Vitamins and minerals functioning as antioxidants with supplementation considerations

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Vitamins and minerals functioning as antioxidants with
supplementation considerations
Lee R. McDowell1, Nancy Wilkinson, Rachel Madison and Tara Felix
Department of Animal Sciences
University of Florida
Introduction
Vitamin E, vitamin C, carotenoids, Se and other trace minerals are important
antioxidant components of animal diets and their roles in animal health and immune
function are indispensable. The present paper will discuss these nutrients in relation to
antioxidant and health considerations and draw conclusions as to vitamin and mineral
supplementation needs.
Free radicals and antioxidants
Free radicals are atoms or molecules containing one or more unpaired electrons,
making them very “reactive” (Surai, 2003). Biologically relevant free radicals are
activated atoms or groups of atoms (usually containing oxygen or nitrogen) with an odd
(unpaired) number of electrons. In a non-radical compound, all orbits are occupied by
two electrons. When a chemical reaction breaks the bonds that hold paired electrons
together, free radicals are produced. Therefore in a ‘free radical’ compound, there is a
single unpaired electron in the outer orbit.
A single excited electron is searching to become part of a paired set and will
steal an electron from another, nearby atom to accomplish this pairing. During this
theft, the original free radical becomes stable while the neighboring atom, by losing an
electron, becomes a free radical itself. This new free radical will then seek out another
atom to steal from, creating a chain reaction.
The extreme reactivity driven by a desire to acquire another electron underlies
their ability to interact with and ultimately damage tissue. Biologically relevant
molecules such as DNA, proteins, lipids and carbohydrates are damaged. Free radical
collective terms are reactive oxygen species (ROS) and reactive nitrogen species
(RNS) and include not only the oxygen or nitrogen radicals, but some non-radical
reactive derivatives of oxygen and nitrogen.
Free radicals (ROS/RNS) are constantly produced during normal physiological
metabolism in tissues. Oxygen is utilized within the mitochondria to generate ATP. The
majority of oxygen is reduced to form water however a small quantity (2-5%) is not
1 Contact at: P.O. Box 110910, Gainesville, FL 32611, (352) 392-7561, FAX (352)392-7652, Email:
mcdowell@animal.ufl.edu
January 30-31, 2007 • Florida Ruminant Nutrition Symposium • Best Western Gateway Grand • Gainesville, FL

completely reduced with the result of oxygen intermediates formed. The super oxide
radical is the main free radical produced in living cells and the electron transport chain
in the mitochondria is considered to be responsible for it. Free radicals generated
naturally during oxidative metabolism if present in excess react with fatty acids to form
fatty acid hydroperoxides which can then induce a chain reaction forming further free
radicals and hydroperoxides: if the chain reaction is not terminated cell damage will
occur. The activation of macrophages in stress conditions is another important source
of free radical generation. Immune cells produce ROS/RNS and use them as an
important weapon to destroy pathogens (Schwarz, 1996).
Under normal conditions the deleterious effects of ROS/RNS are counteracted by
the body’s antioxidant defenses, which are contributed to through dietary intake of key
nutrients (e.g. vitamins and trace minerals). Antioxidants serve to stabilize these highly
reactive free radicals, thereby maintaining the structural and functional integrity of cells.
Therefore, antioxidants are very important to immune defense and health of humans
and animals. Oxidative stress occurs when the production of reactive oxygen
metabolites exceeds the capacity of the antioxidant system of the cell, tissue or body.
Because free radicals are toxic to cells, the body has developed a sophisticated
antioxidant system that depends on antioxidant nutrients (Table 1). Tissue defense
mechanisms against free-radical damage generally includes vitamin C, vitamin E, and
β-carotene (and other carotenoids) as the major vitamin antioxidant sources. In
addition, several metalloenzymes which include glutathione peroxidase (Se), catalase
(Fe), and superoxide dismutase (Cu, Zn, and Mn) are also critical in protecting the
internal cellular constituents from oxidative damage. Only when these metals are
delivered in the diet in sufficient amounts can the animal body synthesize these
antioxidant enzymes. In contrast, deficiency of those elements causes oxidative stress
and damage to biological molecules and membranes.
There is a delicate balance between the amount of free radicals generated in the
body and the antioxidants needed to provide protection against them. An excess of free
radicals, or lack of antioxidant protection, can shift this balance resulting in oxidative
stress. The dietary and tissue balance of all these nutrients (vitamins and trace
elements) are important in protecting tissues against free-radical damage as well as
participating in immune function.
