5 Vitamins

5.1 Introduction
5.2 Fat-soluble vitamins
5.3 The vitamin B complex
5.4 Vitamin C
5.5 Hypervitaminosis

-
5.1 INTRODUCTION
Discovery of vitamins
The discovery and isolation of many of the vitamins were originally achieved
through work on rats given diets of purified proteins, fats, carbohydrates and
inorganic salts. Using this technique, Hopkins in 1912 showed that a synthetic
diet of this type was inadequate for the normal growth of rats, but that when a small
quantity of milk was added to the diet the animals developed normally. This proved
that there was some essential factor, or factors, lacking in the pure diet.
About this time the term ‘vitamines’, derived from ‘vital amines’, was coined by
Funk to describe these accessory food factors, which he thought contained amino-
nitrogen. It is now known that only a few of these substances contain amino-nitrogen
and the word has been shortened to vitamins, a term that has been generally accepted
as a group name.
Although the discovery of the vitamins dates from the beginning of the twentieth
century, the association of certain diseases with dietary deficiencies had been recog-
nised much earlier. In 1753 Lind, a British naval physician, published a treatise on
scurvy, proving that this disease could be prevented in human beings by including
salads and summer fruits in their diet. The action of lemon juice in curing and pre-
venting scurvy had been known, however, since the beginning of the seventeenth
century. The use of cod-liver oil in preventing rickets has long been appreciated, and
Eijkmann knew at the end of the nineteenth century that beri-beri, a disease common
in the Far East, could be cured by giving the patients brown rice grain rather than
polished rice.

70
 Introduction

Vitamins and biochemistry

Vitamins are usually defined as organic compounds that are required in small
amounts for normal growth and maintenance of animal life. But this definition
ignores the important part that these substances play in plants and their importance
generally in the metabolism of all living organisms. Unlike the nutrients covered in
Chapters 2–4, vitamins are not merely building blocks or energy-yielding com-
pounds but are involved in, or are mediators of, the biochemical pathways (Fig. 5.1).
For example, many of the B vitamins act as cofactors in enzyme systems but it is not
always clear how the symptoms of deficiency are related to the failure of the meta-
bolic pathway.
In addition to avoiding explicit vitamin deficiency symptoms (see below) or a
general depression in production due to a subclinical deficiency, some vitamins are
added to the diet at higher levels in order to (1) enhance the quality of the animal
product, e.g. vitamin D for eggshell strength and vitamin E for prolonging the shelf

BOX 5.1 Vitamin supplementation of diets

Most food mixes prepared as supplements for ruminants and horses or as the sole food for pigs,
poultry, dogs and cats are supplemented with vitamins. With other nutrients, such as energy and
protein, it is possible to demonstrate a response to increments in intake, which can be evaluated
against the cost of the increment. This is not possible with vitamins, for which the cost is relatively
small in relation to the consequences of deficiency.Therefore, vitamins are usually supplied at levels
greater than those shown to be required under experimental conditions. This oversupply allows for
uncertainties met under practical conditions (e.g. variable vitamin content and availability in foods,
loss of vitamin potency in storage, range of management practices, quality of the environment,
health status, extra requirements due to stress).This is not to say that such safety margins should be
excessive, since this would be wasteful: in addition, an excess of one vitamin may increase the re-
quirement for another. For example, the fat-soluble vitamins share absorption mechanisms and
compete with each other; thus, an excess of vitamin A will increase the dietary requirements of
vitamins E, D and K.
Originally, vitamins for supplements were isolated from plant products. However, yields from
such sources are low and the vitamins can be expensive. Yields can be increased when vitamins are
produced from microorganisms by fermentation. Nowadays many vitamins are produced in multi-
stage chemical processes that are controllable and the yield is predictable.
For ease of handling in the feed mill the vitamin supplement needs to (1) be free-flowing, (2) not
be dusty and (3) mix homogeneously with other diet ingredients (vitamins are added in minute
amounts but must be thoroughly dispersed throughout the mix); the vitamin must remain stable and
yet be biologically available when consumed by the animal. Some of these criteria are incompatible
and a compromise has to be reached. Oily vitamins are absorbed on to silica; others are coated or
micro-encapsulated and antioxidants are added to prevent breakdown of those vitamins that are
susceptible to oxidation. The manufacturers also make use of stable derivatives of vitamins (e.g. the
acetate form of ␣-tocopherol as opposed to the alcohol form).
Maintenance of vitamin activity in the supplement is affected by temperature, humidity, acidity/
alkalinity, oxygen, ultraviolet light, the presence of some trace minerals (dietary supplements are
usually combinations of vitamins, minerals and trace elements), physical factors such as hammer
milling and the length of time the supplement is stored. For example, choline chloride can destroy
other vitamins during storage.

71
 B12 PP H C Inositol

72
Carbohydrate
metabolism
PP Malonyl-CoA
Nucleic B1 PP
H
acids Cholesterol

B6 Pyruvate
Chapter 5 Vitamins

H
Folic B1 B2 Calpan Acyl-CoA
B12 CoA Acetyl-CoA
B6 Cal-
B1 B2
pan
B2 B6 Calpan

PP B1 PP
Cal-
H pan Aspartate
Oxalacetate Acetate

Cal-
pan PP
Calpan Choline
B6 PP Malate
Citrate H B2 PP
Cal- Chol-
B6
pan ine

H Krebs Fumarate

H K
Cycle CO2 Acetylcholine
PP E Chol- Cal-
Folic PP B6 B12 B1 B12 H
ine pan
Protein
Fat metabolism
metabolism B6
Isocitrate
Folic Chol- PP
ine PP
Glutamate Succinate KEY
B12
B6
B1 Thiamin
α-Ketoglutarate
Cal- Succinyl-CoA Propionate
B2 Riboflavin
pan

B6 Pyridoxine
PP Niacin
Calpan Pantothenic acid
B1 E
B1 H Biotin
B1

Folic Folacin
2H
Choline Choline
C Ascorbic acid
E Vitamin E
K Vitamin K
B6 Energy
output B12 Vitamin B12
IIIIIIII IIIII

Fig. 5.1 Diagram showing the involvement of vitamins in biochemical pathways.

Adapted from Roche Vitec Animal Nutrition and Vitamin News 1: A1–10/2 November 1984.
 Introduction

life of carcasses, or (2) improve health, e.g. vitamin A to improve the health status of
the mammary gland in dairy cows.
Vitamins are required by animals in very small amounts compared with other nu-
trients; for example, the vitamin B1 (thiamin) requirement of a 50 kg pig is only
about 3 mg/day. Nevertheless, a continuous deficiency in the diet results in dis-
ordered metabolism and eventually disease.
Some compounds function as vitamins only after undergoing a chemical change;
such compounds, which include ␤-carotene and certain sterols, are described as
provitamins or vitamin precursors.
Many vitamins are destroyed by oxidation, a process speeded up by the action of
heat, light and certain metals such as iron. This fact is important since the conditions
under which a food is stored will affect the final vitamin potency. Some commercial
vitamin preparations are dispersed in wax or gelatin, which act as a protective layer
against oxidation (for further details of vitamin supplementation of diets, see Box 5.1).
The system of naming the vitamins by letters of the alphabet was most conven-
ient and was generally accepted before the discovery of their chemical nature. Al-
though this system of nomenclature is still widely used with some vitamins, the
modern tendency is to use the chemical name, particularly in describing members of
the B complex.
At least 14 vitamins have been accepted as essential food factors, and a few
others have been proposed. Only those that are of nutritional importance are dealt
with in this chapter.
It is convenient to divide the vitamins into two main groups: fat-soluble and
water-soluble. Table 5.1 lists the important members of these two groups.

Table 5.1 Vitamins important in animal nutrition

Vitamin Chemical name

Fat-soluble vitamins
A Retinol
D2 Ergocalciferol
D3 Cholecalciferol
E Tocopherola
K Phylloquinoneb

Water-soluble vitamins
B complex
B1 Thiamin
B2 Riboflavin
Nicotinamide
B6 Pyridoxine
Pantothenic acid
Biotin
Folic acid
Choline
B12 Cyanocobalamin
C Ascorbic acid
a
A number of tocopherols have vitamin E activity.
b
Several naphthoquinone derivatives possessing vitamin K activity are known.

73
Chapter 5 Vitamins

- 5.2 FAT-SOLUBLE VITAMINS

Vitamin A
Chemical nature
Vitamin A (C20H29OH), known chemically as retinol, is an unsaturated monohydric
alcohol with the following structural formula:

CH3 CH3
H3C CH3
CH2OH

CH3

Vitamin A (all-trans form)

The vitamin is a pale yellow crystalline solid, insoluble in water but soluble in fat
and various fat solvents. It is readily destroyed by oxidation on exposure to air and
light. A related compound with the formula C20H27OH, originally found in fish, has
been designated dehydroretinol or vitamin A2.

Sources
Vitamin A accumulates in the liver and this organ is likely to be a good source; the
amount present varies with species of animal and diet. Table 5.2 shows some typical
liver reserves of vitamin A in different species, although these values vary widely within
each species.
The oils from livers of certain fish, especially cod and halibut, have long been
used as an important dietary source of the vitamin. Egg yolk and milk fat also are
usually rich sources, although the vitamin content of these depends, to a large ex-
tent, upon the diet of the animal from which it has been produced.
Vitamin A is manufactured synthetically and can be obtained in a pure form.

