Ruminants: Vitamin E

Vitamin E has been shown to be essential for the integrity and optimum function of reproductive, muscular, circulatory, nervous and immune systems in animals and humans (Hoekstra, 1975; Sheffy and Schultz, 1979; Bendich, 1993; McDowell, 2000; Traber and Sies, 1996). It is well established that some functions of vitamin E can be fulfilled in part or entirely by selenium or by other antioxidants. Vitamin E requirement is affected by the sulfur-bearing amino acids, cystine and methionine. Vitamin C (ascorbic acid) has been shown to spare vitamin E in tissues by regenerating alpha-tocopherol from its oxidation products. Considerable evidence indicates there may be undiscovered metabolic roles for vitamin E, which may be paralleled biologically by roles of selenium and possible other substances. For example, vitamin E has been shown to potentiate the action of insulin in humans (Cabalero, 1994). The most widely accepted functions of vitamin E are discussed in this section.

Aerobic metabolism generates reactive oxygen species (superoxide anion, hydroxyl radical, hydrogen peroxide, etc.). These species damage cellular proteins, nucleic acids and membrane lipids (Machlin, 1991). Living cells possess multiple defenses against oxidative damage, including the antioxidant enzymes, vitamin E and vitamin C (Sies et al., 1992). These defenses work synergistically to protect the cell from oxidative damage.

Vitamin E has several interrelated functions, most of which can be traced to its role as the primary lipid-soluble antioxidant in cell membranes (Machlin, 1991). Vitamin E is part of the body’s intracellular defense against the adverse effects of reactive oxygen species and free radicals that initiate oxidation of unsaturated phospholipids (Chow, 1979; Sies et al., 1992) and critical sulfhydryl groups of proteins and DNA (Brownlee et al., 1977). This function is closely related to and synergistic with the role of selenium and other antioxidants. Selenium is a cofactor of the enzyme glutathione peroxidase (GSHpx) that neutralizes hydrogen peroxide and hydroperoxides in the aqueous phase (cytosol and mitochondrial matrix). Also involved in these defenses are the copper-zinc-manganese superoxide dismutase (SOD) enzymes and the iron (heme) containing enzyme catalase. These enzymes indirectly prevent oxidation of unsaturated lipids within cell membranes.

Vitamin E provides direct protection of membrane lipids by itself reacting with peroxyl radicals formed during oxidation of PUFA. By doing so, vitamin E breaks the self-propagating chain of oxidative damage to the cell membrane and related structures (Drouchner, 1976; Machlin, 1991; Sies et al., 1992). When lipid hydroperoxides form in the absence of adequate vitamin E, direct cellular damage can result, in which peroxidation of lipids destroys the structural integrity of the cell and eventually cell function. Vitamin E also offers antioxidant protection to aqueous-phase cell components. Vitamin E increased the concentration of reduced glutathione in red blood cells and the activity of hepatic SOD in rats (Lii et al., 1998). Supplemental vitamin E was also shown to reduce to concentration of water-soluble oxidation products in mice challenged with dietary pro-oxidants (fish oil and excess iron).

By neutralizing free radicals and preventing oxidation of lipids within membranes, vitamin E reacts or functions as a chain-breaking antioxidant. Lipid peroxyl radicals are formed when reactive oxygen species abstract a hydrogen atom from an unsaturated fatty acid molecule. The fatty acid-peroxyl radical becomes self-propagating as double bonds of adjacent fatty acids are attacked. This "chain reaction" of membrane oxidation can produce widespread tissue damage if left unchecked. One of the unique properties of vitamin E is its direct incorporation into cell membranes where it interrupts free radical damage at the initiation stage, thus preventing the chain reaction of cell damage.

Muscle damage and muscular dystrophy are common signs of both vitamin E and selenium deficiency (McDowell, 2000). This results in leakage of cellular metabolites such as creatinine and enzymes (i.e. transaminases, dehydrogenases) through damaged membranes into plasma. The more metabolically active tissues (such as skeletal and smooth muscles and liver) have a greater potential for oxidative tissue damage if vitamin E supply is limiting. Erythrocytes and capillary walls are also susceptible to damage in animals with marginal vitamin E status (Machlin, 1991).

