Ruminants: Vitamin A

The basic structure of vitamin A is all-trans retinol (Illus. 1), which gives rise to several isomers, the most important of which are all-trans retinal and all-trans retinoic acid. Vitamin A is one of the least stable vitamins and is highly labile to light and oxidation. Chemically, retinol consists of a substituted cyclohexene ring with an aliphatic side-chain marked by a series of conjugated double bonds. This unique structure allows retinol and its derivatives to function as both a visual pigment and a regulator of cellular growth and differentiation (Olson, 1991).

Vitamin A (retinol) does not occur per se in plants, but its precursors the carotenes occur in several forms. Carotenes are orange-yellow pigments that occur in green leaves and to a lesser extent in corn grain. Four of these compounds, alpha-carotene, beta-carotene, gamma-carotene and cryptoxanthin (the main carotenoid of corn), are of particular importance because of their provitamin A activity. Vitamin A activity of beta-carotene is substantially greater than that of other carotenoids. However, research has consistently shown that pure vitamin A has twice the potency of beta-carotene on a weight-to-weight basis, indicating a maximum conversion efficiency of 50%.

Carotenoids are commonly referred to as "provitamin A" because they can be enzymatically transformed to vitamin A by most species. The primary site of carotene conversion to retinol is the intestinal mucosa. Beta-carotene is cleaved by the enzyme: beta-carotene 15, 15' dioxygenase (Bauernfeind, 1981), yielding one molecule of retinaldehyde, which is enzymatically reduced to retinol. Normal processes of fat digestion and absorption and adequate dietary fat content are required for the absorption and subsequent conversion of beta-carotene to retinol. In dairy cows supplemental fat elevates plasma levels of beta-carotene (Weiss et al., 1994).

Beta-carotene is the most potent vitamin A precursor in plants. The efficiency of carotene transformation varies with species. Some species, such as cats, are unable to convert carotenoids to vitamin A (Bauernfeind, 1981). In dairy cattle, the accepted standard of conversion is that 1 mg of all-trans beta-carotene will provide 400 IU of vitamin A activity, for a 12% conversion efficiency (NRC, 1989, Bauernfeind, 1981). The Jersey and Guernsey breeds absorb a greater proportion of carotene intact from the intestine, leading to the orange-yellow color of their body and milk fat, but they have the capacity to convert carotene to retinol in the liver, lung and other tissues (McGinnis, 1988). Cattle reportedly have the capacity to convert beta-carotene to retinol in the ovary (Sklan, 1983; Schweigert et al., 1988), where beta-carotene may act as a local supply of retinol.

The vitamin A value of a feedstuff is the sum of its carotene and preformed vitamin A content. Vitamin A activity (IU) is defined in terms of the all-trans isomer of retinol. One mg of all-trans retinol equates to 3,333 IU of vitamin A activity (Machlin, 1991). The 13-cis isomer has a relative biological activity of 50% for chicks (Ullrey, 1972). The carotene content of most feedstuffs is quite variable and subject to large losses under less than ideal harvest and storage conditions. Vitamin A and the precursor carotenoids are rapidly destroyed by oxygen, heat, light and acids. Presence of moisture and trace minerals accelerates destruction of vitamin A activity in feeds (Olson, 1991).

Common forms of vitamin A for use in commercial feeds are the acetate, propionate and palmitate esters of retinol. These are normally produced as hardened or cross-linked gelatin beadlets to improve stability. Vitamin A acetate is the most common source used in dry feeds for livestock. Propionate and palmitate esters are generally used in liquid feeds.

A number of factors influence the digestibility of carotene and vitamin A in ruminants. Working with lambs, Donoghue et al. (1983) reported that dietary levels of vitamin A ranging from mildly deficient to toxic levels affect digestion and uptake. Percentage absorption from supplemental levels of 0, 100 and 12,000 µg retinol (0, 333 and 40,000 IU) per kg diet were 91%, 58% and 14%, respectively. Enteric disease states such as Cryptosporidium infection reduce vitamin A absorption in calves (Holland et al., 1992). Wing (1969) reported that the apparent digestibility of carotene in various forages fed to dairy cattle averaged about 78%.

Variables that influence carotene digestibility include month of forage harvest; type of forage (hay, silage, greenchop or pasture); plant species; plant dry matter content; and harvest and storage conditions. In general, carotene digestibility was above average during warmer months and below average during winter. Preruminant calves (Bierer et al., 1995) readily absorb carotenoid compounds, especially beta-carotene, which contributes to liver vitamin A stores (Hoppe et al., 1996). Conversion of beta-carotene to retinol by animals is inversely related to vitamin A status (McDowell, 1992). Conversion of carotene to retinol decreases with increasing intake of carotene or vitamin A and with increasing vitamin A concentrations in liver (van Vliet et al., 1996). There are notable species differences in the absorption and transport of carotenoids by plasma lipoprotein fractions (Yang et al., 1992). The physiological significance of these species differences is not yet understood.