Table 5.2 Some typical values for liver reserves of vitamin

A in different speciesa

Species Vitamin A (µg/g liver)

Pig 30
Cow 45
Rat 75
Man 90
Sheep 180
Horse 180
Hen 270
Codfish 600
Halibut 3000
Polar bear 6000
Soup-fin shark 15000
a
In every species, wide individual variations are to be expected.
Adapted from Moore T 1969 In: Morton R A (ed.) Fat Soluble Vitamins,
Oxford, Pergamon Press, p. 233.

74
 Fat-soluble vitamins

Provitamins
Vitamin A does not exist as such in plants, but it is present as precursors or provita-
mins in the form of certain carotenoids, which can be converted into the vitamin.
At least 600 naturally occurring carotenoids are known, but only a few of these are
precursors of the vitamin.
In plants, carotenoids have yellow, orange or red colours but their colours are fre-
quently masked by the green colour of chlorophyll.When ingested, they are responsible
for many of the varied and natural colours that occur in crustaceans, insects, birds and
fish.They are also found in egg yolk, butterfat and the body fat of cattle and horses, but
not in sheep or pigs. Carotenoids may be divided into two main categories: carotenes
and xanthophylls. The latter include a wide range of compounds, for example lutein,
cryptoxanthin and zeaxanthin, most of which cannot be converted into vitamin A. Of
the carotenes, ␤-carotene is the most important member and this compound forms the
main source of vitaminA in the diets of farm animals. Its structure is shown here:

H3C
CH3 CH3
H3C CH3

H3C CH3
CH3 CH3
CH3

β-Carotene

The long unsaturated hydrocarbon chains in carotenes (and vitamin A) are easily
oxidised to by-products that have no vitamin potency. Oxidation is increased by heat,
light, moisture and the presence of heavy metals. Consequently, foods exposed to air
and sunlight rapidly lose their vitamin A potency, so that large losses can occur during
the sun-drying of crops. For example, lucerne hay has around 15 mg ␤-carotene/kg, but
artificially dried lucerne and grass meals have 95 mg/kg and 155 mg/kg, respectively.
Fresh grass is an excellent source (250 mg/kg DM), but this is halved during ensilage.
Carotenoids and supplemental vitamin A are prone to destruction in the rumen,
especially with high concentrate diets. Recent studies indicate that naturally occur-
ring carotenoids in forages may not be degraded to the same extent as purified prod-
ucts used as supplements. The gelatin preparations of vitamin A, with stabilising
agents, are intended to protect the vitamin from this destruction but still remain
available to be absorbed from the duodenum. In monogastrics the availability varies
between foods. In humans it has been found that oil solutions of carotenoids are
more available than those naturally occurring in foods. This is reflected in the fact
that the efficiency of absorption is largely dependent on the quality and quantity of
fat in the diet. The measurement of availability of carotenoids in foods and factors
that affect it are currently an active area of research in animals and humans.
Conversion of carotene into vitamin A can occur in the liver but usually takes
place in the intestinal mucosa. Theoretically, hydrolysis of one molecule of the C40
compound ␤-carotene should yield two molecules of the C20 compound retinol, but
although central cleavage of this type is thought to occur, it is considered likely that
the carotene is degraded from one end of the chain by step-wise oxidation until only
one molecule of the C20 compound retinol remains.Although the maximum conver-
sion measured in the rat is 2 mg ␤-carotene into 1 mg retinol, authorities differ

75
Chapter 5 Vitamins
regarding the conversion efficiency in other animals with ranges from 3 : 1 to 12 : 1.
Ruminants convert about 6 mg of ␤-carotene into 1 mg of retinol.The corresponding
conversion efficiency for pigs and poultry is usually taken as 11 : 1 and 3 : 1, respec-
tively. Cats do not have the enzyme to convert carotene to vitamin A. Since their
diet comprises meat, which usually contains sufficient vitamin A and low levels of
carotenoids, the conversion pathway is redundant.The vitamin A values of foods are
often stated in terms of international units (iu), one iu of vitamin A being defined as
the activity of 0.3 µg of crystalline retinol.
Metabolism
Vitamin A appears to play two different roles in the body according to whether it is
acting in the eye or in the general system.
In the retinal cells of the eye, vitamin A (all-trans-retinol) is converted into the
11-cis-isomer, which is then oxidised to 11-cis-retinaldehde. In the dark the latter
then combines with the protein opsin to form rhodopsin (visual purple), which is the
photoreceptor for vision at low light intensities. When light falls on the retina, the
cis-retinaldehyde molecule is converted back into the all-trans form and is released
from the opsin.This conversion results in the transmission of an impulse up the optic
nerve. The all-trans-retinaldehyde is converted to all-trans-retinol, which re-enters
the cycle, thus continually renewing the light sensitivity of the retina (Fig. 5.2).
In its second role, in the regulation of cellular differentiation, vitamin A is involved
in the formation and protection of epithelial tissues and mucous membranes. In this
way it has particular importance in growth, reproduction and immune response.Vita-
min A is important in the resistance to disease and promotion of healing through its
effect on the immune system and epithelial integrity. In addition, it acts, along with
vitamins E and C and ␤-carotene, as a scavenger of free radicals (see Box 5.2, p. 83).
The placental transfer of vitamin A to the foetus is limited and the neonate has
low stores of the vitamin and relies on consumption of colostrum to establish ade-
quate tissue stores.
All-trans-retinol
t
11-Cis-retinol
t
11-Cis-retinal
Dark
-- I~ - -_-~ - -
t
Rhodopsin Opsin
I
Light
All-trans-retinal I

1~-------- • i
I
•
Nerve impulse
Fig. 5.2 The role of vitamin A (retinol) in the visual cycle.
76
 Fat-soluble vitamins

Deficiency symptoms
Ability to see in dim light depends upon the rate of resynthesis of rhodopsin; when
vitamin A is deficient, rhodopsin formation is impaired. One of the earliest symp-
toms of a deficiency of vitamin A in all animals is a lessened ability to see in dim
light, commonly known as ‘night blindness’.
It has long been realised that vitamin A plays an important role in combating infec-
tion, and it has been termed the ‘anti-infective vitamin’. In several species, vitamin A
deficiency has been shown to be accompanied by low levels of immunoglobulins,
although the exact function of the vitamin in the formation of these important proteins
is uncertain.
In adult cattle, a mild deficiency of vitamin A is associated with roughened hair
and scaly skin. If it is prolonged the eyes are affected, leading to excessive watering,
softening and cloudiness of the cornea and development of xerophthalmia, which is
characterised by a drying of the conjunctiva. Constriction of the optic nerve canal
may cause blindness in calves. In breeding animals a deficiency may lead to infertility,
and in pregnant animals deficiency may lead to failure of embryo growth, disrupted
organ development, abortion, short gestation, retained placenta or the production of
dead, weak or blind calves. Less severe deficiencies may result in metritis and der-
matitis and calves born with low reserves of the vitamin; it is then imperative that
colostrum, rich in antibodies and vitamin A, should be given at birth, otherwise the
susceptibility of such animals to infection leads to scours and, if the deficiency is not
rectified, they frequently die of pneumonia. The National Research Council of the
United States has increased the recommended allowance for dairy cows in order to
improve the health of the mammary gland and reduce mastitis.
In practice, severe deficiency symptoms are unlikely to occur in adult animals ex-
cept after prolonged deprivation. Grazing animals generally obtain more than ade-
quate amounts of provitamin from pasture grass and normally build up liver reserves.
If cattle are fed on silage or well-preserved hay during the winter months, deficien-
cies are unlikely to occur. Cases of vitamin A deficiency have been reported among
cattle fed indoors on high cereal rations, and under these conditions a high vitamin
supplement is recommended.
In ewes, in addition to night blindness, severe cases of deficiency may result in
lambs being born weak or dead. A deficiency is not common in sheep, however, be-
cause of adequate dietary intakes on pasture.
In pigs, eye disorders such as xerophthalmia and blindness may occur. A defi-
ciency in pregnant animals may result in the production of weak, blind, dead or de-
formed litters. In view of the apparent importance of vitamin A in preventing
reproductive disorders in pigs, it has been suggested that the retinoids may have a
role in embryo development (cell differentation, gene transcription). Alternatively,
they may regulate ovarian steroid production and influence the establishment and
maintenance of pregnancy. In less severe cases of deficiency, appetite is impaired and
growth retarded. Where pigs are reared out of doors and have access to green food,
deficiencies are unlikely to occur, except possibly during the winter. Pigs kept in-
doors on concentrates may not receive adequate amounts of vitamin A in the diet
and supplements may be required.
In poultry consuming a diet deficient in vitamin A, the mortality rate is usually
high. Early symptoms include retarded growth, weakness, ruffled plumage and a stag-
gering gait. In mature birds, egg production and hatchability are reduced. Since most
concentrated foods present in the diets of poultry are low or lacking in vitamin A or

77
Chapter 5 Vitamins

its precursors, vitamin A deficiency may be a problem unless precautions are taken.
Yellow maize, dried grass or other green food, or alternatively cod- or other fish-liver
oils or vitamin A concentrate, can be added to the diet.
In horses, the signs of deficiency include the catalogue of symptoms seen in other
farm animals: night blindness, keratinisation of the skin and cornea, susceptibility to
infection and infertility.
Dogs and cats show similar symptoms. In addition dogs have ataxia and anorexia
and cats have reproductive and developmental disorders.
It has been suggested that, in addition to vitamin A, some species may have a
dietary requirement for ␤-carotene per se. The ovaries of bovine species are known
to contain high concentrations of ␤-carotene during the luteal phase – indeed, it is
an integral component of the mucosal membrane of luteal cells – and it has been
postulated that certain fertility disorders in dairy cattle, such as retarded ovulation
and early embryonic mortality, may be caused by a deficiency of the provitamin in
the diet. In sows, injections of ␤-carotene have reduced embryonic mortality and in-
creased litter sizes. It is suggested that it influences steroidogenesis and, through its
antioxidant properties, it may protect the highly active ovarian cells from damage by
free radicals. Supplementation of the diet of dogs with ␤-carotene resulted in increased
plasma progesterone concentration.