The antioxidant properties of vitamin E explain the well-established observation that dietary tocopherols protect or spare oxidizable nutrients such as vitamin A, vitamin C and the carotenes. Certain deficiency signs of vitamin E (i.e., muscular dystrophy) can be prevented by dietary supplementation with other antioxidant nutrients, such as selenium, which helps validate the antioxidant role of tocopherols. Other deficiency signs respond only to vitamin E (Maynard et al., 1979). Synthetic antioxidants such as ethoxyquin exhibit limited tissue storage, as well as rapid clearance from the body, and thus cannot replace tocopherol. It is clear that diets high in PUFA increase the vitamin E requirement (McDowell, 2000). Vitamin E is depleted during its action as an antioxidant, which explains the frequent observation that the presence of dietary unsaturated fat, especially PUFA, increases vitamin E requirement and can precipitate a vitamin E deficiency.

Alpha-tocopherol may be involved in the formation of structural components of biological membranes, thus exerting a unique influence on architecture of membrane phospholipids (Ullrey, 1981, Chen et al., 1998). It is reported that alpha-tocopherol stimulates the incorporation of 14C from linoleic acid into arachidonic acid in fibroblast phospholipids (Machlin, 1991). Additionally, alpha-tocopherol exerted a marked stimulatory effect on the formation of prostaglandin E from arachidonic acid, while a synthetic antioxidant had no effect (Machlin, 1991). Supplemental vitamin E increased the proportion of linoleic and eicosapentaenoic acids in liver phospholipids of the rat (Chen et al., 1998). These changes in membrane composition may induce changes in functionality or stability. Supplemental vitamin E has been reported to reverse the age-related decline in T lymphocyte function, in part by reducing prostaglandin production (Beharka et al., 1997).

Vitamin E is an inhibitor of platelet aggregation in pigs and humans (McIntosh et al., 1985; Traber and Sies, 1996). Vitamin E inhibits peroxidation of arachidonic acid, which is required for formation of prostaglandins involved in platelet aggregation (Panganamala and Cornwell, 1982; Machlin, 1991). Vitamin E-deficient animals exhibit an elevation in plasma thromboxane that is relieved by supplemental vitamin E (Chen D. Stress, Immune Function and Disease Resistance
Vitamin E clearly enhances immune function (Bendich, 1993; Machlin, 1991; Traber and Sies, 1996; McDowell, 2000). Furthermore, the dietary levels of vitamin E that result in optimum immune function are well above those required to prevent the classical vitamin E deficiency symptoms in humans and animals (Machlin, 1991; Beharka et al., 1997; McDowell, 2000). Lymphocytes and mononuclear cells have the highest vitamin concentration of any circulating cells (Traber and Sies, 1996). Considerable attention is presently being directed to the role that vitamin E, selenium and other antioxidant nutrients (vitamin C, beta carotene, zinc, manganese and copper) play in protecting leukocytes and macrophages during phagocytosis, the mechanism whereby immune cells engulf and kill bacteria and other pathogens. Vitamin E and selenium each play a specific role in protecting immune cells from damage by the oxygen radicals that these cells produce to kill ingested microorganisms (Badwey and Karnovsky, 1980; Machlin, 1991; Sies et al., 1992). For example, lung alveolar macrophages accumulate vitamin E, which enhances their defense against oxygen-free radicals generated during phagocytosis (Pathania et al., 1998).

During stress and disease, there is an increase in production of glucocorticoids, epinephrine, eicosanoids, oxygen radicals, nitric oxide and phagocytic activity of immune cells (Gross and Siegel, 1997; McDowell, 2000). Eicosanoid and corticoid synthesis and phagocytic respiratory bursts of leukocytes are prominent producers of free radicals. Supplemental vitamin E has been shown to enhance immune cell function under these conditions in a variety of species, including humans (McDowell, 2000; Traber and Sies, 1996).