Antioxidant Vitamins and Selenium
The principal antioxidant vitamins for tissue defense against free-radical damage
includes vitamins E, C and β-carotene. Selenium would be the mineral most specifically
related to antioxidant function. There are at least 35 antioxidant Se proteins and include
selenoprotein P, five glutathione peroxidases and three thioredoxin reductases (TrXR1).
Most recent research indicates TrXR1 reduces harmful ROS and facilitates gene
expression of other cytoprotective antioxidants (Nakamura, 2005).

Vitamin E functions as a membrane-bound antioxidant, trapping lipid peroxyl free
radicals produced from unsaturated fatty acids under conditions of ‘oxidative stress’.
Table 1. Vitamins and minerals in antioxidant systemsa
Nutrients Component (location in cell) Function
Vitamin C
Vitamin E
β-carotene
Selenium
Copper and zinc
Manganese and zinc
Copper
Iron
Ascorbic acid (cytosol)
α-tocopherol (membranes)
β-carotene (membranes)
Glutathione peroxidase
(cytosol)
Superoxide dismutase
(cytosol)
Superoxide dismutase
(mitochondria)
Ceruloplasmin (water
phase)
Catalase (cytosol)
Reacts with several types
of ROS/RNS
Breaks fatty acid
peroxidation chain
reactions
Prevents initiation of fatty
acids peroxidation chain
reaction
An enzyme that converts
hydrogen peroxide to water
An enzyme that converts
superoxide to hydrogen
peroxide
An enzyme that converts
superoxide to hydrogen
peroxide
An antioxidant protein, may
prevent copper and iron
from participating in
oxidation reactions
An enzyme (primarily in
liver) that converts
hydrogen peroxide to water
aModified from Weiss (2005)
Orientation of vitamin E within cell membranes appears to be critical to its functionality
(Dunnett, 2003). Vitamin E functions as a chain-breaking antioxidant, neutralizing free
radicals and preventing oxidation of lipids within membranes (McDowell, 2000). Vitamin
E serves as the 1st line of defense against peroxidation of phospholipids. Selenium as
part of glutathione peroxidase (GSH-Px) is the 2nd line of defense as the enzyme
destroys peroxides and hydroperoxides. The various GSH-Px enzymes are
characterized by different tissue specificities and are expressed from different genes. In
general, different forms of GSH-Px perform their protective functions in concert, with
each providing antioxidant protection at different sites of the body.

The principal vitamin E form with antioxidant and immune functions is α-tocopherol.
However, although studies are limited, non α-tocopherol and tocotrienols have important
functions. γ-tocopherol has been shown to be a more effective inhibitor of peroxy
nitrite-induced lipid peroxidation (McCormick and Parker, 2004). Also, γ-tocopherol is
more effective at inhibiting inflammatory reactions. Tocotrienols possess excellent
antioxidant activity in vitro and have been suggested to suppress ROS more efficiently
than tocopherols (Schaffer et al., 2005). Tocotrienols were found to be more effective
for reducing the ageing process and age-related diseases.
In the lipid environment of biological membranes a combination of carotenoids
and other antioxidants, especially tocopherols, may provide better antioxidant protection
than tocopherols alone. Antioxidant properties of carotenoids include scavenging
singlet oxygen and peroxyl radicals, sulfur, thiyl, sulfonyl and NO2 radicals and provide
protection of lipids from superoxide and hydroxyl radical attack (Surai, 2002).
Therefore, carotenoids can actively quench singlet oxygen (O2) and prevent lipid
peroxidation caused by O2 and they can intercept the propagation step of lipid
peroxidation. One molecule of β-carotene is able to quench 1000 molecules of O2
before it reacts chemically and forms products. Although the principal antioxidant
carotenoid is β-carotene, other carotenoids (e.g. lutein, lycopene and zeaxanthin) have
strong antioxidant activities.
Vitamin C is the most important antioxidant in extracellular fluids and can protect
biomembranes against lipid peroxidation damage by eliminating peroxyl radicals in the
aqueous phase before the latter can initiate peroxidation. Vitamin C is located in the
aqueous phase of cells, where it contributes to radical scavenging. Vitamin C is a
potent antioxidant, it easily gives up electrons to provide stability to reactive species
such as ROS.