Vitamin D
Chemical nature
A number of forms of vitamin D are known,although not all of these are naturally occur-
ring compounds.The two most important forms are ergocalciferol (D2) and cholecalcif-
erol (D3).The term D1 was originally suggested by earlier workers for an activated sterol,
which was found later to be impure and to consist mainly of ergocalciferol, which had al-
ready been designated D2.The result of this confusion is that in the group of D vitamins,
the term vitamin D1 has been abolished.The structures of vitamins D2 and D3 are:

CH3 CH3 CH3

CH3
II

CHCH CHCH CHCH3

H2C

HO

Vitamin D2 (ergocalciferol)

CH3 CH3
CH3
CHCH2CH2CH2CHCH3

H2C

HO

Vitamin D3 (cholecalciferol)

78
 Fat-soluble vitamins

The D vitamins are insoluble in water but soluble in fats and fat solvents. The
sulphate derivative of vitamin D present in milk is a water-soluble form of the vita-
min. Both D2 and D3 are more resistant to oxidation than vitamin A, D3 being more
stable than D2.

Sources
The D vitamins are limited in distribution. They rarely occur in plants except in sun-
dried roughages and the dead leaves of growing plants. In the animal kingdom vita-
min D3 occurs in small amounts in certain tissues and is abundant only in some
fishes. Halibut-liver and cod-liver oils are rich sources of vitamin D3. Egg yolk is also
a good source, but cow’s milk is normally a poor source, although summer milk
tends to be richer than winter milk. Colostrum usually contains six to ten times the
amount present in ordinary milk.
Clinical manifestations of avitaminosis D, and other vitamin deficiencies, are fre-
quently treated by injection of the vitamin into the animal.

Provitamins
Reference has been made (p. 49) to two sterols, ergosterol and 7-dehydrocholesterol,
as being precursors of vitamins D2 and D3, respectively.The provitamins, as such, have
no vitamin value and must be converted into calciferols before they are of any use to
the animal. For this conversion it is necessary to impart a definite quantity of energy to
the sterol molecule, and this can be brought about by the ultraviolet light present in
sunlight, by artificially produced radiant energy or by certain kinds of physical treat-
ment. Under natural conditions activation is brought about by irradiation from the
sun. The activation occurs most efficiently with light of wavelength 290–315 nm, so
that the range capable of vitamin formation is small. The amount of ultraviolet radia-
tion that reaches the earth’s surface depends upon latitude and atmospheric conditions:
the presence of clouds, smoke and dust reduces the radiation. Ultraviolet radiation is
greater in the tropics than in the temperate regions, and the amount reaching the more
northern areas in winter may be slight. Since ultraviolet light cannot pass through
ordinary window glass, animals housed indoors receive little, if any, suitable radiation
for the production of the vitamin. Irradiation is apparently more effective in animals
with light-coloured skins. If irradiation is continued for a prolonged period, then the
vitamin may be altered to compounds that can be toxic.
The chemical transformation occurs in the skin and also in the skin secretions,
which are known to contain the precursor. Absorption of the vitamin can take place
from the skin, since deficiency can be treated successfully by rubbing cod-liver oil
into the skin.
Vitamin D requirements are often expressed in terms of international units (iu).
One iu of vitamin D is defined as the vitamin D activity of 0.025 µg of crystalline
vitamin D3.

Metabolism
Dietary vitamins D2 and D3 are absorbed from the small intestine and are trans-
ported in the blood to the liver, where they are converted into 25-hydroxycholecalcif-
erol. The latter is then transported to the kidney, where it is converted into
1,25-dihydroxycholecalciferol, the most biologically active form of the vitamin. This
compound is then transported in the blood to the various target tissues, the intestine,

79
Chapter 5 Vitamins

Food Skin

Cholecalciferol 7-Dehydrocholesterol

(Ultraviolet radiation)

Cholecalciferol

Liver
25-Hydroxycholecalciferol

Kidney
1,25-Dihydroxycholecalciferol

Target tissues

Fig. 5.3 Metabolic pathway showing production of the hormonally active form
of vitamin D.

bones and the eggshell gland in birds. The compound 1,25-dihydroxycholecalciferol

acts in a similar way to a steroid hormone, regulating DNA transcription in the intes-
tinal microvilli, inducing the synthesis of specific messenger RNA (see Chapter 9),
which is responsible for the production of calcium-binding protein. This protein is in-
volved in the absorption of calcium from the intestinal lumen. The various pathways
involved in these transformations are summarised in Fig. 5.3. Cats do not obtain vita-
min D by exposure to sunlight.The natural diet of the cat contains adequate amounts
of vitamin D to meet their requirements.Their metabolism has become adapted such
that 7-dehydroxycholesterol is converted to cholesterol and is not available for
vitamin D synthesis.
The amount of 1,25-dihydroxycholecalciferol produced by the kidney is con-
trolled by parathyroid hormone. When the level of calcium in the blood is low
(hypocalcaemia), the parathyroid gland is stimulated to secrete more parathyroid
hormone, which induces the kidney to produce more 1,25-dihydroxycholecalciferol,
which in turn enhances the intestinal absorption of calcium.
In addition to increasing intestinal absorption of calcium, 1,25-dihydroxychole-
calciferol increases the absorption of phosphorus from the intestine and also en-
hances calcium and phosphorus reabsorption from the kidney and bone.
Recently it has been discovered that 1,25-dihydroxycholecalciferol regulates the
expression of genes and the activity of cells associated with the immune system.

Deficiency symptoms
A deficiency of vitamin D in the young animal results in rickets, a disease of growing
bone in which the deposition of calcium and phosphorus is disturbed; as a result the
bones are weak and easily broken and the legs may be bowed. In young cattle the
symptoms include swollen knees and hocks and arching of the back. In pigs the symp-
toms are usually enlarged joints, broken bones, stiffness of the joints and occasionally

80
 Fat-soluble vitamins

paralysis.The growth rate is generally adversely affected.The term ‘rickets’ is confined

to young growing animals; in older animals vitamin D deficiency causes osteomalacia,
in which there is reabsorption of bone already laid down. Osteomalacia due to vitamin
D deficiency is not common in farm animals, although a similar condition can occur in
pregnant and lactating animals, which require increased amounts of calcium and phos-
phorus. Rickets and osteomalacia are not specific diseases necessarily caused by vita-
min D deficiency; they can also be caused by lack of calcium or phosphorus or an
imbalance between these two elements.
In poultry, a deficiency of vitamin D causes the bones and beak to become soft
and rubbery; growth is usually retarded and the legs become weak. Egg production
is reduced and eggshell quality deteriorates. Most foods of pigs and poultry, with the
possible exception of fishmeal, contain little or no vitamin D, and the vitamin is gen-
erally supplied to these animals, if reared indoors, in the form of fish-liver oils or
synthetic preparations.
The need for supplementing the diets of cattle and sheep with vitamin D is gener-
ally not so great as that for pigs and poultry. Adult ruminants can receive adequate
amounts of the vitamin from hay in the winter months, and from irradiation while
grazing. However, since the vitamin D content of hays is extremely variable, it is possi-
ble that vitamin D supplementation may be desirable, especially with young growing
animals or pregnant animals, on winter diets. There is a considerable lack of informa-
tion about the vitamin D needs of farm animals under practical conditions.
For cattle, sheep and pigs vitamins D2 and D3 have the same potency, but for
poultry vitamin D2 has only about 10 per cent of the potency of D3.
Certain foods, such as fresh green cereals and yeast, have been shown to have
rachitogenic (rickets-causing) properties for mammals, and raw liver and isolated
soya bean protein have a similar effect on poultry. In one study it was shown that in
order to overcome the rachitogenic activity of whole raw soya bean meal, a tenfold
increase in vitamin D supplement was necessary. Heating destroys the rachitogenic
activity.

Vitamin E
Chemical nature
Vitamin E is a group that includes a number of closely related active compounds.
Eight naturally occurring forms of the vitamin are known, and these can be divided
into two groups according to whether the side chain of the molecule, as shown
below, is saturated or unsaturated.
The four saturated vitamins are designated ␣-, ␤-, ␥- and ␦-tocopherol. Of these
the ␣-form is the most biologically active and most widely distributed.

CH3
CH3 CH3
H3C O
CH2(CH2 CH2 CH CH2)3H

HO
CH3

α-Tocopherol

81
Chapter 5 Vitamins

The ␤-, ␥- and ␦-forms have only about 45, 13 and 0.4 per cent of the activity of
the ␣-form, respectively. The unsaturated forms of the vitamin have been desig-
nated ␣-, ␤-, ␥- and ␦-tocotrienols. Of these only the ␣-form appears to have any
significant vitamin E activity, and then only about 13 per cent of its saturated
counterpart.
The ␣-tocopherol molecule has three centres where stereoisomers can occur. The
naturally occurring molecule is the D-␣-tocopherol (or RRR-␣-tocopherol) configura-
tion and has the highest vitamin activity. Synthetic DL-␣-tocopherol acetate (also
called all racemic ␣-tocopherol acetate) is used as a vitamin E supplement and com-
prises all eight possible stereoisomers; only one molecule in eight is in the RRR
form. The vitamin activity of the four stereoisomers in the L forms is considerably
lower than the four that make up the D forms; in the latter the RRR form is the most
active.

