British Journal of Nutriiion (1992), 68, 283-291
Production of chickens with marginal vitamin A deficiency
BY CLIVE E. WEST', S. REINDER SIJTSMA'~2~3*,
JAN H. W. M. ROMBOUT* AND AKKE J. VAN DER ZIJPP3$
' Department o f Human Nutrition, Wageningen Agricultural University, PO Box 8129, 6700 EV
Wageningen, * Department of Experimental Animal Morphology and Cell Biology and Department
HARRY P. F. PETERS't,
of Animal Husbandry, Wageningen Agricultural University, PO Box 338, 6700 AH Wageningen,
(Received 19 February 1991 -Accepted 1 August 1991)
Marginally vitamin A-deficient I-d-old chickens capable of remaining healthy for at least 6 weeks were
produced using a two-generation model. In this model, hens fed on diets with a limited vitamin A content
were used to obtain l-d-old chickens which were marginally deficient in vitamin A. Only hens with a
narrow range of plasma retinol values (0-60-0%5 pmol/l) were satisfactory for this purpose. Above this
range the I-d-old chickens were not marginally vitamin A deficient. Below this range egg production and
hatchability were affected to some extent depending on the degree of vitamin A deficiency. Even when
egg production and hatchability remained at a high level in such birds, the l-d-old chickens produced
were not sufficiently strong to survive the first weeks of life. The advantages of the two-generation model
for producing marginally vitamin A-deficient chickens are the increased uniformity and predictability of
the chickens with respect to body-weight, general health and vitamin A status. However, it does take
about 3 months to produce such chickens.
Vitamin A deficiency: Chicken: Animal model
Animal models have often been used in vitamin A research (Scrimshaw et al. 1968; Sporn
et al. 1984). However, many problems can arise in producing vitamin A deficiency in
animals (Underwood, 1984; Beisel, 1988). Sometimes animals have to be fed diets deficient
in vitamin A for a long time before the deficient state is reached and often large variations
in vitamin A status can be observed within the same diet group. In addition, diets free of
vitamin A can produce sudden and uncontrollable vitamin A deficiency with consequent
loss of appetite and concomitant protein%nergy malnutrition. Signs of vitamin A
deficiency are sometimes irreversible, and together with increased sensitivity to infection,
can lead to death. A few models have been described in which many of these problems have
been overcome (Stowe et al. 1980; Davis & Sell, 1983; Nauss et al. 1985; Pueng-
tomwatanakul & Sirisinha, 1986; Smith et al. 1987). One system which can be used is a
two-generation model in which parent animals are made marginally deficient in vitamin A
(Stowe et al. 1980; Nauss et al. 1985; Smith et al. 1987). Their progeny are also marginally
vitamin A deficient and can be used in studies requiring young animals.
The aim of the present study was twofold : first, to produce l-d-old chickens marginally
deficient in vitamin A for a long period without health problems; second, to document the
advantages of such a two-generation model over a one-generation model based on 1-d-old
chickens with an adequate vitamin A status.
Present addresses * Hendrix Voeders, PO Box I, 5830 MA Boxmeer; t Department of Medical Physiology and
Sports Medicine, University of Utrecht, Vondellaan 24, 3521 GG Utrecht; $Research Institute for Animal
Production 'Schoonoord', PO Box 501, 3700 AM Zeist, The Netherlands.