Mechanisms of Antioxidant Functions
Antioxidants are effective via various mechanisms including: 1) preventive
antioxidants, 2) free radical scavengers, 3) sequestration of elements by chelation and
4) quenching active oxygen species (Dunnet, 2003).
• Preventive antioxidants – These antioxidants suppress formation of free radicals;
for example catalase (Fe containing) and glutathione peroxidase (Se containing),
two antioxidant enzymes, decompose hydrogen peroxide, preventing the
formation of oxygen radicals.
• Free radical scavengers: These antioxidants confer stability to the ‘reactive’
species by donation of an electron and become oxidized themselves to form a
more stable radical. For example α-tocopherol (vitamin E) scavenges peroxyl
radicals and is converted to a tocopherol radical. Illustrating antioxidant
interactions, the vitamin E becomes “re-activated” by ascorbic acid donating an
electron which in turn forms an ascorbate radical in the process.

• Sequestration of metal by chelation - Although trace minerals are important
dietary constituents, they can act as pro-oxidants (promote free radical
formation). Since trace minerals such as Fe and Cu can propagate the formation
of more reactive radicals they are kept bound to transport proteins such as
transferrin or ceruloplasmin, as this renders them less available to contribute to
radical or pro-oxidant formation.
• Quenching of active oxygen species - Antioxidants can convert active oxygen
species to more stable forms, for example, carotenoids and vitamin E stabilize
singlet oxygen radicals, forming less reactive hydrogen peroxide.
Antioxidant-pro-oxidant Balance in the Body and Stress Conditions
In equilibrium, free radical generation is neutralized by the antioxidant system.
Natural and synthetic antioxidants in the feed as well as optimal levels of Mn, Cu, Zn,
Fe and Se help to maintain the efficient levels of endogenous antioxidants in the
tissues. Optimal nutrient composition allows the food antioxidants to be efficiently
absorbed and metabolized. Optimal temperature, humidity and other environmental
conditions are also needed for effective protection against free radical production.
Prevention of different diseases by antibiotics and other drugs is an integral part of the
efficient antioxidant defense as well.
Different stress conditions are associated with overproduction of free radicals
and cause oxidative stress i.e., a disturbance in the pro-oxidant-antioxidant balance
leading to potential tissue damage. Stress conditions can be generally divided into
three main categories (Table 2). The most important part is nutritional stress conditions
including high levels of PUFAs, deficiencies of vitamin E, Se, Zn or Mn, Fe-overload,
hypervitaminosis A and presence of different toxins and toxic compounds. Other stress
factors include environmental conditions: increased temperature or humidity, radiation,
etc.
The third group of stress factors is internal and includes various bacterial or viral
diseases as well as allergy. All animals protect themselves from invasion of
microorganisms, parasites, fungi, viruses and any foreign molecules. Immunity
phagocytosis is the major mechanism by which microbes are removed from the body
and is especially important for defense against extracellular microbes. The mechanism
of the phagocytic process is the killing of microbes by bombarding them with oxidants
(superoxide and hydroxyl radicals, hydrogen peroxide, nitric oxide, etc.). Without
adequate antioxidant nutrient reserves, cellular machinery will be damaged by the free
radicals, thereby reducing the effectiveness of the immune cell. When antioxidant
capacity is limited, the lifespan of immune cells is reduced and an infection can become
established or severity of an infection can increase (Weiss, 2005).
Intensified production increases stress and subclinical disease level conditions
because of higher densities of animals in confined areas. Stress and disease

conditions in ruminants may increase the basic requirement for certain vitamins and
minerals. A number of studies indicate that nutrient levels that are adequate for growth,
Table 2. Stress conditions that generate free radical productiona
Nutritional Environmental Internal
Toxins, high PUFA
Deficiencies-Vitamins A and E
Carotenes and Trace Minerals
Iron or Copper overload
Temperature
Humidity
Radiation
Diseases-bacterial, viral and fungal
Allergy
Mycotoxins
aAdapted from Surai (2002).
feed efficiency, gestation and lactation may not be adequate for normal immunity and
for maximizing the animal’s resistance to disease (Cunha, 1985). Diseases or parasites
affecting the gastrointestinal tract will reduce intestinal absorption of vitamins, both from
dietary sources and those synthesized by microorganisms. If they cause diarrhea or
vomiting this will also decrease intestinal absorption and increase needs. Vitamin A
deficiency is often seen in heavily parasitized animals that supposedly were receiving
an adequate amount of the vitamin.