The adrenal glucocorticoids, cortisol and corticosterone, and thyroid hormone (T3) are secreted in response to stress, including adverse environmental conditions, disease, food or water deprivation or social competition (Gross and Siegel, 1997). As these hormones are absorbed from plasma there is an increase in the ratio of "polymorphic" leukocytes (i.e. neutrophils and related cells) to lymphocytes (T and B lymphocytes) in plasma (Gross and Siegel, 1997). The change in this "P/L" ratio is a measure of the severity of the stress. Corticosterone treatment, a model of stress response, increases lipid peroxidation, decreases the activity of antioxidant enzymes and decreases growth rate and feed efficiency in laboratory rats (Ohtsuka et al., 1998). In the same study, feeding high levels of supplemental vitamin E significantly reduced lipid peroxidation, increased antioxidant enzyme activity and largely reversed the depression in growth rate and feed efficiency caused by corticosterone (Ohtsuka et al., 1998).

Vitamin E has been implicated in stimulation of serum antibody synthesis, particularly immunoglobulin G (IgG) (Tengerdy, 1980; Machlin, 1991). As discussed previously, the protective effects of vitamin E on animal health appear to involve reversal of the oxidative and immunosuppressive effects of glucocorticoids (Golub and Gershwin, 1985). Vitamin E also supports immune function by effects on arachidonic acid metabolism and subsequent synthesis of prostaglandins, thromboxanes and leukotrienes. The production of these compounds increases under stress conditions. Thromboxane and interleuken II appear to exert a negative feedback effect on leukocyte function (Hadden, 1987).

Supplemental vitamin E and selenium enhance the immune response against several types of pathogenic organisms. Increasing dietary vitamin E increases both antibody titers and phagocytosis of pathogens in calves (Cipriano et al., 1982; Reddy et al., 1985b, 1987b); lambs (Reffett et al., 1988; Finch and Turner, 1989; Turner and Finch, 1990); and dairy cows (Weiss et al., 1997; Weiss, 1998; Politis et al., 1995, 1996). For example, immune response was maximized in calves receiving 125 IU per day of vitamin E compared to calves receiving lower levels of dietary vitamin E (Reddy et al., 1987a). Garber et al. (1995) reported that mitogen-stimulated lymphocyte proliferation of dairy steers was maximized by feeding 1,000 IU of vitamin E daily as compared to either lower or higher levels of supplementation. Vitamin E and selenium deficiency reduced in vitro lymphocyte proliferation of calves, while repletion restored both numbers and function of lymphocytes (Pollock et al., 1994). Vitamin E and selenium exerted specific and joint effects on immune cell function. Stabel et al. (1992) reported that vitamin E increased immunoglobulin M (IgM) production by blood monocytes in vitro, and increased interleukin-1 gene expression by monocytes isolated from vitamin E-supplemented steers.

Vitamin E and selenium supplementation of dairy cows resulted in reduced rates and duration of intramammary infections and incidence of clinical mastitis (Smith et al., 1984, 1985, 1997). Vitamin E and selenium enhance host defenses by improving phagocytic cell function (Erskine et al., 1989; Hogan et al., 1992; Politis et al., 1995, 1996). Both vitamin E and the selenium-dependent glutathione peroxidase (GSHpx) protect phagocytic cells and surrounding tissues from oxidative attack by free radicals produced by the respiratory burst of neutrophils and macrophages during phagocytosis (Baboir, 1984; Baker and Cohen, 1983; Machlin, 1991). Hogan et al. (1990, 1992) reported that vitamin E supplementation of diets increased intracellular kill of Staphylococcus aureus and Escherichia Coli bacteria by neutrophils. Cows supplemented starting four weeks prior to calving through eight weeks postpartum with 3,000 IU vitamin E per day, in the presence of adequate selenium (0.3 ppm), had increased neutrophil and macrophage function and reduced somatic cell count compared to controls (Politis et al., 1995, 1996) ( Figure 1, 2). Supplemental vitamin E was shown to specifically stimulate phagocytosis of Staph. aureus by bovine neutrophils (Ndiweni and Finch, 1995). Both vitamin E and selenium increased neutrophil chemotaxis and superoxide production in these studies.