N U T 68
C. E. WEST A N D OTHERS
MATERIALS A N D METHODS
Animals, diets and experimental design
White Leghorn laying hens (Lohmann Selected Leghorn strain), aged approximately 18
weeks, were obtained from a commercial breeder (Verbeek, Barneveld). On arrival, the
hens were housed individually in a room controlled for temperature (20"), relative humidity
(4&50 %) and light-dark cycles (I6 h dimmed light - 8 h darkness). A commercial diet
(Opfokvoer 2, Rijnzate, Wageningen) was fed for 1 week. After this period the birds were
allocated randomly to five dietary groups, each of twelve hens. The first four groups (P-0,
P-300, P-600 and P-1500) were fed on purified diets varying in vitamin A content (0, 300,
600 or 1500 retinol equivalents (RE)/kg respectively) and the fifth group (N- 1500) was fed
on a diet based on non-purified components with the same amount of vitamin A as in the
fourth group (Table 1). This fifth group was added in order to test for possible differences
between purified and non-purified diets. Water and diets were provided ad lib. and the hens
had free access to oyster shell grit. The laying hens were studied for a total period of 19
weeks during which time feed consumption, body-weight, egg production and plasma
retinol concentration were measured. Hatchability and health status of I-d-old chickens
were determined three times during the experiment (weeks 8, 13 and 18).
In this experiment, two groups were formed from progeny of marginally vitamin A-
deficient hens. These hens were fed on diets containing either 300 or 600 RE/kg for at least
13 weeks, resulting in plasma retinol levels of 0.35-0.50 pmol/l and 0.60-0.85 pmol/l
respectively. Both groups of chickens were fed on a purified diet limited in vitamin A
(120 RE/kg; Table I). In addition, female I-d-old chickens (Lohmann Selected Leghorn
strain) were obtained from a commercial hatchery (Verbeek, Barneveld). These chickens
were allocated to a further three groups. One of those groups was also fed on a purified diet
limited in vitamin A (120 RE/kg); another was fed on a purified diet free of vitamin A; a
third group was also fed on the purified diet free of vitamin A for 2 weeks and then fed on
the diet limited in vitamin A (120 RE/kg). The 1-d-old chickens, twelve birds/group and
one group/cage, were housed as described previously (Sijtsma et a/. 1989~). They were
studied for a period of 6 weeks during which health status and plasma retinol levels were
Sampling of blood
Blood from a wing was collected in heparinized tubes; after centrifugation, plasma was
stored at -20".
Vitamin A in plusma
In Expt 1, retinol levels were determined in the plasma of hens at weeks 0, 5, 8, 1 I, 13, 15,
17 and 19, and in plasma from their I-d-old progeny. In Expt 2, retinol analyses in the
plasma of chickens were carried out at 0, 2, 3.54, and 5-6 weeks. A reversed-phase high-
performance liquid chromatographic (HPLC) method modified from that of Driskell et al.
(1982) was used, as described previously (Sijtsma et al. 1989a, b).
Vitamin A content of diets
In order to determine the content and stability of vitamin A in diets used in Expt 1, analyses
were carried out at 1 and 10 weeks after manufacture as described by Manz & Philipp
MARGINAL VITAMIN A DEFICIENCY IN CHICKENS
Table 1. Composition (g/kg) of the purified and non-purijied diets*
Diet . . .
Vitamin-mineral premix§, 7
- - _ _ _ ~ -
* Diets were manufactured in pelleted form following the guidelines of the National Research Council (1984)
by the Institute of Animal Nutrition and Physiology (IGMB-TNO, Wageningen). The metabolizable energy of the
purified hen and chicken diets, and the non-purified hen diet was 12.54 (3000), 13.38 (3200) and 12.44 (2975) MJ
(kcal)/kg feed, respectively.
t Soya-bean isolate: Purina Protein 500 E, Ralston Purina, St Louis, MO, USA, containing 880 g protein/kg
$ The laying hens had free access to oyster shell grit.
9 The vitamin and mineral premix (10 g/kg purified diet) was prepared with dextrose and contained (mg/kg
diet): thiamin 2.5, riboflavin 5.5, pantothenic acid 15.0, nicotinic acid 50.0, pyridoxine 5.0, biotin in hens 0.10 and
in chickens 015, folic acid in hens 0.45, and in chickens 075, choline chloride in hens 1000 and in chickens 1850,
cyanocobalamin 0.01 5, inositol 100, p-aminobenzoic acid 50.0, cholecalciferol 0.075, m-a-tocopherol 30.0,
menadione 50, L-ascorbic acid 50.0, FeSO,. 7H,O 400, MnO, 150, CuSO,. 5H,O 100, ZnSO,. H,O 200,
Na,SeO,.SH,O 0.3, KI 5, ethoxyquin 100. Vitamin A was added as retinyl acetate and retinyl palmitate, Rovimix
A 500 (156.6 mg retinol/g; F. Hoffmann-La Roche, Basel, Switzerland) in hens: 0, 300, 600 or 1500 retinol
equivalents/kg diet, and in chickens: 0 or 120 retinol equivalents/kg diet.