Sources
Vitamin E, unlike vitamin A, is not stored in the animal body in large amounts for
any length of time and consequently a regular dietary source is important. Fortu-
nately, the vitamin is widely distributed in foods. Green fodders are good sources
of ␣-tocopherol, young grass being a better source than mature herbage. The
leaves contain 20–30 times as much vitamin E as the stems. Losses during hay-
making can be as high as 90 per cent, but losses during ensilage or artificial dry-
ing are low.
Cereal grains are also good sources of the vitamin, but the tocopherol composi-
tion varies with species.Wheat and barley grain resemble grass in containing mainly
␣-tocopherol, but maize contains, in addition to ␣-tocopherol, appreciable quantities
of ␥-tocopherol. During the storage of moist grain in silos, the vitamin E activity can
decline markedly. Reduction in the concentration of the vitamin from 9 to 1 mg/kg
DM has been reported in moist barley stored for 12 weeks.
Animal products are relatively poor sources of the vitamin, although the amount
present is related to the level of vitamin E in the diet.
The vitamin E values of foods are often stated in terms of international units, one
iu of vitamin E being defined as the specific activity of 1 mg of synthetic all-racemic
␣-tocopherol acetate. It is generally accepted that 1 mg of RRR-␣-tocopherol is
equivalent to 1.49 iu vitamin E and 1 mg RRR-␣-tocopherol acetate is equivalent to
1.36 iu vitamin E. However, recent evidence suggests that the equivalence of all-
racemic to RRR forms is related to species, age and the criteria used to assess them
and that it may be as high as 2 : 1.

Metabolism
Vitamin E functions in the animal mainly as a biological antioxidant; in associa-
tion with the selenium-containing enzyme glutathione peroxidase and other vita-
mins and trace-element-containing enzymes, it protects cells against oxidative
damage caused by free radicals. Free radicals are formed during cellular metabo-
lism and, as they are capable of damaging cell membranes, enzymes and cell
nuclear material, they must be converted into less reactive substances if the ani-
mal is to survive. This protection is particularly important in preventing oxidation
of polyunsaturated fatty acids, which function as primary constituents of subcellu-
lar membranes and precursors of prostaglandins. Oxidation of unsaturated fatty

82
 Fat-soluble vitamins

acids produces hydroperoxides, which also damage cell tissues, and more lipid
free radicals, so that prevention of such oxidation is of vital importance in main-
taining the health of the living animal. The animal has complementary methods
of protecting itself against oxidative damage: scavenging of radicals by vitamin E
and destruction of any peroxides formed by glutathione peroxidase (see
Box 5.2).

BOX 5.2 Free radicals and antioxidants

Antioxidants are required to protect the animal’s cells from damage due to the presence of free rad-
icals. These are highly reactive molecules containing one or more unpaired electrons and can exist
independently (e.g. superoxide, O*2 -, and hydroxyl, OH*). Their high reactivity is a result of their
trying to lose or gain an electron to achieve stability.Within cells hydrogen peroxide (H2O2) can eas-
ily break down, especially in the presence of transition ions (e.g. Fe2+), to produce the hydroxyl rad-
ical, which is the most reactive and damaging of the free radicals:

H2O2 + Fe2+ → OH* + OH- + Fe3+

Free radicals are generated during normal cellular metabolism owing to leakage from the elec-
tron transport chain in mitochondria and leakage from peroxidation of polyunsaturated fatty acids
in the pathway of conversion of arachidonic acid to prostaglandins and related compounds. Also
O2*- plays an essential role in the extracellular killing of microorganisms by activated phagocytes,
and activation of this system can lead to further leakage.
All classes of biological molecules are vulnerable to free radical damage, but especially lipids,
proteins and DNA. Cell membranes are an important target because of the enzyme systems con-
tained within them. Lipids are the most susceptible; oxidative destruction of polyunsaturated fatty
acids can be extremely damaging, since it proceeds as a self-perpetuating chain reaction. The more
active cells, such as muscle cells, are at greatest risk of damage because they depend on the utilisa-
tion of lipids as energy sources.
To maintain cell integrity the animal’s cells require protection mechanisms and these are pro-
vided by the antioxidant system, which involves a group of vitamins and enzymes containing trace
elements working in series.The initial line of defence is by the enzymes superoxide dismutase (con-
taining copper), glutathione peroxidases (containing selenium) and catalase. Superoxide dismutase
eliminates superoxide radicals formed in the cell and prevents the reaction of the radical with bio-
logical membranes or their participation in the production of more powerful radicals. Glutathione
peroxidase detoxifies lipid hydroperoxides that are formed in the membrane during lipid peroxida-
tion. Catalase can also break down hydrogen peroxide.
If large amounts of radicals are produced the enzyme systems will be insufficient to prevent
damage and the second antioxidant system is brought into action. Antioxidants break the chain re-
action by scavenging peroxyl radicals and thus interfere with the propagation steps in the lipid per-
oxidation process. Vitamin E is the main antioxidant but the carotenoids, vitamin A and vitamin C
are also involved. In mammalian cells vitamin E is located in the mitochondria and endothelial
reticulum. It donates a hydrogen atom to the free radical to form a stable molecule, thereby break-
ing the chain. The amount of vitamin E in the cell membranes is low and it must be regenerated so

(Continued)

83
Chapter 5 Vitamins

NAD(P)H

α-Tocopheroxyl TRXox
radical Reductases
GSSG

NAD(P)
Vitamin E
Thiol cycle
cycle

ROOH,
ROH TRXred
α-Tocopherol Ascorbate
GSH

ROO•, RO• Vitamin C

cycle

PUFA
Semi-ascorbyl
radical

Peroxide and other

radicals

Dehydroascorbate

Fig. 5.4 The regeneration of vitamin E.

After Rooke J A, Robinson J J and Arthur J R 2004 Journal of Agricultural Science 142: 253–62.

that there is sufficient to act against other radicals. The regeneration is carried out by reaction with
vitamin C and the ascorbate radical in turn is reduced by NADH-dependent enzymes (Fig. 5.4). It
has been reported that vitamin C also acts as an antioxidant in extracellular fluid, where it operates
as a scavenger preventing the initiation of lipid peroxidation. It contributes up to one-quarter of the
total antioxidant activity in plasma.

Vitamin E also plays an important role in the development and function of the
immune system. In recognition of this the National Research Council requirements
for dairy cows have been increased to reduce the incidence of mastitis. In studies
with several species, supplementation of diets with the vitamin provided some pro-
tection against infection with pathogenic organisms.
Recent research has indicated that vitamin E is also involved in the regulation of
cell signalling and gene expression.
Like vitamin A, it was thought that the transfer of vitamin E across the placenta
was limited, with the neonate relying on colostrum to meet its requirements. More
recent evidence in sheep indicates that placental transfer does occur, with increased
muscle and brain concentrations in lambs born from ewes fed higher levels.
Nonetheless, colostrum is a very important source of vitamin E for the new born.

84
 Fat-soluble vitamins

Deficiency symptoms
The most frequent and, from a diagnostic point of view, the most important manifes-
tation of vitamin E deficiency in farm animals is muscle degeneration (myopathy).
Nutritional myopathy, also known as muscular dystrophy, frequently occurs in cattle,
particularly calves, when they are turned out on to spring pasture. It is associated
with low vitamin E and selenium intakes during the in-wintering period and possi-
bly the relatively high concentration of polyunsaturated fatty acids in the young
grass lipids. The requirement for the vitamin increases with increasing concentra-
tions of polyunsaturated fatty acids in the diet. The myopathy primarily affects the
skeletal muscles and the affected animals have weak leg muscles, a condition man-
ifested by difficulty in standing and, after standing, a trembling and staggering gait.
Eventually, the animals are unable to rise, and weakness of the neck muscles pre-
vents them from raising the head. A popular descriptive name for this condition is
‘white muscle disease’, owing to the presence of pale patches or white streaks in the
muscles.The heart muscle may also be affected and death may result. Serum creatine
phosphokinase and glutamic oxaloacetic transaminase levels are elevated in animals
deficient in vitamin E.
Nutritional myopathy also occurs in lambs, with similar symptoms to those of
calves. The condition is frequently referred to as ‘stiff lamb disease’. Dietary supple-
ments of vitamin E given to pregnant ewes have resulted in increased birth weight
and improved vigour and viability of neonatal lambs through quicker times to stand
and suck. The National Research Council has recently increased the dietary recom-
mendation for vitamin E several fold owing to its beneficial effects on prolonging the
shelf life of lamb at retail.
In pigs, the two main diseases associated with vitamin E and selenium deficiency
are myopathy and cardiac disease. Nutritional myopathy affects in particular young
fast-growing pigs, but it may occur at any age.The pigs demonstrate an uncoordinated
staggering gait or are unable to rise. In contrast to other animals, it is the pig’s heart
muscle that is more often affected. Sudden cardiac failure occurs; on post-mortem
examination, large amounts of fluid are found around the heart and lungs and the
lesions of the cardiac muscles are seen as haemorrhagic and pale areas.This condition
is commonly known as ‘mulberry heart disease’. Sometimes the liver is also affected
and it becomes enlarged and mottled. Supplemental vitamin E has improved litter
size in pigs, probably through its antioxidant properties protecting arachidonic acid
and maintaining the functional integrity of the reproductive organs.
Vitamin E deficiency in chicks may lead to a number of distinct diseases: myopa-
thy, encephalomalacia and exudative diathesis. In nutritional myopathy the main
muscles affected are the pectorals, although the leg muscles also may be involved.
Nutritional encephalomalacia, or ‘crazy chick disease’, is a condition in which the
chick is unable to walk or stand and is accompanied by haemorrhages and necrosis
of brain cells. Exudative diathesis is a vascular disease of chicks characterised by a
generalised oedema of the subcutaneous fatty tissues, associated with an abnormal
permeability of the capillary walls. Both selenium and vitamin E appear to be in-
volved in nutritional myopathy and in exudative diathesis, but the element does not
seem to be important in nutritional encephalomacia. It should be stressed that sele-
nium itself is a very toxic element and care is required in its use as a dietary additive.
The toxic nature of selenium is discussed in Chapter 7.
In horses, vitamin E deficiency results in the previously mentioned problems, i.e.
lameness and muscle rigidity (‘tying up’) associated with skeletal and heart muscles.