Neutrophil function in dairy cattle is suppressed around the time of calving (Guidry et al., 1976; Kehrli et al., 1989; Gilbert et al., 1993a), as are several other indices of immune function (Mallard et al., 1998). High levels of vitamin E (3,000 IU per day) essentially prevented suppression of blood neutrophil and macrophage function in dairy cows in the study by Politis et al. (1995). Gilbert et al. (1993b) found that impaired neutrophil function was associated with retained fetal membranes in dairy cows. This link is of interest in light of the studies showing that supplemental vitamin E during the dry period results in a decreased incidence of retained placenta; (Julien et al., 1976a, b; Harrison et al. 1984; Aréchiga et al., 1994; Miller et al., 1997; Erskine et al., 1997; Kim et al., 1997).

The immune response in sheep has been improved with supplemental vitamin E. Vitamin E improved disease resistance in lambs challenged with chlamydia (Stephens et al., 1979). Reffett et al. (1988) reported that vitamin E and selenium independently increased the immune response of lambs challenged with a viral pathogen. Myopathic lambs exhibit low lymphocyte responses when deficient in vitamin E and selenium (Finch and Turner, 1989; Turner and Finch 1990). The poor lymphocyte responses of the lambs with nutritional myopathy were rapidly reversed by intramuscular administration of vitamin E-selenium, with the prophylaxis most effective during the first five weeks of life (Finch and Turner, 1989). In a study of 1,300 lambings, supplementing 330 IU of vitamin E daily for 21 days prior to lambing significantly reduced lamb mortality for ewes lambing early in the season and increased the total weight of lambs weaned per ewe (Hatfield et al., 1999). Late-lambing ewes did not show a significant response to vitamin E. Supplementing ewes for 28 days prior to and 28 days after lambing has been shown to significantly increase vitamin E concentration in colostrum in the serum of their nursing lambs (Njeru et al., 1994). Dairy ewes injected twice during the dry period with vitamin E and selenium (5 mg and 0.1 mg/kg body weight) had significantly lower somatic cell count, increased erythrocyte glutathione peroxidase activity and enhanced neutrophil function compared to controls (Morgante et al., 1999). Injection of 900 IU vitamin E per week to ewes in late pregnancy increased the survival and growth rate of lambs from birth through weaning (Ali et al., 1999).

Early attempts to establish a practical role for vitamin E in ruminant reproduction were inconclusive (NRC, 1996). In one experiment, four generations of male and female dairy cattle were fed low-vitamin E diets (Guilickson et al., 1949). Although growth, reproduction and milk production were normal, several cattle died suddenly of apparent heart failure between 21 months and 5 years of age. In the bull, supplemental vitamin E did not affect sperm or semen characteristics or fertility (Salisbury, 1944). However, large doses of vitamins A, D, E, and C have been reported to favorably affect some characteristics of semen and sperm (Kozicki, 1981). Velásquez-Pereira et al. (1998a) reported that feeding 4,000 IU per day of supplemental vitamin E to Holstein bulls reversed the negative effects of feeding gossypol (14 mg free gossypol per day as cottonseed meal) on semen quality and reproductive performance ( Table 1). Supplemental vitamin E enhanced semen characteristics, plasma testosterone and breeding performance of gossypol-fed bulls above that of the control group, which was not fed gossypol. This suggests a potential benefit of vitamin E for breeding bulls regardless of diet. LaFlamme and Hidiroglou (1991) reported that pregnancy rate was improved (70% versus 33%) in beef heifers that had been fed supplemental vitamin E and selenium starting from eight months of age (weaning) and continuing for six months until breeding.