11 The vitamin and mineral premix (10 g/kg non-purified diet) was prepared with soya-bean flour and contained
(mg/kg diet): riboflavin 5.5, pantothenic acid 15.0, nicotinic acid 30.0, pyridoxine 2.0, biotin 0.05, folic acid 0.1,
choline chloride 500, cyanocobalamin 0.01 5, cholecalciferol 0,075, DL-a-tocopherol 30.0, menadione 5.0, L-
ascorbic acid 50.0, FeSO, .7H,O 400, MnO, 150, CuSO, .5H,O 100, ZnSO,. H,20 200, Na,SeO,. 5H,O 0.3, KI 2,
ethoxyquin 100. Vitamin A was added as retinyl acetate and retinyl palmitate, Rovimix A 500, 1500 retinol
7 Calculation of the retinol and carotene content was based on the amount of vitamin A added to the diet and
on feedstuff analysis tables respectively (Agricultural Research Council, 1975; Allen, 1984; National Research
Council, 1984). All the carotene present was assumed to be p-carotene and the values were divided by 6 to convert
to retinol equivalents. In the N-1500 diet, the p-carotene content was calculated to be 60 /Lg RE/kg, while in the
other diets no p-carotene was present. Analysis of the hens’ diets 1 week after preparation showed that vitamin
A content was more than expected: 420, 720, 1830 and 2040,ug RE/kg for P-300, P-600, P-1500, and N-1500
respectively. After 10 weeks of storage the content decreased to 240, 570, 1790 and 1620 pg RE/kg for P-300, P-
600, P-1500 and N-I500 respectively. No vitamin A was detected in the P-O diet, 1 or 10 weeks after manufacture.
Tapioca (650 g/kg)
Soya-bean flour (50 g/kg)
Vitamin-mineral premix jl,l
All statistical comparisons among treatment groups were performed by one-way analysis
of variance after testing for normality. Differences between group means were evaluated by
Tukey’s range test (Winer, 1971).
C. E. WEST A N D OTHERS
Table 2. Expt 1. Hatchability o f eggs laid by hens fed on diets varying in viramin A
Proportion of eggs laid
Dietary group.. .
P-300 P-600 P- 1 500
P- 1 500
Embryos surviving, 8 d
Embryos surviving, 17 d
Embryos surviving, 8 d
Embryos surviving, 17 d
Embryos surviving, 8 d
Embryos surviving, 17 d
~ _ _ _ _ _
100 (1 10)
P-0, P-300, P-600, P-1500, purified diets containing 0, 300, 600 and 1500 retinol equivalents (RE)/kg
respectively; N-l 500, non-purified diet containing I 500 RE/kg.
* Eggs collected over 11 d were hatched in three successive periods starting in weeks 8, 13 and 18. For
t The numbers of eggs incubated are shown in parentheses. Numbers exclude eggs that were cracked or
contained double yolks.
$ Hens were removed before the third egg collection period.
description of diets, see Table 1.
GeneraI health o f laying hens
Health status clearly deteriorated in the P-0 and P-300 groups. Morphological signs of
vitamin A deficiency were seen in three hens that died in these groups. After 14 weeks it was
decided, for ethical reasons, to remove birds from the P-0 group. Significant differences in
body-weight among the groups could not be observed throughout the experiment (values
not shown). From weeks 11-12, feed consumption and egg production of the P-0 group
were significantly lower than those of the P-1500 group. Hens fed on a purified diet
adequate in vitamin A and their counterparts fed on a non-purified diet with the same
vitamin A level were similar with respect to body-weight, feed consumption and egg
production (values not shown).