Mycotoxins in feed can substantially decrease antioxidant nutrient assimilation
from the feed and increase their requirements to prevent damaging effects of free
radicals and toxic products of their metabolism. It is now increasingly recognized that at
least 25% of the world’s grains are contaminated with mycotoxins (Surai, 2002).
Mycotoxins are known to cause digestive disturbances such as vomiting and diarrhea
as well as internal bleeding, and interfere with absorption of dietary vitamins A, D, E and
K. Environmental pollutants (e.g. heavy metals, pesticides, fungicides, herbicides, etc.)
can cause oxidative stress.
Animal Studies of Antioxidant and the Immunity Role of Vitamins and Trace
Minerals
Antioxidant vitamins generally enhance different aspects of cellular and non-
cellular immunity. The antioxidant function of these vitamins could, at least in part,
enhance immunity by maintaining the functional and structural integrity of important
immune cells. A compromised immune system will result in reduced animal production
efficiency through increased susceptibility to diseases, thereby leading to increased
animal morbidity and mortality.
One of the protective effects of vitamin C may partly be mediated through its
ability to reduce circulating glucocorticoids. The suppressive effect of corticoids on
neutrophil function in cattle has been alleviated with vitamin C supplementation.
Vitamin C and E supplementation resulted in a 78% decrease in the susceptibility of
lipoproteins to mononuclear cell-mediated oxidation (Rifici and Khachadurian, 1993).
Ascorbic acid is reported to have a stimulating effect on phagocytic activity of
leukocytes, on function of the reticuloendothelial system, and on formation of
antibodies. Ascorbic acid levels are very high in phagocytic cells with these cells using
free radicals and other highly reactive oxygen containing molecules to help kill

pathogens that invade the body. In the process, however, cells and tissues may be
damaged by these reactive species. Ascorbic acid helps to protect these cells from
oxidative damage.
Considerable attention is directed to the role vitamin E and Se play in protecting
leukocytes and macrophages during phagocytosis, the mechanism whereby animals
immunologically kill invading bacteria. Both vitamin E and Se may help these cells to
survive the toxic products that are produced in order to effectively kill ingested bacteria
(Badwey and Karnovsky, 1980). Macrophages and neutrophils from vitamin E-deficient
animals have decreased phagocytic activity.
Since vitamin E acts as a tissue antioxidant and aids in quenching free-radicals
produced in the body, any infection or other stress factors may exacerbate depletion of
the limited vitamin E stores from various tissues. With respect to immunocompetency,
dietary requirements may be adequate for normal growth and production; however,
higher levels have been shown to positively influence both cellular and humoral immune
status of ruminant species. The former two responses are generally used as criteria for
determining the requirement of a nutrient. During stress and disease, there is an
increase in production of glucocorticoids, epinephrine, eicosanoids, and phagocytic
activity. Eicosanoid and corticoid synthesis and phagocytic respiratory bursts are
prominent producers of free radicals which challenge the animal antioxidant systems.
Vitamin E has been implicated in stimulation of serum antibody synthesis, particularly
IgG antibodies (Tengerdy, 1980). Vitamin E supplementation increased α-tocopherol
status and immune response of weaned piglets (Lauridsen and Jensen, 2005).
The effects of vitamin E and Se supplementation on protection against infection
by several types of pathogenic organisms, as well as antibody titers and phagocytosis
of the pathogens have been reported for calves and lambs (Reffett et al., 1988). As an
example, calves receiving 125 IU of vitamin E daily were able to maximize their immune
responses compared to calves receiving low dietary vitamin E (Ready et al., 1987).
Bovine respiratory disease (BRD) is the most economically important disease
affecting feedlot cattle, causing approximately 75% of morbidity and over 50% of
mortality. Rivera et al. (2002) suggest that vitamin E supplementation may enhance
recovery from BRD. Interestingly when given as an adjuvant at vaccination, vitamin E is
highly effective in enhancing antibody titers, implying that this may be an effective way
of obtaining immunological response following vaccination. Cows given a single
subcutaneous injection of 3,000 IU of vitamin E one week before expected calving
tended to have reduced risk of retained placenta (LeBlanc et al., 2002)
Antioxidants, including vitamin E, play a role in resistance to viral infection.
