous experiments, when the chickens were kept on a diet devoid of vitamin A or containing a certain a m o u n t of it, in this series o f experiments t he chickens were fed as usual or were given a single dose (30 000 I.U. of retinyl palmitate) of vitamin A per os on the day of reimm u n i z a t i o n or on one o f the following days. On the 5th day after reimmunizat i o n th e chickens were bled and the sera a n t i b o d y c o n t e n t was determined. T he results o f these investigations (Fig. 2) show that, firstly, the additional dose o f vitamin A consumed by the chickens in the rei m m uni zat i on period considerably increases the a n t i b o d y c o n t e n t in the serum and, secondly, the degree of vitamin A stimulation of the a n t i b o d y synthesis depends on which day the vitamin was administered. The m a x i m u m effect was observed when the vitamin was given not on t he day of reimmunization, but on the 3rd day after it. On th e 5th day after the second administration of the antigen, t he chickens which had received vitamin A on the 3rd da y after reimmunization proved to have a c o n c e n t r a t i o n of antibodies in the serum (1150 + 55 pg a n t i b o d y / m l of serum) 1.7 times higher than those which had received the same dose of the vitamin o n the day of r e i m m u n i z a t i o n (683 + 28 pg/ml), and 4.6 times higher than the i m m u n e response of t he chickens which had n o t received vitamin A at all (253 + 33 pg/ml). In the chickens which were given vitamin A on the 4th day, the a n t i b o d y c o n t e n t (745 +- 42 pg/ml) also increased as compared to t h a t in the
>. CJ
~IOOO Z 0 3
z ~ 50o Z CONTRO¢
CHICKS
100 DAYS
I
L
[
I
1
2
:5
4
AFTER REIMMUNIZATION WHEN RETINVL
PALMITATE
WAS
ADMINISTERED
F i g . 2. D e p e n d e n c e o f t h e a n t i b o d y c o n t e n t i n c h i c k e n s e r u m o n p e r o r a l a d m i n i s t r a t i o n o f v i t a m i n A in t h e p e r i o d o f r e i m m u n i z a t i o n . A n t i b o d y c o n t e n t o n t h e 5 t h d a y a f t e r r e i m r n u n i z a t i o n . E a c h p o i n t corresponds to the results obtained for 6 or 7 chickens. Vertical lines show S.D.
211 TABLE I I N C O R P O R A T I O N O F [CARBINOL-14C]RETINOL I N T H E S P L E E N O F C O N T R O L A N D R E I M M U N I Z E D CHICKENS. A L L CHICKENS W E R E I M M U N I Z E D W I T H H U M A N S E R U M A L B U M I N (n = 6).
I n c o r p o r a t i o n time
c p m / g w e t tissue
(h)
Control
R eimmunization
3
6 0 0 -+ 8
24
316 + 5
7 6 1 0 -+ 19 P < 0.001 366 + 3 P < 0.001
controls, but was much lower than in the chickens which were given vitamin A on the third day.
Accumulation of retinol in the spleen of control and reimmunized chickens This series of experiments was performed on chickens immunized with human serum albumin. Unimmunized chickens were used as control. Radioactive retinol (11 pCi per chicken) was injected intraperitoneally. The chickens were bled on the 5th postreimmunization day and the radioactivity of vitamin A extracted from the spleen was assayed. The control and reimmunized chickens were given labelled retinol 24 and 3 h before slaughter. As is seen from the data of Table I, within 3 h retinol is incorporated in the spleen of the reimmunized chickens 13 times more intensively than in the spleen of the control chickens. In 24 h, the difference in the labelled vitamin A in the spleen of the control and reimmunized animals is not so pronounced; and compared to the 3 h incorporation is not higher, but even lower. But though this difference is small in the control group, in the reimmunized chickens it is significant. This observation testifies to a considerable (as compared to the control chickens) rate o f utilization of retinol in the spleen of reimmunized chickens.
Effect of retinyl palmitate on in vitro antibody synthesis These experiments on the effect of vitamin A on the immune response of chickens were carried out with spleen cells. To this end, chickens were used on T A B L E II E F F E C T O F R E T I N Y L P A L M I T A T E O N IN V I T R O A N T I B O D Y S Y N T H E S I S I N S P L E E N C E L L S O F C H I C K E N S . A L L C H I C K E N S W E R E I M M U N I Z E D W I T H H U M A N S E R U M A L B U M I N (n = 10).