85
Chapter 5 Vitamins

The red blood cells become fragile and the release of myoglobin from damaged mus-
cle cells gives rise to coffee-coloured urine.

Vitamin K
Vitamin K was discovered in 1935 to be an essential factor in the prevention of
haemorrhagic symptoms in chicks.The discovery was made by a group of Danish sci-
entists, who gave the name ‘koagulation factor’ to the vitamin, which became short-
ened to the K factor and eventually to vitamin K.

Chemical nature
A number of forms of vitamin K are known to exist. All compounds exhibiting vita-
min K activity possess a 2-methyl-1,4-naphthoquinone ring (menadione), which
animals are unable to synthesise but plants and bacteria can.

CH3

Menadione (2-methyl-1,4-naphthoquinone)

The form of the vitamin present in plants is 2-methyl-3-phytyl-1,4-naphtho-

quinone, generally referred to as phylloquinone or vitamin K1.
The compound originally isolated from putrified fishmeal and designated vitamin
K2 is now known to be only one of a series of K vitamins with unsaturated side
chains synthesised by bacteria and referred to as menaquinones. The predominant
vitamins of the menaquinone series contain six to ten isoprenoid (CH2:CCH3:
CH:CH2) side-chain units. Menadione is the synthetic form of the vitamin and is
designated as vitamin K3.
Vitamins K are relatively stable at ordinary temperatures but are rapidly des-
troyed on exposure to sunlight.

Sources
Phylloquinone is present in most green leafy materials, with lucerne, cabbage and
kale being good sources. The amounts present in foods of animal origin are usually
related to the diet, but egg yolk, liver and fishmeal are generally good sources.
Menaquinones are synthesised by bacteria in the digestive tract of animals.

Metabolism
Vitamin K is necessary for the synthesis of prothrombin in the liver. In the blood-clotting
process, prothrombin is the inactive precursor of thrombin, an enzyme that converts
the protein fibrinogen in blood plasma into fibrin, the insoluble fibrous protein that
holds blood clots together. Prothrombin normally must bind to calcium ions before it
can be activated. If the supply of vitamin K is inadequate, then the prothrombin mole-
cule is deficient in ␥-carboxyglutamic acid, a specific amino acid responsible for

86
 The vitamin B complex

calcium binding. Proteins containing ␥-carboxyglutamic acid, dependent on vitamin K

for their formation, are also present in bone, kidney and other tissues.

Deficiency symptoms
Symptoms of vitamin K deficiency have not been reported in ruminants, horses and
pigs under normal conditions, and it is generally considered that bacterial synthesis
in the digestive tract supplies sufficient vitamin for the animal’s needs. A number of
microorganisms are known to synthesise vitamin K, including Escherichia coli. Med-
icines that affect the bacteria in the gut may depress the production of vitamin K. A
disease of cattle called ‘sweet clover disease’ is associated with vitamin K. Sweet
clover (Melilotus albus) naturally contains compounds called coumarins which,
when the crop is preserved as hay or silage, may be converted by a variety of fungi,
such as the Aspergillus species, to dicoumarol. This compound lowers the prothrom-
bin content of the blood and thereby impairs the blood-clotting process. The disease
can be overcome by administering vitamin K to the animals. For this reason di-
coumarol is sometimes referred to as an ‘anti-vitamin’.
The symptoms of vitamin K deficiency in chicks are anaemia and a delayed clotting
time of the blood; birds are easily injured and may bleed to death. It is doubtful
whether, in birds, microbially synthesised vitamin K is available by direct absorption
from the digestive tract, because the site of its formation is too distal to permit absorp-
tion of adequate amounts except by ingestion of faecal material (coprophagy).

-
5.3 THE VITAMIN B COMPLEX
The vitamins included under this heading are all soluble in water and most of them
are components of coenzymes (see Table 5.3). Although the mechanism of action in
this role is known, the connection between the observed deficiency symptoms and
the failure of the metabolic pathways is not always clear.
Unlike the fat-soluble vitamins, members of the vitamin B complex, with the ex-
ception of cyanocobalamin, are not stored in the tissues in appreciable amounts and a

Table 5.3 Some coenzymes and enzyme prosthetic groups involving the B vitamins

Vitamin Coenzyme or prosthetic group Enzyme or other function

Thiamin Thiamin pyrophosphate (TPP) Oxidative decarboxylation
Riboflavin Flavin mononucleotide (FMN) Hydrogen carrier
Riboflavin Flavin adenine dinucleotide Hydrogen carrier
(FAD)
Nicotinamide Nicotinamide adenine Hydrogen carrier
dinucleotide (NAD)
Nicotinamide Nicotinamide adenine dinucleotide Hydrogen carrier
phosphate (NADP)
Pyridoxine Pyridoxal phosphate Transaminases,
decarboxylases
Pantothenic acid Coenzyme A (CoA) Acyl transfer
Folic acid Tetrahydrofolic acid One carbon transfer
Biotin Biotin Carbon dioxide transfer
Cyanocobalamin Methylcobalamin Isomerases, dehydrases

87
Chapter 5 Vitamins

regular exogenous supply is essential. In ruminants, all the vitamins in this group can
be synthesised by microbial action in the rumen and generally this will provide satis-
factory amounts for normal metabolism in the host and secretion of adequate quanti-
ties into milk. For example, it has been estimated that the amount of thiamin
synthesised in the rumen is equal to the thiamin requirement. However, under certain
conditions, deficiencies of thiamin and cyanocobalamin can occur in ruminants. In
horses, the B vitamins synthesised by the microbial population of the gut plus those
vitamins occurring in the food can meet the requirements of most adult animals.

Thiamin
Chemical nature
Thiamin (vitamin B1) is a complex nitrogenous base containing a pyrimidine ring
joined to a thiazole ring. Because of the presence of a hydroxyl group at the end of
the side chain, thiamin can form esters.The main form of thiamin in animal tissues is
the diphosphate ester, commonly known as thiamin pyrophosphate (TPP). The vita-
min is very soluble in water and is fairly stable in mildly acidic solution but readily
decomposes in neutral solutions.

H3C N S CH2.CH2.OH
NH2

N CH2 N CH3

Cl

Thiamin chloride

Sources
Thiamin is widely distributed in foods. It is concentrated in the outer layers of seeds,
the germ, and in the growing areas of roots, leaves and shoots. Fermentation prod-
ucts, such as brewer’s yeast, are rich sources.Animal products rich in thiamin include
egg yolk, liver, kidney and pork muscle. The synthetic vitamin is available, usually
marketed as the hydrochloride.

Metabolism
Thiamin pyrophosphate (or thiamin diphosphate) is a coenzyme involved in (1) the
oxidative decarboxylation of pyruvate to acetyl coenzyme A (enzyme: pyruvate
dehydrogenase), (2) the oxidative decarboxylation of ␣-ketoglutarate to succinyl
coenzyme A (␣-ketoglutarate dehydrogenase) in the tricarboxylic acid cycle, (3) the
pentose phosphate pathway (transketolase) and (4) the synthesis of branched-chain
amino acids such as valine (branched-chain ketoacid dehydrogenase) in bacteria,
yeasts and plants.
Thiamin triphosphate is involved in the activation of the chloride ion channel in
the membranes of nerves, possibly by phosphorylation of the channel protein.

Deficiency symptoms
Early signs of thiamin deficiency in most species include loss of appetite, emaciation,
muscular weakness and a progressive dysfunction of the nervous system. In pigs, ap-
petite and growth are adversely affected and the animals may vomit and have respi-
ratory troubles.

88
 The vitamin B complex

Chicks reared on thiamin-deficient diets have poor appetites and consequently

are emaciated.After about 10 days they develop polyneuritis, which is characterised
by head retraction, nerve degeneration and paralysis.
Many of these deficiency conditions in animals can be explained in terms of the
role of TPP in the oxidative decarboxylation of pyruvic acid. On a thiamin-deficient
diet animals accumulate pyruvic acid and its reduction product lactic acid in their tis-
sues, which leads to muscular weakness. Nerve cells are particularly dependent on
the utilisation of carbohydrate and for this reason a deficiency of the vitamin has a
particularly serious effect on nervous tissue. Since acetyl coenzyme A is an impor-
tant metabolite in the synthesis of fatty acids (see p. 220), lipogenesis is reduced.The
pentose phosphate pathway is also impaired by a deficiency of thiamin but there is
little effect on the activity of the citric acid cycle.
Because thiamin is fairly widely distributed in foods and, in particular, because
cereal grains are rich sources of the vitamin, pigs and poultry are in practice unlikely
to suffer from thiamin deficiency.
In ruminants, microbial synthesis of the vitamin in the digestive tract, together
with that present in the diet, will normally provide adequate amounts of thiamin to
satisfy the animal’s requirements. However, under certain conditions, bacterial thi-
aminases can be produced in the rumen, which destroy the vitamin, thereby causing
the deficiency condition known as cerebrocortical necrosis (CCN). This condition is
characterised by circling movements, head pressing, blindness and muscular tremors.
There are two types of thiaminase: one splits the molecule in two and the other sub-
stitutes an N-containing ring for the thiazole ring.The resulting compound is absorbed
and blocks the reactions involving thiamin. It has been suggested that lactic acidosis
caused by feeding with rapidly fermentable foods may be an important factor in the
production of thiaminases. Young animals appear to be the most susceptible.
Thiaminase is present in bracken (Pteridium aquilinum), and thiamin deficiency
symptoms have been reported in horses consuming this material. Raw fish also con-
tains the enzyme, which destroys the thiamin in foods with which the fish is mixed.
The activity of the thiaminase is, however, destroyed by cooking.