Vitamin E deficiency allows a normally benign virus to cause disease (Beck et al.,
1994). In mice, enhanced virulence of a virus resulted in myocardial injury that was
prevented with vitamin E adequacy. A Se or vitamin E deficiency leads to a change in
viral phenotype, such that an avirulent strain of a virus becomes virulent and a virulent
strain becomes more virulent (Beck, 1997).

Carotenoids have been shown to have biological actions independent of vitamin
A (Koutsos, 2003). Recent animal studies indicate that certain carotenoids with
antioxidant capacities, but without vitamin A activity, can enhance many aspects of
immune functions, can act directly as antimutagens and anticarcinogens, can protect
against radiation damage, and can block the damaging effects of photosensitizers.
Also, carotenoids can directly affect gene expression and this mechanism may enable
carotenoids to modulate the interaction between B cells and T cells, thus regulating
humoral and cell-mediated immunity (Koutsos, 2003).
Vitamin A and β-carotene have important roles in protection against numerous
infections including mastitis. Potential pathogens are regularly present in the teat
orifice, and under suitable circumstances can invade and initiate clinical mastitis. Any
unhealthy state of the epithelium would increase susceptibility of a mammary gland to
invasion by pathogens. There are reports of improved mammary health in dairy cows
supplemented with β-carotene and vitamin A during the dry and lactating periods (Chew
and Johnston, 1985).
Polymorphonuclear neutrophils (PMN) are the major line of defense against
bacteria in the mammary gland. β-carotene supplementation seems to exert a
stabilizing effect on PMN and lymphocyte function during the period around dry off
(Tjoelker et al., 1990). Daniel et al. (1991) reported that β-carotene enhanced the
bactericidal activity of blood and milk PMN, against S. aureus but did not affect
phagocytosis. Vitamin A either had no effect or suppressed bactericidal activity and
phagocytosis. Control of free radicals is important for bactericidal activity but not for
phagocytosis. The antioxidant activity of vitamin A is not important; it does not quench
or remove free radicals. β-carotene, on the other hand, does have significant
antioxidant properties and effectively quenches singlet oxygen free radicals (Mascio et
al., 1991).
Supplemental levels of vitamin E higher than recommended by the dairy cattle
NRC (2001) have been beneficial in the control of mastitis. Smith and Conrad (1987)
reported that intramammary infection was reduced 42.2 % in vitamin E-Se
supplemented versus unsupplemented controls. The duration of all intramammary
infections in lactation was reduced 40 to 50% in supplemented heifers. Clinical mastitis
was negatively related to plasma Se concentration and concentration of vitamin E in the
diet.
Somatic cell counts (SCC) are a primary indicator of mastitis and milk quality in
dairy herds. The PMN are a major defensive mechanism against infection in the bovine
mammary gland. A known consequence of vitamin E and Se deficiency is impaired
PMN activity and postpartum vitamin E deficiencies are frequently observed in dairy
cows. Dietary supplementation of cows with Se and vitamin E results in a more rapid
PMN influx into milk following intramammary bacterial challenge and increased
intracellular kill of ingested bacteria by PMN. Subcutaneous injections of vitamin E
approximately 10 and 5 d before calving successfully elevated PMN α-tocopherol

concentrations during the periparturient period and negated the suppressed intracellular
kill of bacteria by PMN that commonly is observed around calving (Smith et al., 1987).
Plasma α-tocopherol decreased at calving for cows fed dietary treatments with
low or intermediate concentrations of vitamin E, but not for cows fed high vitamin E
treatment (Weiss et al., 1997). High dietary vitamin E increased concentration of α-
tocopherol in blood neutrophils at parturition. The high vitamin E treatment was 1,000
IU/d of vitamin E during the first 46 d of the dry period, 4,000 IU/d during the last 14 d of
the dry period, and 2,000 IU/d during lactation.
The percentage of quarters with new infections at calving was not different
(32.0%) between cows receiving treatments that contained low and intermediate
concentrations of vitamin E but was reduced (11.8%) in cows receiving the high vitamin
E treatment. Clinical mastitis affected 25.0, 16.7 and 2.6% of quarters during the first 7
d of lactation for cows receiving the low, intermediate, and high vitamin E treatments,
respectively. Cows with plasma concentrations of α-tocopherol <3.0 µg/ml at calving
were 9.4 times more likely to have clinical mastitis during the first 7 d of lactation than
were cows with plasma concentrations of α-tocopherol >3.0 µg/ml (Weiss et al., 1997).