Statistical indices
M ± P
W i t h o u t retinyl palmitate
With retinyl palmitate
Before incubation 0zg/g of w e t tissue)
A f t e r incubation (/~g/g o f w e t tissue)
Increment (%)
Before incubation (~g/g o f w e t tissue)
A f t e r incubation ~ug/g o f w e t tissue)
Increment
59.0
84.1
42.5
59.0
162.0 4.6
174.6
0.37
2.9 ~0.001
0.37
'(0.001
(%)
Statistical indices
M +P
M +P
G r o u p s of c h i c k e n
Control
Vitamin A deficient
48.0 1.4
56.0 2.9
52.5 1.9
Biochimica et Biophysica Acta, 475 (1977) 207--216
© Elsevier/North-Holland Biomedical Press
BBA 98861 ANTIBODY SYNTHESIS STIMULATION BY VITAMIN A IN CHICKENS
Z.K. LEUTSKAYA a and D. FAIS b a Laboratory of Helmintology of the U.S.S.R. Academy of Sciences and b Laboratory of Bioorganic Chemistry of Moscow State University, Moscow (U.S.S.R.)
(Received July 26th, 1976) (Revised manuscript received October 25th, 1976) Summary The effect of vitamin A on the immune response of chickens on primary and secondary immunization has been studied. It is demonstrated that the antibody content in chickens depends on the dose of vitamin A in the diet. In chickens fed a high dose of vitamin A in the diet the antibody content in the serum is 2 to 5 times as high as in chickens which have not been given vitamin A. An additional dose of vitamin A administered perorally any day following reimmunization increases the content of antibodies; the effect is most pronounced when vitamin A is given on the third day after reimmunization. In the period of maximal synthesis of antibodies (during the fourth day after reimmunization) labelled retinol is incorporated into the spleen of reimmunized chickens 13 times as actively as in the spleen of control chickens. In the in vitro experiments with antibody synthesis b y spleen cells it has been demonstrated that addition of retinyl palmitate to the incubation medium enhances antibody synthesis during incubation. Addition of retinyl palmitate to the spleen cells of vitamin A-deficient chickens restores the synthesis up to the level observed in the control cells. The suggestion is discussed that the effect of vitamin A on the immune process is realized at the level of immunoglobulin synthesis.
Introduction Vitamin A was recognized as being anti-infectious as early as in 1928 [1], b u t what underlied this definition was for a long time unknown. One of the authors of this paper demonstrated in 1964 that vitamin A deficiency in the diet decreases the antibody level in blood serum of chickens [2]. Then the same author [3,4] demonstrated the existence of a correlation b e t w e e n the level of vitamin A in chick liver and the quantity of antibodies synthesized. Recently results were published b y several authors [5--8] demonstrating that
208 the effect of vitamin A in the immunization process is due to its role as an adjuvant. Analysis of our previous data and the results of the recent investigations show that the effect of vitamin A on the immune response lies not so much in its adjuvanticity, but rather in its participation in antibody synthesis. Materials and Methods
Animals and diets. Use was made of White Leghorn chicks, 2 months old (average b o d y weight 400 g) at the beginning of immunization and 5 m o n t h s old (average b o d y weight 1100 g) at the m o m e n t of reimmunization. All the chicks were given the usual food which allowed normal growth in chickenraising farms until the 5th immunization injection, after which the chicks were kept on the synthetic vitamin A and carotine-deficient diet [9]. The chickens were divided into experimental groups as described in Results. Immunization o f animals. Human serum albumin or salt extraction of Ascaridia galli nematodes were used as the antigen. The chickens were immunized with 10 intraperitoneal injections of 1 ml of the antigen in physiological saline solution. The antigen was administered every two days with a 5--6 day interval after the fifth injection. Human serum albumin was administered in increasing concentrations (1-2.5 mg/ml) [10]. The concentration of the antigen from Ascaridia was 2.8 mg protein per ml of the extract [11]. Reimmunization of chickens was performed two m o n t h s after the first immunization by a single intraperitoneal injection of the respective antigen. Antibody assay. Isolation of antibodies was carried out by the immunochemical m e t h o d of Heidelberger [12] and McDuffie and Kabat [13] as modified by Gurvich [14]. The protein content was determined by the method of Lowry
[15]. Incorporation o f [~4C]retinol. Each chicken was given intraperitoneal 11 pCi retinol [14C] carbinol (6.4 Ci/mol, Radiochemical Centre, Amersham) dissolved in 1 ml of physiological saline solution containing 0.1% Tween-40 and 20% ethanol. Vitamin A from spleen tissue was extracted as described by Glover et al. [16]. The reagents were purified and prepared for extraction according to the method of Roels and Mahadevan [17]. Final chlorophorm extracts of vitamin A were applied to membrane filters and radioactivity of vitamin A was measured in a toluene scintillator using a Nuclear Chicago Mark I counter. Incubation o f spleen cells. Chickens were immunized and reimmunized as described above. On the fourth day after reimmunization the chickens were bled. All further operations were carried out in cold as described [18]. Spleens were excized, placed into cold physiological saline solution, freed from connective tissue, and the pulp was pipetted into cell suspension. The cell suspension was filtered through 4 gauze layers, washed twice with physiological saline solution and divided into 500 mg samples. The samples were incubated in 4 ml of Eagle medium [19] in an atmosphere of 5% CO2 and 95% 02 for 3 h at 37°C under shaking. Retinyl palmitate was added at a concentration of 5 pg per sample in 0.02 ml of 1% ethyl alcohol. To control samples equal amounts of ethyl alcohol were added. The samples were fixed with n-butanol and treated as described by Peters and Anfinsen [10] for antibody assay.
