Vitamins and minerals functioning as antioxidants with supplementation considerations

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. 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. 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.

Introduction

Vitamin E, vitamin C, carotenoids, selenium (Se) and other trace minerals are important antioxidant components of animal diets which are indispensable in animal health and immune function. The focus of this paper is supplementation levels for these nutrients in relation to antioxidant and health considerations.

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 of free radicals driven by the need to acquire another electron underlies their ability to interact with and ultimately damage tissue. 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 incompletely reduced resulting in the formation of oxygen intermediates.

The super oxide radical is the main free radical produced in living cells; it is produced in the electron transport chain in the mitochondria. Free radicals generated naturally during oxidative metabolism can, if present in excess, react with fatty acids to form fatty acid hydroperoxides. The fatty acid hydroperoxides 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 supplied by 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. 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 include vitamin C, vitamin E, and beta-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. The body can synthesize antioxidant enzymes only when these metals are ingested in sufficient amounts. 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 which protect tissues from free-radical damage include vitamins E, C and beta-carotene. Selenium (Se) would be the mineral most specifically related to antioxidant function. There are at least 35 antioxidant Se proteins, including 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’. 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.

Table 1. Vitamins and minerals in antioxidant systems (a)

Nutrients 
Component (location in cell) Function
Vitamin C
Ascorbic acid (cytosol) Reacts with several types of ROS/RNS
Vitamin E  alpha-tocopherol (membranes)
Breaks fatty acid peroxidation chain reactions
beta-carotene
beta-carotene (membranes) Prevents initiation of fatty acids peroxidation chain reaction
Selenium
Glutathione peroxidase (cytosol) An enzyme that converts hydrogen peroxide to water
Copper and zinc  Superoxide dismutase (cytosol)
An enzyme that converts superoxide to hydrogen peroxide
Manganese and zinc Superoxide dismutase (mitochondria) An enzyme that converts superoxide to hydrogen peroxide
Copper Ceruloplasmin (water phase) An antioxidant protein, may prevent copper and iron from participating in oxidation reactions
Iron Catalase (cytosol) An enzyme (primarily in liver) that converts hydrogen peroxide to water

(a) Modified from Weiss (2005)

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 alpha-tocopherol. However, limited studies suggest non alpha-tocopherol and tocotrienols have important functions. gamma-tocopherol has been shown to be a more effective inhibitor of peroxy nitrite-induced lipid peroxidation (McCormick and Parker, 2004). Also, gamma-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 in 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).

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 beta-carotene is able to quench 1000 molecules of O2 before it reacts chemically. Although the principal antioxidant carotenoid is beta-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, easily giving up electrons to stabilize reactive species such as ROS.

Mechanisms of Antioxidant Functions

Antioxidants work 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 – 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 - confer stability to the ‘reactive’ species by donating an electron and become oxidized themselves to form a more stable radical. For example alpha-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, rendering 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 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 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 is nutritional stress 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 phagocytic process kills 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).

Table 2. Stress conditions that generate free radical production (a)

Nutritional
Environmental Internal
Toxins Temperature Diseases-bacterial, viral and fungal
high PUFA Humidity Allergy
Deficiencies-Vitamins A and E, carotenes and trace minerals Radiation Mycotoxins
Iron or Copper overload    

(a) Adapted from Surai (2002).

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, feed efficiency, gestation and lactation may not be adequate for normal immunity or maximizing 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. Diarrhea or vomiting will decrease intestinal absorption and increase needs. Vitamin A deficiency is often seen in heavily parasitized animals receiving properly fortified diets.

Mycotoxins in feed can substantially decrease uptake of antioxidant nutrients from feed and increase levels requireed to prevent damage by free radicals and their metabolites. It is estimated 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 Antioxidants and the Immunity Role of Vitamins and Trace Minerals

Antioxidant vitamins generally enhance different aspects of cellular and noncellular 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 disease susceptibility, animal morbidity, and mortality.

Vitamin C protective effect 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 which use free radicals and other highly reactive oxygen containing molecules to help kill pathogens. In the process, however, cells and tissues may be damaged by these reactive species. Ascorbic acid provides protection from oxidative damage.