Based on neutrophil function and clinical mastitis, the minimal plasma vitamin E level for
peripartum dairy cows is 3.0 to 3.5 µg/mL.
Baldi et al. (2000) reported significantly lower SCC (log 10) in cows fed 2,000
IU/d supplemental vitamin E versus 1,000 IU/d from 14 d pre-calving through 7 d post-
calving in diets with 0.3 ppm added Se. Plasma and milk vitamin E levels were
increased in cows fed 2,000 vs 1,000 IU/d. Taken together, these studies substantiate
the cooperative roles of vitamin E and Se in maintaining mammary immune function and
suggest that 2,000 to 4,000 IU vitamin E/d during the peripartum period can be
beneficial to dairy cows in terms of udder health and milk quality (Seymour, 2001).
Additional Benefits of Vitamin E Supplementation
Supplementing vitamin E in well balanced diets has been shown to increase
humoral immunity for ruminant (Hoffmann-LaRoche, 1994) and monogastric species
(Wuryastuti et al., 1993). These results suggest that the criteria for establishing
requirements based on overt deficiencies or growth do not consider optimal health.
Gill et al. (1986) supplemented newly received feedlot cattle with 1,600 IU
vitamin E (as dl-α-tocopheryl acetate) per head per day for the first 21 days and 800 IU
vitamin E for the remaining 7 days of a 28 day trial. Average daily gain and gain to feed
ratios were improved by 23.2 and 28.6%, respectively, for vitamin E supplemented
stressed cattle. The number of sick pen days per head was reduced by 15.6%, and
morbidity was reduced by 13.4% with vitamin E supplementation.
Baldi et al. (2000) supplemented cows with 1,000 or 2,000 IU vitamin E starting
14 d prepartum through 7 d postpartum and reported significant reduction in days open
in cows fed 2,000 IU/d (111 vs 84 d) and fewer services per conception (1.3 vs. 2.2).

Kim et al. (1997) used 120 Holstein cows to test the effects of injecting 500 IU vitamin E
and 40 mg Se at 20 d prepartum on reproductive performance. The combination of
vitamin E and Se significantly reduced the incidence of retained placenta (30 vs 13.3%)
and days to first service (103 vs 60 d). Olson and Seidel (2000) reported that vitamin E
enhanced the development of bovine embryos both in vitro and after their transfer to
recipient cows.
Selenium source for heat stressed dairy cows (Florida) were fed supplemental
Se as yeast Se or as selenite for 81 days (beginning 26 d prepartum) (Thatcher, 2006).
The benefits of organic Se were: 1) elevated plasma Se, 2) phagocytosis and killing
activity of neutrophils increased, immunocompetence increased at parturition, milk yield
increased, uterine health improved and 2nd service pregnancy rate increased.
Research has shown a beneficial response for vitamin E supplementation on
male reproduction for bulls fed high concentrations of gossypol. Velasquez-Pereira et
al. (1998) reported that bulls which received 14 mg free gossypol/kg body weight had a
lower (P < 0.05) percentage of normal sperm than those which also received
supplemental vitamin E, 31 vs 55%, respectively (Table 2). Likewise, sperm production
per gram of parenchyma and total daily sperm production were higher (P < 0.05) when
gossypol treated animals also received vitamin E. Bulls receiving gossypol exhibited
more sexual inactivity (P < 0.05) than bulls in other treatments. Vitamin E
supplementation to bulls receiving gossypol improved number of mounts in the first test
and time of first service in the second test. The final conclusion of the Florida data is
that vitamin E is effective in reducing or eliminating important gossypol toxicity effects
for male cattle.