Riboflavin
Chemical nature
Riboflavin (vitamin B2) consists of a dimethyl-isoalloxazine nucleus combined with
ribitol. Its structure is shown here:

HO OH OH
I I I
CH2 - C C C CH2OH

I I
H I
H I
H

CH3 N N O

CH3XX~Y N
NH

Riboflavin

89
Chapter 5 Vitamins

It is a yellow crystalline compound, which has a yellowish-green fluorescence in

aqueous solution. Riboflavin is only sparingly soluble in water; it is heat-stable in
acid or neutral solutions, but it is destroyed by alkali. It is unstable to light, particu-
larly ultraviolet light.

Sources
Riboflavin occurs in all biological materials. The vitamin can be synthesised by all
green plants, yeasts, fungi and most bacteria, although the lactobacilli are a notable
exception and require an exogenous source. Rich sources are yeast, liver, milk (espe-
cially whey) and green leafy crops. Cereal grains are poor sources.

Metabolism
Riboflavin is an important constituent of the flavoproteins. The prosthetic group of
these compound proteins contains riboflavin in the form of the phosphate (flavin
mononucleotide, FMN) or in a more complex form as flavin adenine dinucleotide
(FAD). There are several flavoproteins that function in the animal body; they
are all concerned with chemical reactions involving the transport of hydrogen.
Further details of the importance of flavoproteins in carbohydrate and amino
acid metabolism are discussed in Chapter 9. Flavin adenine dinucleotide plays a
role in the oxidative phosphorylation system (see Fig. 9.2 on p. 196) and forms the
prosthetic group of the enzyme succinic dehydrogenase, which converts succinic
acid to fumaric acid in the citric acid cycle. It is also the coenzyme for acyl-CoA
dehydrogenase.

Deficiency symptoms
In pigs, deficiency symptoms include poor appetite, with consequent retardation in
growth, vomiting, skin eruptions and eye abnormalities. Riboflavin is essential in the
diet of sows to maintain normal oestrus activity and prevent premature parturition.
Chicks reared on a riboflavin-deficient diet grow slowly and develop ‘curled toe
paralysis’, a specific symptom caused by peripheral nerve degeneration, in which the
chicks walk on their hocks with the toes curled inwards. In breeding hens, a defi-
ciency reduces hatchability. Embryonic abnormalities occur, including the character-
istic ‘clubbed down’ condition in which the down feather continues to grow inside
the follicle, resulting in a coiled feather.
The vitamin is synthesised in the rumen and deficiencies in animals with func-
tional rumens are unlikely to occur. However, riboflavin deficiencies have been
demonstrated in young calves and lambs. Symptoms include loss of appetite, diar-
rhoea and lesions in the corners of the mouth.

Nicotinamide
Chemical nature
Another member of the B vitamin complex, nicotinamide is the amide derivative of
nicotinic acid (pyridine 3-carboxylic acid) and is the form in which it functions in the

90
 The vitamin B complex

body.The relationship between nicotinic acid, nicotinamide and the amino acid tryp-
tophan, which can act as a precursor, is shown here:

COOH CONH2 CH2 CH COOH

C
C
NH2
N N N
H

Nicotinic acid Nicotinamide Tryptophan

Nicotinamide is a stable vitamin and is not easily destroyed by heat, acids, alkalis
or oxidation.

Sources
Nicotinic acid can be synthesised from tryptophan in the body tissues; since ani-
mals can convert the acid to the amide-containing coenzyme (see below), it fol-
lows that if the diet is adequately supplied with proteins rich in tryptophan, then
the dietary requirement for the vitamin itself should be low. However, the effi-
ciency of conversion of tryptophan into nicotinamide is poor. Studies with chicks
have shown that the amino acid was converted into the vitamin at a ratio of only
45 : 1 on a weight basis and with some foods, such as soya bean meal, the conver-
sion ratio may be even greater. Because of this it is generally considered that an
exogenous source of the vitamin is also necessary. Although cats possess the
enzymes for the conversion of tryptophan to nicotinic acid, the activity of an
enzyme in a competing pathway is very high and no nicotinic acid is synthesised.
Cats do not need to produce nicotinic acid because their natural diet is well
supplied with NAD and NADH. Rich sources of the vitamin are liver, yeast,
groundnut and sunflower meals. Although cereal grains contain the vitamin, much
of it is present in a bound form that is not readily available to pigs and poultry.
Milk and eggs are almost devoid of the vitamin, although they contain the precur-
sor tryptophan.

Metabolism
Nicotinamide functions in the animal body as the active group of two important
coenzymes: nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine
dinucleotide phosphate (NADP).These coenzymes are involved in the mechanism of
hydrogen transfer in living cells (see Chapter 9): NAD is involved in the oxidative
phosphorylation system, the tricyclic acid (TCA) cycle and the metabolism of many
molecules, including pyruvate, acetate, ␤-hydroxy-butyrate, glycerol, fatty acids and
glutamate; NADPH is the hydrogen acceptor in the pentose phosphate pathway.

Deficiency symptoms
In pigs, deficiency symptoms include poor growth, anorexia, enteritis, vomiting
and dermatitis. In fowls, a deficiency of the vitamin causes bone disorders,

91
Chapter 5 Vitamins

feathering abnormalities, and inflammation of the mouth and upper part of the
oesophagus.
Deficiency symptoms are particularly likely in pigs and poultry if diets with a
high maize content are used, since maize contains very little of the vitamin or of
tryptophan.
It has been suggested that through its effects on (1) rumen fermentation (some
experiments have shown increased microbial growth and increased propionic acid
production) and (2) cell metabolism (increased utilisation of carbohydrate and re-
duced lipid mobilisation), nicotinic acid may be a useful supplement to dairy cows,
particularly in situations of subclinical ketosis. However, the experimental evidence
is not consistent. Nicotinic acid does not always give positive responses in the rumen
and increases in blood concentrations were not observed in all experiments. Current
recommendations do not advocate the supplementation of dairy cow diets in order
to increase milk yield and composition.

Vitamin B6
Chemical nature
The vitamin exists in three forms, which are interconvertible in the body tissues.The
parent substance is known as pyridoxine, the corresponding aldehyde derivative as
pyridoxal and the amine as pyridoxamine. The term vitamin B6 is generally used to
describe all three forms.

CH2OH CHO CH2NH2

HO CH2OH HO CH2OH HO CH2OH

H3C N H 3C N N
H3C

Pyridoxine Pyridoxal Pyridoxamine

The amine and aldehyde derivatives are less stable than pyridoxine and are
destroyed by heat.

Sources
The vitamin is present in plants as pyridoxine, whereas animal products may also
contain pyridoxal and pyridoxamine. Pyridoxine and its derivatives are widely dis-
tributed: yeast, pulses, cereal grains, liver and milk are rich sources.

Metabolism
Of the three related compounds, the most actively functioning is pyridoxal in the
form of the phosphate. Pyridoxal phosphate plays a central role as a coenzyme in
the reactions by which a cell transforms nutrient amino acids into mixtures of
amino acids and other nitrogenous compounds required for its own metabolism.

92
 The vitamin B complex

These reactions involve the activities of transaminases and decarboxylases (see

p. 210), and over 50 pyridoxal phosphate-dependent enzymes have been identified.
In transamination, pyridoxal phosphate accepts the ␣-amino group of the amino
acid to form pyridoxamine phosphate and a keto acid. The amino group of pyri-
doxamine phosphate can be transferred to another keto acid, regenerating pyri-
doxal phosphate. The vitamin is believed to play a role in the absorption of amino
acids from the intestine.

Deficiency symptoms
Because of the numerous enzymes requiring pyridoxal phosphate, a large variety of
biochemical lesions are associated with vitamin B6 deficiency. These lesions are con-
cerned primarily with amino acid metabolism, and a deficiency affects the animal’s
growth rate. Convulsions may also occur, possibly because a reduction in the activity
of glutamic acid decarboxylase results in an accumulation of glutamic acid. In addi-
tion, pigs reduce their food intake and may develop anaemia. Chicks on a deficient
diet show jerky movements; in adult birds, hatchability and egg production are ad-
versely affected. In practice, vitamin B6 deficiency is unlikely to occur in farm ani-
mals because of the vitamin’s wide distribution.

Pantothenic acid
Chemical nature
Pantothenic acid, another member of the vitamin B complex, is an amide of pantoic
acid and ␤-alanine and has the following formula:

CH3 OH
I I
HOCH2 C CH CONHCH2CH2COOH

CH3

Pantothenic acid

Sources
The vitamin is widely distributed; indeed, the name is derived from the Greek
pantothen, ‘from everywhere’, indicating its ubiquitous distribution. Rich sources are
liver, egg yolk, groundnuts, peas, yeast and molasses. Cereal grains and potatoes are
also good sources of the vitamin. The free acid is unstable. The synthetically prepared
calcium pantothenate is the commonest product used commercially.