Vitamin E and Se play vital roles in protecting leukocytes and macrophages during phagocytosis, the immune mechanism which kills invading bacteria. Both vitamin E and Se may help these cells to survive the toxic products that are produced when ingested bacteria are killed (Badwey and Karnovsky, 1980). Macrophages and neutrophils from vitamin E-deficient animals have decreased phagocytic activity.

Since vitamin E acts to quench free-radicals produced in the body, infection or other stress factors may deplete the limited tissue stores of vitamin E. Dietary requirements adequate for normal growth and production may not support maximal cellular and humoral immunity in ruminants. Unfortunately, nutrient requirements are generally based on growth and production. During stress and disease, there is an increase in production of glucocorticoids, epinephrine, eicosanoids, and phagocytic activity and concurrent production of free radicals. Further, Vitamin E has been implicated in stimulation of serum antibody synthesis, particularly IgG antibodies (Tengerdy, 1980).

Supplemental vitamin E and Se have been shown to provide protection against infection by several types of pathogenic organisms, as well as improve antibody titers and phagocytosis of pathogens (Reffett et al., 1988). For 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). Supplemental vitamin E may enhance recovery from bovine respiratory disease (BRD, Rivera et al., 2002). 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.

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). 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 antioxidant carotenoids which lack vitamin A activity, can enhance immune function, act directly as antimutagens and anticarcinogens, protect against radiation damage, and block the damaging effects of photosensitizers. Also, carotenoids can directly affect gene expression which 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 beta-carotene have important roles in protection against numerous infections including mastitis. Improved mammary health in dairy cows supplemented with beta-carotene and vitamin A during the dry and lactating periods has been reported (Chew and Johnston, 1985). Beta-carotene supplementation appears to stabilize PMN and lymphocyte function, both key components in defense against infection, during the period around dry off (Tjoelker et al., 1990). Beta-carotene enhanced the bactericidal activity of blood and milk PMN against S. aureus but did not affect phagocytosis (Daniel et al., 1991). Control of free radicals is important for bactericidal activity but not for phagocytosis. The antioxidant activity of vitamin A is negligible; it does not quench or remove free radicals. Beta-carotene, on the other hand, does have significant antioxidant properties and effectively quenches singlet oxygen free radicals (Mascio et al., 1991).

Supplementing vitamin E at higher than recommended levels (Dairy cattle NRC, 2001) has improved 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. A known consequence of vitamin E and Se deficiency is impaired PMN activity. Further, 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 alpha-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). Currently, it is suggested that peripartum dairy cows receive 2,000 to 4,000 IU vitamin E/d for optimal udder health (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. Extra vitamin E supplementation has been shown to improve gains and feed efficiencies in stressed feedlot cattle (Gill et al., 1986). Reductions in morbidity and sick pen stays have been demonstrated with vitamin E supplementation of feedlot cattle. In dairy cows, supplementation with 1,000 or 2,000 IU vitamin E starting 14 d prepartum through 7 d postpartum significant reduced days open (111 vs 84 d) and services per conception (1.3 vs. 2.2) in cows fed 2,000 IU/d.

Vitamin E supplementation has improved reproductive parmeters of bulls fed high concentrations of gossypol. Velasquez-Pereira et al. (1998) concluded that vitamin E is effective in reducing or eliminating important gossypol toxicity effects for male cattle.

Optimum Vitamin and Mineral Allowances

Most nutritionists usually consider NRC requirements for vitamins and minerals to be minimum requirements sufficient to prevent clinical deficiency signs; they may be adjusted upward as experience dictates. The concept of optimum vitamin nutrition suggests that optimal productivity occurs at higher levels of supplementation than those required to prevent deficiencies (Roche, 1979).

Allowances of vitamins and minerals are levels from all feed sources required to compensate for factors influencing the needs of animals. These “influencing factors” 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.

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 benefited 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 improved hardness and positive conformational changes in bovine hooves. Future research may find a need for carnitine supplementation. Feeding a complete B-vitamin mixture to cattle 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). Under stress conditions of feedlots, the microbial population in the rumen is apparently 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 nonprotein 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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Authors

University of Florida

University of Florida