Table 3. Relationship of gossypol and vitamin E on semen characteristics of dairy
bullsa
Treatment
Item TRT1b TRT2c TRT3d
Normal, % 64.7 ± 6.4h 31.4 ± 7.4i 54.6 ± 6.4h
Abnormal,% 4.4 ± 1.3h 13.4 ± 1.5i 4.8 ± 1.2h
DSPG (x 106/g)e 14.5 ± 1.0h 10.2 ± 1.0i 17.6 ± 1.0h
DSP (x 109)f 3.2 ± 3.0h 2.2 ± 3.0i 4.1 ± 3.0h
aLeast square means + SEM.
bDiet based on SMB, corn and 30 IU vitamin E/kg of supplement.
cDiet containing 14 mg free gossypol/kg BW/d and 30 IU vitamin E/kg of supplement.
dDiet containing 14 mg free gossypol/kg/BW/d and 4,000 IU vitamin E/bull/d.
eDaily sperm production per gram of parenchyma.
fDaily sperm production total.
h,iMeans in row with different superscript differ P < 0.05.
Many attempts have been made to control lipid oxidation in meats through the
use of antioxidants. Dietary supplementation of vitamin E, and intravenous infusion of
vitamin C immediately before harvest, are efficacious techniques for increasing the
concentration of these vitamins in beef skeletal muscle (Schaefer et al., 1995). Meat

with elevated levels of either and probably both of these antioxidant vitamins possesses
greater stability of oxymyoglobin and lipid, which results in less discoloration and
rancidity. Vitamin E would seem to be the most practical since it is administered
dietetically. Increased dietary levels of vitamin E result in higher tissue α-tocopherol
concentrations and greater stability of these tissues toward lipid oxidation (Roeber et al.,
2001).
Dramatic effects of vitamin E supplementation (500 IU per head daily) to finishing
steers on the stability of beef color have been observed (Faustman et al., 1989). Loin
steaks of control steers discolored two to three days sooner than those supplemented
with vitamin E. Supplemental dietary vitamin E extended the color shelf life of loin
steaks from 3.7 to 6.3 days. This was most likely due to the increased α-tocopherol
content of the loin tissue of the supplemented animals, which was approximately 4-fold
greater than controls (Faustman et al., 1989). Vitamin E supplementation of feedlot
cattle at 1,000 IU daily for at least 100 d increased color of beef products, increased α-
tocopherol concentration in beef cuts and subsequently increased its retail case life
(Roeber et al., 2001).
Color is an extremely critical component of fresh red meat appearance and
greatly influences the customer perception of meat quality. Steaks from cattle
supplemented with vitamin E were preferred over control steaks by 91% of Japanese
survey participants (n=10,941), and 58% of all participants identified muscle color as the
most important factor in selecting beef products (Sanders et al., 1997).
Optimum Vitamin and Mineral Allowances
The National Research Council (NRC) requirements for vitamins and minerals
are usually close to minimum levels required to prevent deficiency signs and for
conditions of health and adequate performance, provided sufficient amounts of all other
nutrients are supplied. Most nutritionists usually consider NRC requirements for
vitamins and minerals to be close to minimum requirements sufficient to prevent clinical
deficiency signs and they may be adjusted upward according to experience within
industry in situations where higher levels are needed. The concept of optimum vitamin
nutrition under commercial production conditions is illustrated in Figure 1 (Roche, 1979).
Allowances of vitamins and minerals are those total levels from all sources fed to
compensate for factors influencing the needs of animals. These “influencing factors”
(Figure 2) include those that may lead to inadequate levels of dietary vitamins and
minerals. Influencing factors can include stress, infectious diseases, parasites,
biological variations, diet composition, bioavailability of sources, stability and nutrient
interrelationships. Mineral levels vary in feed ingredients because of crop location,
fertilization, plant genetics, plant disease and weather. For example, corn and other
grains will vary in their concentration of vitamins and minerals depending on varieties,
soils, levels of fertilization, maturity at harvest, processing methods and storage.
Intensive cropping practices and use of new crop varieties may result in reduced levels
of these nutrients in many feedstuffs. In forage crops, factors that favor production of

lush, green plants also favor production of higher levels of vitamins and minerals, as
most plants decline in their vitamin and mineral content as they mature. Harvesting
conditions, processing and storage will affect the vitamin and mineral content of both
feedstuffs and supplements.
Vitamin Supplementation Most Needed By Ruminants
Supplementation allowances need to be set at levels that reflect different
management systems and that are high enough to take care of fluctuations in
environmental temperatures, energy content of feed, or other factors that might
influence feed consumption or the vitamin requirements in other ways. Grazing
ruminants generally only need supplemental vitamin A, if pastures are low in carotene,
and possibly vitamin E (influenced by Se status). Vitamin D is provided by ultraviolet
light activity on the skin, while all other vitamins can be provided by ruminal or intestinal
microbial synthesis.