In dairy cattle, supplementation with selenium or both selenium and vitamin E reduced the incidence of retained placenta in herds where prevalence of retained placenta was high, or when selenium or vitamin E were marginal in the diet (Hurley and Doane, 1989). Supplemental vitamin E-selenium has also been reported to reduce metritis, cystic ovaries (Harrison et al., 1984) and time of uterine involution in cows with metritis (Harrison et al., 1986). Miller et al. (1997) summarized seven years of experimental data (n = 602 cows) in which comparisons were made between cows fed approximately 200 IU of vitamin E per day, and cows fed the same ration with 1,000 IU per day of supplemental vitamin E for the last 42 days of the dry period. Statistical analysis revealed a highly significant effect of supplemental vitamin E in reducing the incidence of retained placenta (27.4% in controls versus 12.6% in vitamin E-supplemented; P < 0.0001) (Figure 3). Cows fed 1,000 IU of supplemental vitamin E per day were 2.6 times less likely to develop retained placenta. In a more recent trial with 126 cows, feeding 1,000 IU per day of supplemental vitamin E during the dry period significantly reduced the interval to first estrus (Miller et al., 1997). Vitamin E increased plasma fast-acting antioxidant capacity and may increase plasma estradiol through protection of the cytochrome P-450 dependent enzyme system required for estradiol synthesis (Miller et al., 1993, 1997). These authors hypothesize that the ratio of corticosterone to estradiol in plasma is an indicator of stress level and predisposition toward reproductive disorders in dairy cattle.

In a recent study, Campbell and Miller (1998) fed 144 cows (64 primigravid heifers and 80 cows) either 0 or 1,000 IU of supplemental vitamin E per day in the presence or absence of excess iron and 800 mg added zinc for the last 42 days prepartum. Supplementation was discontinued after calving. Plasma vitamin E levels were low for all treatments (1.0 to 1.5 µg/ml), compared to levels considered minimal for periparturient cows (3.0 to 3.5 µg/ml), based on neutrophil function and udder health (Weiss, 1998). Despite the low levels of plasma vitamin E, cows supplemented with 1,000 IU vitamin E before calving had a significant reduction in days to first estrus, days to first breeding and days open. Days open were reduced by 32 overall. Retained placenta was not affected by any treatment. Therefore, 1,000 IU of supplemental vitamin E fed for 42 days prior to calving had significant beneficial effects on reproduction after calving.

In Ohio research, incidence of retained placenta was reduced from a mean of 51.2% in control cows to 8.8% in cows injected with a combination of selenium and vitamin E (Julien et al., 1976b). Harrison et al. (1984) reported 17.5% retained placenta for control dairy cows, with no incidence for cows receiving both selenium and vitamin E. (Neither vitamin E nor selenium was as effective alone.) From the same study, control versus selenium administration reduced cystic ovaries (47% versus 19%) and incidence of metritis (84% versus 60%).

Other research found no effect of a prepartum injection of selenium and vitamin E on the incidence of retained placenta (Gwazdauskas et al. 1979; Kappel et al., 1984; Hidiroglou et al., 1987; Schingoethe et al., 1982). However, more recent studies have reported positive effects of vitamin E or vitamin E-selenium injection on reproduction in dairy and beef cattle. Aréchiga et al. (1994), using 198 Holstein cows in Florida, found that a single injection of 50 mg selenium and 680 IU vitamin E at 21 days prepartum significantly reduced retained placenta (10.1% versus 3%), increased first service pregnancy rate (41% versus 25%), reduced services per conception and reduced days open (141 versus 121 days). Erskine et al. (1997), reported results of a trial with 420 Holstein cows, in which a single injection of approximately 4,000 IU vitamin E at 14 days prepartum significantly reduced retained placenta (6.4% versus 12.5%), metritis (3.9% versus 8.8%) and increased serum vitamin E up to 14 days after injection. Kim et al. (1997) compared cows injected 20 days prior to calving with: placebo, selenium (40 mg), vitamin E (500 IU), or both selenium and vitamin E. They reported significant reductions in retained placenta (13.3% versus 30%) and days to first service (59.5 versus 102.7) in cows injected with both selenium and vitamin E. Table 2 summarizes results of studies on the effect of vitamin E and selenium on reproduction in dairy cattle.

Miller et al. (1993, 1997) reported that udder edema of dairy heifers was significantly reduced by supplementing diets with 1,000 IU of vitamin E daily for the last 42 days of gestation.

There is limited evidence that vitamin E is involved in biological oxidation-reduction reactions and may influence the biosynthesis of DNA within cells. Vitamin E appears to enhance the activity of microsomal cytochrome P-450 (Chen et al., 1998), which has multiple roles in detoxification and cell biosynthesis (Lehninger, 1982). Conflicting data exist on the role of vitamin E and DNA stability (Antunes and Takahashi, 1998; Umegaki et al., 1997; Pincheira et al., 1999).