Hatchability was not affected in the first period (Table 2) and the I-d-old chickens
produced were in a healthy condition. However, in the second period, hatchability was
poor in the P-0 group. Many embryos died during the first 8 d of incubation, while others
were too weak to break out of the shell. In the third period, hatchability tended to be lower
in the P-300 group than in the other groups. Differences in weight of eggs or body-weight
of 1-d-old chickens could not be observed among the diet groups in all periods measured,
while weight of eggs and body-weight of 1-d-old chickens increased slightly from the first
to the third period (values not shown).
MARGINAL VITAMIN A DEFICIENCY IN CHICKENS
0 2 4
Duration of dietary treatment (weeks)
10 12 14 16 18
Fig. I. Expt I. Plasma retiiiol concentration of laying hens fed on diets varying in vitamin A content. For
description of diets, see Table 1. Points represent ineans with their standard errors varying from 1 to 10% for ten
to twelve hens. (O--. O), P-0; (+), P-300; (0-O), P-600; ( . - .
Marginal vitamin A status (0.35-0.70 /tmol/l); above this area, vitamin A status is normal and below this area it
is deficient (< 0.35 pol/l). P-0, P-300. P-600, P-1500, purified diets containing 0, 300, 600 and 1500 retinol
equivalents (RE)/kg respectively; N- 1500, non-purified diet containing 1500 RE/kg.
.-a). P-1500; (*-*),
Table 3. Evpt 1. Plasma retinol concentration in I-d-old chickens derived as progeny from
hens fed on diets varying in vitamin A content
(Mean values with their standard errors for the progeny of six hens/group Plasma samples from all
1-d-old chickens from one hen were pooled)
_ _ _ _ _ _ _ _ _ - _ _ _ - _ _ _ _
Retinol concentration @mol/l)
- _ _ _ -
_ - - - - - - - -
_ - - - - ___ -
P-0, P-300. P-600, P-1500, purified diets containing 0, 300, 600 and I500 retinol equivalents (RE)/kg
respectively, N- 1500. non-purified diet containing 1500 RE/kg , ND, not determined
Means within a column with unlike superscript letters were significdntly different (P < 005, Tukey)
* For description of diets, see Tdble 1
_ _ _ _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ _ _
_ _ _ - _
Plasma retinol concentration
At the start of the experiment mean plasma retinol levels of the 18-week-old hens ranged
from 2.4 to 3.0 pmol/l (Fig. 1). These levels decreased in all groups, including the P- 1500
and N-1500 groups throughout the experiment. The fall in plasma retinol levels was
negatively related to the level of vitamin A in the diet. Plasma retinol concentrations in 1-
d-old chickens derived from these hens are shown in Table 3. In the first hatching period
plasma retinol levels were similar in all groups (between 0.86 and 1.0.5,~~mol/l).
progeny of the P-300 group the concentrations were significantly lower than in the progeny
of all other groups after the second period (0.1 1 pmol/l compared with 0.89, 1.05 and
1.14 ,umol/l). Similar results were obtained after the third period for the progeny of the P-
600 group. Plasma retinol concentrations in the progeny of the P- I500 and N- I500 groups
were stable throughout the experiment.
C . E. WEST AND OTHERS
Fig. 2. Expt 2. Plasma retinol concentration at different stages in chicks from hens fed on diets containing 300
or 600 (B-m)
retinol equivalents (RE)/kg or from commercial hens (0)
containing 0 (- -) RE/kg or 120 (-)
RE/kg. For details of methods, see p. 284. Values are means with their
standard errors represented by vertical bars. (0).