Metabolism
Pantothenic acid is a constituent of coenzyme A, which is the important coenzyme
in fatty acid oxidation, acetate metabolism, and cholesterol and steroid synthesis. It
forms the prosthetic group of acyl carrier protein in fatty acid synthesis. Chemically,

93
Chapter 5 Vitamins

coenzyme A is 3-phospho-adenosine-5-diphospho-pantotheine. The importance of

this coenzyme in metabolism is discussed in Chapter 9.

NH2

N
N
CH O O
II 11
N O CH2 - O -P- O - P - O
N
I
OH OH
H H H H C H

H3C C CH3
OH O I
HO C H
I
-

Pantotheine
HO P O C O
I
OH H N

H C H

H C H

C O

H N

H C H

H C H

S H

Coenzyme A

Deficiency symptoms
Deficiency of pantothenic acid in pigs causes slow growth, diarrhoea, loss of hair,
scaliness of the skin and a characteristic ‘goose-stepping’ gait; in severe cases,
animals are unable to stand. In the chick, growth is retarded and dermatitis occurs.
In mature birds, hatchability is reduced. Pantothenic acid, like all the B complex
vitamins, can be synthesised by rumen microorganisms; Escherichia coli, for ex-
ample, is known to produce this vitamin. Pantothenic acid deficiencies are consid-
ered to be rare in practice because of the wide distribution of the vitamin,
although deficiency symptoms have been reported in commercial herds of
Landrace pigs.

Folic acid
Chemical nature
This B complex vitamin was first discovered in the 1930s when it was found that a
certain type of anaemia in human beings could be cured by treatment with yeast

94
 The vitamin B complex

or liver extracts. The active component in the extracts, which was also shown to be
essential for the growth of chicks, was found to be present in large quantities in
green leaves and was named folic acid (Latin folium, a leaf).
The chemical name for folic acid is pteroylmonoglutamic acid. It is made up of
three moieties: p-aminobenzoic acid, glutamic acid and a pteridine nucleus.

NH2 N N
COOH

N CH2 NH CONHCHCH2CH2COOH
N

OH

Pteroylmonoglutamic acid

Several active derivatives of the vitamin are known to occur, these containing up
to 11 glutamate residues in the molecule. The monoglutamate form is readily ab-
sorbed from the digestive tract but the polyglutamates must be degraded by en-
zymes to the monoglutamate form before they can be absorbed.

Sources
Folic acid is widely distributed in nature; green leafy materials, cereals and ex-
tracted oilseed meals are good sources of the vitamin. Folic acid is reasonably stable
in foods stored under dry conditions, but it is readily degraded by moisture, partic-
ularly at high temperatures. It is also destroyed by ultraviolet light.

Metabolism
After absorption into the cell, folic acid is converted into tetrahydrofolic acid, which
functions as a coenzyme in the mobilisation and utilisation of single-carbon groups
(e.g. formyl, methyl) that are added to, or removed from, such metabolites as histi-
dine, serine, glycine, methionine and purines. It is involved in the synthesis of RNA,
DNA and neurotransmitters.

Deficiency symptoms
A variety of deficiency symptoms in chicks and young turkeys have been re-
ported, including poor growth, anaemia, poor bone development and poor egg
hatchability. Folic acid deficiency symptoms rarely occur in other farm animals be-
cause of synthesis by intestinal bacteria. Injections of folic acid in sows has in-
creased litter size. In one experiment, dietary supplements resulted in higher foetal
survival, thought to be related to prostaglandin activity, but the response has not
been substantiated in other experiments, possibly because of the variable content
of folic acid in foods. Its role in nucleic acid metabolism concurs with the view
that supplements might be beneficial at times of growth and differentiation of em-
bryonic tissue.

95
Chapter 5 Vitamins

Biotin
Chemical nature
A part of the vitamin B complex, biotin is chemically 2-keto-3,4-imidazolido-2-
tetrahydrothiophene-n-valeric acid. Its structure is:

C
HN NH

HC CH

H2C CH (CH2)4 COOH

S

Biotin

Sources
Biotin is widely distributed in foods: liver, milk, yeast, oilseeds and vegetables are rich
sources. However, in some foods, much of the bound vitamin may not be released dur-
ing digestion and hence may be unavailable. Studies with chicks and pigs have shown
that the availability of biotin in barley and wheat is very low, whereas the biotin in
maize and certain oilseed meals, such as soya bean meal, is completely available.

Metabolism
Biotin serves as the prosthetic group of several enzymes that catalyse the transfer of
carbon dioxide from one substrate to another. In animals there are three biotin-
dependent enzymes of particular importance: pyruvate carboxylase (carbohydrate
synthesis from lactate), acetyl coenzyme A carboxylase (fatty acid synthesis) and
propionyl coenzyme A carboxylase (the pathway of conversion of propionate to
succinyl-CoA). The specific role of these enzymes in metabolism is discussed in
Chapter 9.

Deficiency symptoms
In pigs, biotin deficiency causes foot lesions, alopecia (hair loss) and dry scaly skin.
In growing pigs, both growth rate and food utilisation are adversely affected. In
breeding sows, a deficiency of the vitamin can adversely influence reproductive per-
formance.
In poultry, biotin deficiency causes reduced growth, dermatitis, leg bone abnormali-
ties, cracked feet, poor feathering, and fatty liver and kidney syndrome (FLKS).This last
condition, which mainly affects 2- to 5-week-old chicks, is characterised by a lethargic
state with death frequently following within a few hours. On autopsy, the liver and
kidneys, which are pale and swollen, contain abnormal depositions of lipid.
Although ruminants and horses do not have a requirement for dietary biotin,
microbial production in the gut normally being adequate, feeding biotin has
improved hoof structure and strength.
Biotin deficiency can be induced by giving animals avidin, a protein present in the
raw white of eggs, which combines with the vitamin and prevents its absorption
from the intestine. Certain bacteria of the Streptomyces spp. that are present in soil

96
 The vitamin B complex

and manure produce streptavidin and stravidin, which have a similar action to the
egg white protein. Heating inactivates these antagonist proteins.

Choline
Chemical nature
The chemical structure of choline is:

CH3
+
CH3 N CH2CH2OH

CH3

Choline

Sources
Green leafy materials, yeast, egg yolk and cereals are rich sources of choline.

Metabolism
Unlike the other B vitamins, choline is not a metabolic catalyst but forms an essen-
tial structural component of body tissues. It is a component of lecithins, which play
a vital role in cellular structure and activity. It also plays an important part in lipid
metabolism in the liver, where it converts excess fat into lecithin or increases the util-
isation of fatty acids, thereby preventing the accumulation of fat in the liver. Choline
is a component of acetylcholine, which is responsible for the transmission of nerve
impulses. Finally, choline serves as a donor of methyl groups in transmethylation
reactions that involve folic acid or vitamin B12. Although other compounds, such as
methionine and betaine, can also act as methyl donors, they cannot replace choline
in its other functions.
Choline can be synthesised in the liver from methionine; the exogenous require-
ment for this vitamin is therefore influenced by the level of methionine in the diet.

Deficiency symptoms
Deficiency symptoms, including slow growth and fatty infiltration of the liver, have
been produced in chicks and pigs. Choline is also concerned with the prevention of
perosis or slipped tendon in chicks.The choline requirement of animals is unusually
large for a vitamin, but in spite of this, deficiency symptoms are not common in
farm animals because of its wide distribution and its high concentrations in foods,
and because it can be readily derived from methionine.

Vitamin B12
Chemical nature
Vitamin B12 has the most complex structure of all the vitamins.The basic unit is a cor-
rin nucleus, which consists of a ring structure comprising four five-membered rings
containing nitrogen. In the active centre of the nucleus is a cobalt atom.A cyano group
is usually attached to the cobalt as an artefact of isolation and, as this is the most sta-
ble form of the vitamin, it is the form in which the vitamin is commercially produced.

97
Chapter 5 Vitamins

CONH2

CH2

CH2 CH3
H CH3
CH2.CONH2
NH2.CO.CH2
CH2.CH2.CONH2
H3C A CN B
N N
H3C
Co
N N
CH3
D C
NH2.CO.CH2 CH3
CO.CH2.CH2 CH3 CH3
CH2.CH2.CONH2
NH

CH2

CH.CH3 CH3

<XX
N
0
O O
"-/
P N CH3
O
~"-O H
O
I
C - - -C
H

C
ii'
H
'\
H
CH
I~/
H2COH O

Vitamin B12 (cyanocobalamin)

In the animal, the cyanide ion is replaced by a variety of ions, e.g. hydroxyl
(hydroxocobalamin), methyl (methylcobalamin) and 5-deoxyadenosyl (5-deoxyadeno-
sylcobalamin), the last two forms acting as coenzymes in animal metabolism.

Sources
Vitamin B12 is considered to be synthesised exclusively by microorganisms and its pres-
ence in foods is thought to be ultimately of microbial origin.The main natural sources
of the vitamin are foods of animal origin, liver being a particularly rich source. Its lim-
ited occurrence in higher plants is still controversial, since many think that its presence
in trace amounts may result from contamination with bacteria or insect remains.