Both vitamin E and selenium provide protection against toxicity of certain heavy metals (Whanger, 1981). Vitamin E is highly effective in reducing toxicity of silver, arsenic, nickel and lead, and shows slight effects against cadmium and mercury toxicity. Heavy metals produce oxidative damage to tissues, and thus vitamin E can exert a protective antioxidant effect.

Vitamin E can be effective against other toxic substances. For example, treatment with vitamin E gave protection to weanling pigs against monensin-induced skeletal muscle damage (Van Vleet et al., 1977). Mycotoxins must be detoxified by the cytochrome P-450 system, the activity of which appears related to vitamin E status. Adsorbants (bentonites, calcium aluminosilicates) used in research to alleviate symptoms of mycotoxicosis have been shown to reduce plasma vitamin E concentrations (Plank et al., 1990), suggesting that vitamin E levels may need to be increased if these products are fed. Velásquez-Pereira et al. (1998a) found that 4,000 IU of supplemental vitamin E per day significantly reduced bull sperm abnormalities caused by feeding 14 mg per day free gossypol occurring naturally in cottonseed meal. Likewise, Velásquez-Pereira et al. (1999) reported that feeding 4,000 IU vitamin E per day alleviated negative effects of gossypol on the growth and health of dairy calves, and increased calf performance compared to the positive control ration.

Vitamin E and selenium have synergistic roles in protecting cells and tissues from oxidative damage. Selenium has a sparing effect on vitamin E and delays onset of deficiency signs. Likewise, vitamin E and sulfur amino acids partially protect against or delay onset of several forms of selenium deficiency syndromes. Vitamin E prevents fatty acid hydro-peroxide formation, sulfur amino acids are precursors of glutathione and selenium is an essential component of glutathione peroxidase (Smith et al., 1974).

Tissue necrosis occurs in most species as a result of vitamin E-selenium deficiency, primarily due to oxidative damage. Peroxides and related oxygen radicals are highly destructive to tissues and can increase susceptibility to disease. It now appears that vitamin E in cellular and subcellular membranes is the first line of defense against peroxidation of vital membrane phospholipids (Figure 4). Selenium, as cofactor of glutathione peroxidase, is a second line of defense against oxygen radicals and their oxidation products. Vitamin C, beta-carotene, copper, iron, manganese and zinc also play important roles in the cellular antioxidant system, either directly or as cofactors in the antioxidant enzymes superoxide dismutase and catalase.

Other functions attributed to vitamin E (Scott et al., 1982; Machlin, 1991) include:

• phosphorylation reactions, especially of high-energy phosphate compounds, such as creatinine phosphate and adenosine triphosphate (ATP);
• support of the cytochrome P-450 system;
• a role in synthesis of vitamin C (ascorbic acid);
• a role in synthesis of ubiquinone;
• a role in sulfur amino acid metabolism; and
• potentiation of insulin action (Cabalero, 1994).

Pappu et al. (1978) reported that vitamin E plays a role in vitamin B12 metabolism. A deficiency of vitamin E interfered with conversion of vitamin B12 to its coenzyme 5'-deoxyadenosylcobalamin and therefore reduced conversion of methylmalonyl-CoA to succinyl-CoA. Turley and Brewster (1993) reported that vitamin E deficiency increases urinary excretion of methylmalonic acid, which is symptomatic of vitamin B12 deficiency. They also reported that conversion of cyanocobalamin to its active form was reduced by vitamin E deficiency.

In rats, vitamin E deficiency has been reported to inhibit vitamin D metabolism in the liver and kidneys with decreased formation of active vitamin D metabolites and decreases in the concentration of the hormone-receptor complexes in the target tissue. Liver vitamin D 25-hydroxylase activity decreased by 39%, 25-OHD-1-alpha-hydroxylase activity in the kidneys decreased by 22%, and 24-hydroxylase activity by 52% (Sergeev et al., 1990).

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