Marginal vitamin A status (0.35-0.70 /mol/l) : above this area,
vitamin A status is normal and below this area it is deficient (< 0.35 /tmol/l). For details of diets. see Table 1.
and fcd on purified diets
Vitumin A content of diets
Analysis of the diets 1 week after manufacturing revealed that all diets contained more
vitamin A than expected (see Table 1). This could not be the result of bad mixing of the
vitamin and mineral premix through the diets, because at least five samples were taken from
each diet before pooling them to one sample for analysis. After 10 weeks of storage the
content of vitamin A had decreased in all diet groups but the decrease was relatively most
pronounced in the P-300 group (Table I).
Plusmu retinol concentration in chickens
In the progeny of marginally vitamin A-deficient laying hens (plasma retinol levels between
0.60 and 0.85 pmol/l) plasma retinol concentrations remained rather stable (Fig. 2) at a
level that could be considered as marginally deficient (between 0.35 and 0.70 pmol/l;
Interdepartmental Committee on Nutrition for National Defence, 1963). Marginal vitamin
A levels could also be obtained when chickens, progeny of normal hens, were fed on a diet
without vitamin A during the first 2 weeks and then with 120 RE/kg. However, the
variation in plasma retinol levels within this group was much higher than within the group
which were progeny of marginally vitamin A-deficient hens. Chickens, progeny of
marginally vitamin A-deficient hens (plasma retinol levels between 0.35 and 0.50 pmoI/l),
did not remain healthy for more than a few days. Gain in body-weight was extremely low
and after 2 weeks only five of twelve birds were still alive. Similar findings were observed
in the progeny of normal hens fed on a diet without vitamin A. In the third week four of
M A R G I N A L VITAMIN A DEFICIENCY IN C H I C K E N S
these birds died. Chickens which were progeny of normal hens and fed on a diet with
120 RE/kg were not marginally deficient in vitamin A after 5 weeks.
The aim of the present study was to develop a method for producing chickens which
remained not only marginally vitamin A-deficient but also healthy. Since a two-generation
model was used for this purpose, the advantages and disadvantages compared with other
methods have been investigated. The first signs of vitamin A deficiency, such as loss of
appetite, decreased egg production and ruffled feathers, appeared after I 1 weeks in the hens
in Expt 1 which were completely deprived of vitamin A, when plasma retinol levels were
about 0.75 pmoI/l. This is in accordance with earlier reports that a period ranging from 2
to 5 months is necessary to induce similar signs of vitamin A deficiency in hens (Lowe et
ul. 1957; Titus, 1961 ; Ewing, 1963; Sebrell& Harris, 1967; Gratzl& Kohler, 1968; Morton,
1970; Scott et a/. 1982; Davis & Sell, 1983; Richter et ul. 1990). Sudden death, which was
observed in three hens fed on a diet deprived or deficient in vitamin A, has also been
described as a sign of vitamin A deficiency (Sebrell & Harris, 1967), and it might be the
result of lower resistance to infections (Scrimshaw rt ul. 1968; Sporn et ul. 1984;
Underwood, 1984; Beisel, 1988). In two of these birds post-mortem examination revealed
overt morphological signs of vitamin A deficiency, such as white plaques in the oesophagus
and pale kidneys.
Hatchability was seriously affected when hens were maintained on vitamin A-deficient
diets for 13 weeks (Expt I). Reduced hatchability has often been described as a sign of
vitamin A deficiency (Barger, 1950; Ewing, 1963; Scott et ul. 1982). Egg production and
hatchability were optimal in hens fed on diets with more than 600 RE/kg throughout the
experiment. Other investigators have reported threshold values for maximal egg production
and hatchability between 900 and 1400 RE/kg diet (Hill et ul. 1961 ; Reid et ul. 1965;
Plasma retinol concentrations were used as an indicator of vitamin A status in our
experiment. Plasma retinol concentration does reflect liver stores after moderate to severe
depletion. It is also a good indicator of circulating vitamin A available to extrahepatic
tissues (Wright & Hall, 1979; Olson, 1984; Wittpenn et ul. 1988). After approximately 8-1 1
weeks on the experimental diets, plasma retinol concentration in hens reflected the levels
of vitamin A in the diets. This indicated that vitamin A stores in liver were moderately to
severely depleted by that time. The decrease in plasma retinol levels in hens from the P- 1500
and N-1500 groups could be explained by the change of diet; the commercial diet
contained 50% more vitamin A than these diets. Plasma retinol levels in the I-d-old
chickens reflected the vitamin A intake of their mothers, provided that these hens were fed
on the various diets for at least 13 weeks. Similar results have been reported previously
(Ewing, 1963; Joshi et 01. 1973; Scott et ul. 1982; Beynen ct al. 1989).