Metabolism
Before vitamin B12 can be absorbed from the intestine it must be bound to a highly
specific glycoprotein, termed the intrinsic factor, which is secreted by the gastric mu-
cosa. In humans, the intrinsic factor may be lacking, which leads to poor absorption
of the vitamin and results in a condition known as pernicious anaemia.
The coenzymic forms of vitamin B12 function in several important enzyme sys-
tems. These include isomerases, dehydrases and enzymes involved in the biosynthesis

98
 Vitamin C

of methionine from homocysteine. Of special interest in ruminant nutrition is the role

of vitamin B12 in the metabolism of propionic acid into succinic acid. In this pathway,
the vitamin is necessary for the conversion of methylmalonyl coenzyme A into suc-
cinyl coenzyme A (see p. 203).

Deficiency symptoms
Adult animals are generally less affected by a vitamin B12 deficiency than are young
growing animals, in which growth is severely retarded and mortality high.
In poultry, in addition to the effect on growth, feathering is poor and kidney dam-
age may occur. Hens deprived of the vitamin remain healthy, but hatchability is
adversely affected.
On vitamin B12-deficient diets, baby pigs grow poorly and show lack of coordina-
tion of the hind legs. In older pigs, dermatitis, a rough coat and suboptimal growth
result. Intestinal synthesis of the vitamin occurs in pigs and poultry. Organisms that
synthesise vitamin B12 have been isolated from poultry excreta; this fact has an im-
portant practical bearing on poultry housed with access to litter, where a majority, if
not all, of the vitamin requirements can be obtained from the litter.
Vitamin B12 and a number of biologically inactive vitamin B12 analogues are synthe-
sised by microorganisms in the rumen and, in spite of poor absorption of the vitamin
from the intestine, the ruminant normally obtains an adequate amount of the vitamin
from this source. However, if levels of cobalt in the diet are low, a deficiency of the vita-
min can arise and cause reduced appetite, emaciation and anaemia (see p. 125). If cobalt
levels are adequate, then except with very young ruminant animals, a dietary source of
the vitamin is not essential. Horses also are supplied with sufficient B12 from microbial
fermentation when sufficient cobalt is supplied. Parasitised horses have responded to
vitamin B12 supplementation, presumably as a result of impaired digestive activity.

Other growth factors included in the vitamin B complex

A number of other chemical substances of an organic nature have been included in
the vitamin B complex.These include inositol, orotic acid, lipoic acid, rutin, carnitine
and pangamic acid, but it is doubtful whether these compounds have much practical
significance in the nutrition of farm animals.

-
5.4 VITAMIN C
Chemical nature
Vitamin C is chemically known as L-ascorbic acid and has the following formula:
O C

HO C
O
HO C

H C

HO C H

CH2OH

L-Ascorbic acid

99
Chapter 5 Vitamins

The vitamin is a colourless, crystalline, water-soluble compound with acidic and

strong reducing properties. It is heat-stable in acid solution but is readily decom-
posed in the presence of alkali. The destruction of the vitamin is accelerated by
exposure to light.

Sources
Well-known sources of this vitamin are citrus fruits and green leafy vegetables.
Synthetic ascorbic acid is available commercially.

Metabolism
Ascorbic acid plays an important part in various oxidation–reduction mechanisms in
living cells.The vitamin is necessary for the maintenance of normal collagen metabo-
lism. It also plays an important role in the transport of iron ions from transferrin,
found in the plasma, to ferritin, which acts as a store of iron in the bone marrow, liver
and spleen. As an antioxidant, ascorbic acid works in conjunction with vitamin E in
protecting cells against oxidative damage caused by free radicals (see Box 5.2). The
vitamin is required in the diet of only a few vertebrates – humans, other primates,
guinea pigs, the red-vented bulbul bird and the fruit-eating bat (both native to India)
and certain fishes. Some insects and other invertebrates also require a dietary source
of vitamin C. Other species synthesise the vitamin from glucose, via glucuronic acid
and gulonic acid lactone; the enzyme L-gulonolactone oxidase is required for the syn-
thesis, and species requiring ascorbic acid are genetically deficient in this enzyme.

Deficiency symptoms
The classic condition in humans arising from a deficiency of vitamin C is scurvy,
characterised by oedema, emaciation and diarrhoea. Failure in collagen formation
results in structural defects in bone, teeth, cartilage, connective tissues and muscles.
Resistance to infection is reduced.
Since farm animals can synthesise vitamin C, deficiency symptoms normally do
not arise. However, it has been suggested that under certain conditions, e.g. climatic
stress in poultry, the demand for ascorbic acid becomes greater than can be provided
for by normal tissue synthesis, and a dietary supplement may then be beneficial.

- 5.5 HYPERVITAMINOSIS
Hypervitaminosis is the name given to pathological conditions resulting from an
overdose of vitamins. Under ‘natural’ conditions it is unlikely that farm animals will
receive excessive doses of vitamins, although when synthetic vitamins are added to
diets there is always the risk that abnormally large amounts may be ingested if errors
are made during mixing. There is experimental evidence that toxic symptoms can
occur if animals are given excessive quantities of vitamin A or D.
Clinical signs of hypervitaminosis A in young chicks kept under experimental
conditions and given very high doses of vitamin A include loss of appetite, poor
growth, diarrhoea, encrustation around the mouth and reddening of the eyelids. In
pigs, toxic symptoms include rough coat, scaly skin, hyperirritability, haemorrhages
over the limbs and abdomen, periodic tremors and death.

100
 Summary

Excessive intake of vitamin D causes abnormally high levels of calcium and phos-
phorus in the blood, which results in the deposition of calcium salts in the arteries
and organs. Symptoms of hypervitaminosis D have been noted in cattle and calves.
In the UK, the maximum amount of vitamin D supplement added to diets for farm
animals is controlled by legislation.
Depression in growth and anaemia caused by excessive doses of menadione (vita-
min K) have been reported.

SUMMARY
Vitamins are involved in metabolic pathways as coenzymes, and some act as protectors in antioxidant
and immune systems. The sources and functions of individual vitamins, and the disorders caused by
their deficiencies, are summarised below.

Vitamin Source Actions Deficiency symptoms

A, retinol Fish-liver oil Sight, Blindness,
epithelial tissues epithelial infection
D3, cholecalciferol Fish-liver oil, Calcium absorption Rickets
sun-dried roughage
E, a-tocopherol Green foods, cereals Antioxidant Muscle degeneration,
liver damage
K, menadione Green foods, egg yolk Prothrombin Anaemia, delayed
synthesis clotting
B1, thiamin Seeds Carbohydrate and Poor growth,
fat metabolism polyneuritis
B2, riboflavin Green foods, milk Carbohydrate and Poor growth, curled toe
amino acid paralysis
metabolism
Nicotinamide Yeast, liver, tryptophan Hydrogen transfer Poor growth, dermatitis
(NAD and NADP)
B6, pyridoxine Cereals, yeast Amino acid metabolism Poor growth, convulsions
Pantothenic acid Liver, yeast, cereals Acetate and fatty acid Poor growth, scaly
metabolism skin, ‘goose-stepping’
(coenzyme A) in pigs
Folic acid Green foods, cereals, Metabolism of single Poor growth, anaemia,
oilseed meals carbon compounds poor hatchability
Biotin Liver, vegetables Carbon dioxide transfer Foot lesions, hair loss,
FLKS
Choline Green foods, cereals, Component of lecithin Poor growth, fatty liver,
methionine perosis
B12, cyanocobalamin Microorganisms, liver Propionate metabolism Poor growth, anaemia,
poor coat/feathering
C, ascorbic acid Citrus fruits, leafy Oxidation–reduction Reduced resistance to
vegetables reactions infection

FLKS = fatty liver and kidney syndrome.

101
Chapter 5 Vitamins

FURTHER READING
Bender, D A 1992 Nutritional Biochemistry of the Vitamins, Cambridge, Cambridge
University Press.
Bieber-Wlaschny M 1988 Vitamin requirements of the dairy cow. In: Garnsworthy P C (ed.)
Nutrition and Lactation in the Dairy Cow, London, Butterworth, pp. 135–56.
Chew B P 1995 The influence of vitamins on reproduction in pigs. In: Garnsworthy P C and
Cole D J A (eds) Recent Advances in Animal Nutrition 1995, Nottingham, Nottingham
University Press, pp. 223–39.
Debier, C and Larondelle, Y 2005 Vitamins A and E: metabolism, roles and transfer to
offspring. British Journal of Nutrition 93: 153–74.
Latscha T 1990 Carotenoids: Their Nature and Significance in Animal Feeds, Basel,
Hoffmann–La Roche.
Morris J G 2002 Idiosyncratic nutrient requirements of cats appear to be diet-induced evolu-
tionary adaptations. Nutrition Research Reviews 15: 153–68.
National Research Council 1994 Nutrient Requirements of Poultry, 9th edn,Washington, DC,
National Academy Press.
National Research Council 1998 Nutrient Requirements of Swine, 10th edn,Washington, DC,
National Academy Press.
National Research Council 2000 Nutrient Requirements of Beef Cattle, 7th edn, Washington,
DC, National Academy Press.
National Research Council 2001 Nutrient Requirements of Dairy Cattle, 7th edn,Washington,
DC, National Academy Press.
National Research Council 2006 Nutrient Requirements of Dogs and Cats, Washington, DC,
National Academy Press.
National Research Council 2007 Nutrient Requirements of Horses, 6th edn, Washington, DC,
National Academy Press.
National Research Council 2007 Nutrient Requirements of Small Ruminants, Washington,
DC, National Academy Press.
Whitehead C C 1986 Requirements for vitamins. In: Fisher C and Boorman K N (eds)
Nutrient Requirements of Poultry and Nutrition Research, London, Butterworth,
pp. 173–89.

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