Ruminants housed under more strict confinement conditions generally require
vitamins A and E and may require vitamin D if deprived of sunlight. Additional
supplemental vitamin E would be needed to stabilize the meat color of finishing animals.
Under specific conditions, relating to stress and high productivity, ruminants may be
benefitted by supplemental B vitamins, particularly thiamin and niacin. Biotin deficiency
has been linked to lameness in cattle (Budras et al., 1997). Increased plasma biotin
levels have been associated with hardness and positive confirmational changes in
bovine hooves. Future research may find a need for carnitine supplementation. Adding
a complete B-vitamin mixture to cattle diets entering the feedlot during the first month
can reduce stress and increase gains. In one study, supplemental B vitamins given
feedlot calves tended to reduce morbidity of animals (Zinn et al., 1987). Apparently
under stress conditions of feedlots, the microbial population in the rumen is not
synthesizing certain B vitamins at adequate levels.
Mineral Supplementation Most Needed by Ruminants
Grazing Ruminants
Ruminants that depend primarily on pasture have different mineral
supplementation needs than those receiving concentrates. Phosphorus is generally
most limiting, with free-choice mineral supplements containing Ca, P, common salt and
selected trace minerals (McDowell and Arthington, 2005). The trace elements most
likely to be deficient for grazing livestock are Cu, Co, I, Se and Zn. Iron is generally not
required unless animals are losing blood due to parasites; Mn deficiency is restricted to
few regions of the world. Animals in some tropical regions may benefit from S
supplementation particularly if non-protein nitrogen (e.g., urea) supplements are fed.
Magnesium supplementation is warranted when grass tetany is a problem. Potassium
may be needed in winter or the dry season when forage K is less than required.

Ruminants Receiving Concentrates
The best means of providing minerals to ruminants receiving concentrates would
be to combine the minerals with the concentrate diets, including Ca, P and a trace
mineralized salt (NaCl plus, Co, Cu, I, Fe, Mn, Se and Zn). Chromium has been shown
to be beneficial to stressed calves (e.g., transport stress). The less dietary forage, the
higher the level of Ca required. Some studies would recommend K supplementation for
high-producing dairy cows and finishing ruminants when high quality K-rich forages are
limited; Mg is often added to high concentrate diets. Sulfur often is needed when non-
protein nitrogen (e.g., urea) is used. To prevent milk fever, dairy cattle need special
diets that regulate the Ca and P concentration and increase acidity. Normally the Ca:P
ratio is less important than that of monogastric diets, assuming the P concentration is
adequate.
Diets that limit forage in relation to concentrates need some mineral salts as
buffers as well as individual elements. High-producing dairy cows, finishing cattle and
sheep often receive high concentrate diets. Feeding large amounts of concentrate with
small amounts of forage or with forages that have been fermented or finely chopped will
decrease ruminant saliva output and increase the acid load of these ruminants.
Conclusion/Implications
Oxidative stress occurs when production of free radicals exceeds the capacity of
the antioxidant system of body cells. Certain nutrients serve as antioxidants or are
components of antioxidant enzymes. Antioxidants include vitamins C and E,
carotenoids, and antioxidant enzymes containing Se, Cu, Mn, Fe or Zn. Stress
conditions that generate free radical production can be classified as nutritional (e.g.,
high PUFA and or excesses of certain minerals and vitamins), environmental (e.g.,
temperature and humidity) and internal (e.g., bacterial, viral and fungal diseases).
Antioxidant nutrients play important roles in animal health by inactivating harmful free
radicals produced through normal cellular activity and from various stressors. A
compromised immune system will result in reduced animal production efficiency through
increased susceptibility to disease, thereby leading to increased animal morbidity and
mortality. A number of factors influence vitamin and mineral requirements and
utilization and include: physiological make-up and production function; confinement
rearing without pasture; stress, disease and adverse environmental conditions; vitamin
antagonists, use of antimicrobial drugs and body vitamin reserves. Under commercial
ruminant production conditions, vitamin and mineral allowances higher than NRC
requirements may be needed to allow optimum performance. Generally, the optimum
vitamin and mineral supplementation level is the quantity that achieves the best growth
rate, feed utilization, health (including immune competency), and provides adequate
body reserves.

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Figure 1
Figure 2