The results from the present experiment indicate that plasma retinol levels of hens should
be between 0.60 and 0.85 pmol/l in order to obtain marginally vitamin A-deficient I-d-old
chickens which remain marginally deficient for at least 6 weeks without health problems.
Above this range, chickens will not be marginally deficient: below this range, although
hatching will be normal the I-d-old chickens will be too weak to survive. Marginal vitamin
A deficiency could also be induced in commercially obtained I-d-old chickens within a few
weeks and these birds could be kept marginally deficient for at least another few weeks by
feeding them on a diet free of added vitamin A during the first 2 weeks and then on a diet
deficient in vitamin A (120 RE/kg). However, the variation in plasma retinol level between
birds was unacceptably high. In the studies reported here, some of the birds were deficient
C. E. WEST AND OTHERS
after 2 weeks, while others had normal values after 6 weeks. When commercially obtained
chickens were fed on a diet completely devoid of vitamin A, plasma retinol levels were
almost negligible after 3 weeks. In addition, when such I-d-old chickens were fed on a diet
deficient in vitamin A (120 RE/kg), plasma retinol levels were still far above deficiency
levels after 2 weeks. Previous reports have shown that I-d-old chickens, derived from hens
with an adequate intake of vitamin A and receiving a diet completely devoid of vitamin A,
had marginally deficient levels of vitamin A from the third week (Nockels er a/. 1984) and
showed signs of deficiency from the sixth week (Scott et al. 1982; Nockels et ul. 1984).
The non-purified diet adequate in vitamin A produced results comparable with the
purified diet with the same amount of vitamin A with respect to body-weight, feed
consumption, egg production, plasma retinol concentration and hatchability. However, it
is difficult to control the carotene content of non-purified diets. Therefore, it is preferable
to use purified diets for producing marginally vitamin A-deficient chickens. Storage of the
diets for a period of 10 weeks produced a reduction in vitamin A content, especially in the
diet with 300 RE/kg. Reduced vitamin A levels in diets after storage have been described
earlier by Fullerton et ul. (1982). They reported that the vitamin A content of purified diets
was more sensitive to oxidation than that of natural diets. Therefore, it is recommended
that new diets should be manufactured every month.
In conclusion, the results of the present paper indicate that it is possible to produce
marginally vitamin A-deficient 1-d-old chickens that are sufficiently healthy to survive and
grow normally for at least 6 weeks after hatching. The advantages of the two-generation
model in producing marginally vitamin A-deficient chickens over one-generation models
are a lower variation in vitamin A status within a treatment group and a more stable and
controllable vitamin A status in such birds. A disadvantage is the long period before l-d-
old chickens are available. It should also be noted that the range of plasma retinol values
in hens which allows the production of satisfactory chickens is very narrow. Moreover, it
is possible that these values are not applicable to all strains of chickens. We have found that
the marginally vitamin A-deficient chickens obtained are particularly suitable as starting
material for studying the interaction between vitamin A status and Newcastle disease virus
infection (Sijtsma et al. 1989a, h).
The authors wish to thank J. G. A. J. Hautvast and A. Hoogerbrugge for their useful
discussions, and M. G. B. Nieuwland, R. W. Terluin, J. W. M. Haas, and all co-workers
for their help in conducting these experiments. Thanks are also due to P. M. H. Abma for
assistance in preparation of the manuscript and to F. Hoffmann-La Roche & Co., Ltd,
Mijndrecht for kindly providing the vitamin A preparations.
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