209
Results
Effect of retinyl palmitate on antibody synthesis upon primary immunization At the beginning o f immunization all the chickens were given their usual food. After the fifth injection of the antigen the chickens were divided into three groups in the following way. The first group was given a synthetic diet devoid of vitamin A (vitamin A-deficient group). The second and the third group received an equal amount of the same diet devoid of vitamin A b u t containing retinyl palmitate, the second group 1000 I.U. daily (control group) and the third group 4000 I.U. daily (surplus vitamin A group). This methodological scheme was adopted to avoid metabolic disturbances that occur in the chickens if they are kept on the vitamin A-deficient diet for a long period. 6--8 chickens from each of the three groups were bled on the 7th, 14th and 21st postimmunization day to test the sera antibody content. Fig. 1 shows the results of antibody assay in the blood serum of the chickens immunized by the antigen from Ascaridia. The highest antibody content in the sera of the chickens from all three groups was registered on the 14th postimmunization day; this is in accord with the literature data for intraperitoneal antigen injections. As seen in Fig. 1, the chickens of the vitamin A-deficient group, as compared to those of the control group, display a lower antibody content in the serum. This difference is more pronounced on the seventh day. In the chickens of the surplus vitamin A group there is a higher antibody yield not only as compared to the vitamin A-deficient group (5 times as high on the 7th day, 1.9 times as high on the 14th day and 2.7 times on the 21st day), b u t also with respect to the control chickens (1.4--2 times higher). Similar results were obtained with the chickens immunized with human serum albumin (not shown in the figure).
A~
t66
m!
IO0-B :E
_~
7-TH DAY
2O0
uJ u'J
J46
z
o ~100 207
IO0-B 10
I 7
I t4
_~
2t-TH DAY
t 78 21 DAYSAFTER IMMUNIZATION
Fig. 1. E f f e c t o f v i t a m i n A o n t h e a n t i b o d y c o n t e n t i n c h i c k e n s e r u m o n p r i m a r y i m m u n i z a t i o n . Ascaridia e x t r a c t w a s u s e d as t h e a n t i g e n . A, v i t a m i n A - d e f i c i e n t g r o u p ; B , c o n t r o l g r o u p ; C, s u r p l u s v i t a m i n A . E a c h p o i n t c o r r e s p o n d s to t h e r e s u l t s o b t a i n e d f o r 6 - - 8 c h i c k e n s . V e r t i c a l lines s h o w S . D .
210
Effect of retinyl palmitate on antibody synthesis upon reimmunization T he n e x t series of exper i m ent s was c o n d u c t e d with chickens immunized and reimmunized with h u m a n serum albumin. It is k n o w n that, as a result of reimmunization, the synthesis occurs in a much m ore intensive fashion than after t h e primary immunization, and t he a n t i b o d y c o n t e n t in the serum reaches the m a x i m u m sooner. In contrast to the previous experiments, when the chickens were kept on a diet devoid of vitamin A or containing a certain a m o u n t of it, in this series o f experiments t he chickens were fed as usual or were given a single dose (30 000 I.U. of retinyl palmitate) of vitamin A per os on the day of reimm u n i z a t i o n or on one o f the following days. On the 5th day after reimmunizat i o n th e chickens were bled and the sera a n t i b o d y c o n t e n t was determined. T he results o f these investigations (Fig. 2) show that, firstly, the additional dose o f vitamin A consumed by the chickens in the rei m m uni zat i on period considerably increases the a n t i b o d y c o n t e n t in the serum and, secondly, the degree of vitamin A stimulation of the a n t i b o d y synthesis depends on which day the vitamin was administered. The m a x i m u m effect was observed when the vitamin was given not on t he day of reimmunization, but on the 3rd day after it. On th e 5th day after the second administration of the antigen, t he chickens which had received vitamin A on the 3rd da y after reimmunization proved to have a c o n c e n t r a t i o n of antibodies in the serum (1150 + 55 pg a n t i b o d y / m l of serum) 1.7 times higher than those which had received the same dose of the vitamin o n the day of r e i m m u n i z a t i o n (683 + 28 pg/ml), and 4.6 times higher than the i m m u n e response of t he chickens which had n o t received vitamin A at all (253 + 33 pg/ml). In the chickens which were given vitamin A on the 4th day, the a n t i b o d y c o n t e n t (745 +- 42 pg/ml) also increased as compared to t h a t in the
>. CJ
~IOOO Z 0 3
z ~ 50o Z CONTRO¢
CHICKS
100 DAYS
I
L
[
I
1
2
:5
4
AFTER REIMMUNIZATION WHEN RETINVL
PALMITATE
WAS
ADMINISTERED
F i g . 2. D e p e n d e n c e o f t h e a n t i b o d y c o n t e n t i n c h i c k e n s e r u m o n p e r o r a l a d m i n i s t r a t i o n o f v i t a m i n A in t h e p e r i o d o f r e i m m u n i z a t i o n . A n t i b o d y c o n t e n t o n t h e 5 t h d a y a f t e r r e i m r n u n i z a t i o n . E a c h p o i n t corresponds to the results obtained for 6 or 7 chickens. Vertical lines show S.D.
211 TABLE I I N C O R P O R A T I O N O F [CARBINOL-14C]RETINOL I N T H E S P L E E N O F C O N T R O L A N D R E I M M U N I Z E D CHICKENS. A L L CHICKENS W E R E I M M U N I Z E D W I T H H U M A N S E R U M A L B U M I N (n = 6).
I n c o r p o r a t i o n time
c p m / g w e t tissue
(h)
Control
R eimmunization
3
6 0 0 -+ 8
24
316 + 5
7 6 1 0 -+ 19 P < 0.001 366 + 3 P < 0.001
controls, but was much lower than in the chickens which were given vitamin A on the third day.
Accumulation of retinol in the spleen of control and reimmunized chickens This series of experiments was performed on chickens immunized with human serum albumin. Unimmunized chickens were used as control. Radioactive retinol (11 pCi per chicken) was injected intraperitoneally. The chickens were bled on the 5th postreimmunization day and the radioactivity of vitamin A extracted from the spleen was assayed. The control and reimmunized chickens were given labelled retinol 24 and 3 h before slaughter. As is seen from the data of Table I, within 3 h retinol is incorporated in the spleen of the reimmunized chickens 13 times more intensively than in the spleen of the control chickens. In 24 h, the difference in the labelled vitamin A in the spleen of the control and reimmunized animals is not so pronounced; and compared to the 3 h incorporation is not higher, but even lower. But though this difference is small in the control group, in the reimmunized chickens it is significant. This observation testifies to a considerable (as compared to the control chickens) rate o f utilization of retinol in the spleen of reimmunized chickens.
Effect of retinyl palmitate on in vitro antibody synthesis These experiments on the effect of vitamin A on the immune response of chickens were carried out with spleen cells. To this end, chickens were used on T A B L E II E F F E C T O F R E T I N Y L P A L M I T A T E O N IN V I T R O A N T I B O D Y S Y N T H E S I S I N S P L E E N C E L L S O F C H I C K E N S . A L L C H I C K E N S W E R E I M M U N I Z E D W I T H H U M A N S E R U M A L B U M I N (n = 10).
Statistical indices
M ± P
W i t h o u t retinyl palmitate
With retinyl palmitate
Before incubation 0zg/g of w e t tissue)
A f t e r incubation (/~g/g o f w e t tissue)
Increment (%)
Before incubation (~g/g o f w e t tissue)
A f t e r incubation ~ug/g o f w e t tissue)
Increment
59.0
84.1
42.5
59.0
162.0 4.6
174.6
0.37
2.9 ~0.001
0.37
'(0.001
(%)
Statistical indices
M +P
M +P
G r o u p s of c h i c k e n
Control
Vitamin A deficient
48.0 1.4
56.0 2.9
52.5 1.9