Several studies indicate that appreciable amounts of vitamin A are degraded in the rumen. Studies with various diets have reported pre-intestinal vitamin A disappearance values ranging from 40% to 70% (Ullrey, 1972). Rode et al. (1990) compared microbial degradation of vitamin A (retinyl acetate) in steers fed concentrate, hay or straw diets. Estimated effective rumen degradation of biologically active vitamin A was 67% for cattle fed high-concentrate diets, compared 16% and 19% for animals fed hay and straw diets, respectively. Weiss et al. (1995) reported 72% in vitro rumen degradation of retinyl esters with a 50% concentrate ration and 16% to 20% degradation when diets of greater than 75% forage were fed. On the other hand, beta-carotene degradation was 23% in sheep, in vivo, across a wide range of diet starch levels. Weiss (1998) interpreted these data to mean that in ruminants fed rations with 50% or more concentrate, retinyl esters would be only 50% as available on a weight basis compared to beta-carotene. However, preformed vitamin A (e.g., retinyl acetate) has approximately eight times the vitamin A activity per mg compared to beta-carotene in cattle (3,000 IU/mg vs. 400 IU/mg), and this must taken into account in making any comparison. Interestingly, Florida workers (Aréchiga et al., 1998), reported that milk production was significantly increased in each of three separate experiments in which cows were fed 400 mg of beta-carotene per head daily, despite being supplemented with 200,000 to 250,000 IU of vitamin A per day. The rations averaged 28.8% and 28% forage on a dry matter basis, although fibrous byproduct feeds made up a significant portion of the rations. Ruminal destruction of vitamin A may have been a factor in this study. The results suggest that beta-carotene supplementation of dairy cows may be warranted under certain dietary or climatic conditions.

The primary site of vitamin A and carotene absorption is the proximal jejunum (Frye et al., 1991; Olson, 1991). Normal pancreatic, liver and bilary function and adequate fat intake are required for absorption of vitamin A and its precursors.

Dietary retinyl esters are hydrolyzed to retinol by pancreatic esterase in the small intestine; they are absorbed as the free alcohol in association with lipid micelles and then re-esterified to form retinyl palmitate in the mucosa. The retinyl esters are transported via the lymphatic system, mainly in association with chylomicrons, to the liver where they are hydrolyzed to retinol and re-esterified for storage in parenchymal cells. Hydrolysis of the stored retinyl esters liberates retinol that is combined with retinol-binding protein for secretion into the bloodstream. Small amounts of retinyl palmitate are absorbed directly into portal blood and are adsorbed to plasma lipoproteins. In vitamin A toxicity, plasma retinyl palmitate concentrations are elevated while retinol concentrations remain normal (Herdt and Stowe, 1991).

Retinol is released from the hepatocyte complexed with retinol binding protein (RBP) and transported in this form to the tissues. The retinol-RBP complex is itself associated with transthyretin, a thyroid hormone carrier. Therefore, there is a metabolic interdependence between vitamin A and thyroid function. Iodine deficiency impairs vitamin A metabolism, and vitamin A deficiency impairs thyroid function (Olson, 1991). Goitrogenic feedstuffs in the diet may increase the vitamin A requirement or potentiate cattle responses to supplemental beta-carotene.

The circulating RBP-retinol complex reaches the target cells and retinol is released at an RBP receptor site on the cell surface. Retinol is transported through the cell membrane where it combines with cellular retinol binding protein (CRBP). CRBP transports retinol to its intracellular sites of action, including the cell nucleus (Olson, 1991). Retinol is irreversibly converted to retinoic acid during metabolism. The main excretory pathway for vitamin A is by elimination as glucuronide conjugates of retinoic acid in the bile prior to fecal excretion. Recently, 9,13 di-cis retinoic acid has been reported to be the predominant retinoic acid isomer in plasma during the periparturient (-14 to +14 days) period in dairy cows (Horst et al., 1995). The significance of this compound in vitamin A metabolism is being investigated (Nonnecke et al., 1997).

Retention and storage of vitamin A is widespread in animals, indicating the critical importance of this vitamin to survival. Liver normally contains about 90% of the total body stores of vitamin A. The rest is stored in the kidneys, lungs, adrenal glands, bone marrow and blood, with small amounts also found in other organs and tissues. In rats, retinol is preferentially sequestered in bone marrow during vitamin A deficiency (Twining et al., 1997), illustrating the primary importance of retinol in the generation of immune cells and erythrocytes.

Vitamin A alcohol (retinol) is a nearly colorless, fat-soluble, long-chain, unsaturated compound with five bonds (Illus. 1). Since it contains double bonds, vitamin A can exist in different isomeric forms. Vitamin A and the precursors, carotenoids, are rapidly destroyed by oxygen, heat, light and acids. Presence of moisture and trace minerals reduces vitamin A activity in feeds (Olson, 1984).

Precursors of vitamin A, the carotenes, occur as orange-yellow pigments, mainly in green leaves and to a lesser extent in corn. Four of these carotenoids, alpha-carotene, beta-carotene, gamma-carotene, and cryptoxanthin (the main carotenoid of corn), are of particular importance because of the provitamin A activity. Vitamin A activity of beta-carotene is substantially greater than that of other carotenoids. However, biological tests have consistently shown that pure vitamin A has twice the potency of beta-carotene on a weight-to-weight basis. Only one molecule of vitamin A is formed from one molecule of beta-carotene. Lycopene is an important carotenoid for its antioxidant function but does not possess the beta-ionone ring structure (required for vitamin A activity) and, therefore, is not a precursor of vitamin A. In humans, beta-carotene and lycopene are the predominant carotenoids in tissue (Ribaya-Mercado et al., 1995).

A number of factors influence digestibility of carotene and vitamin A. Working with lambs, Donoghue et al. (1983) reported that dietary levels of vitamin A ranging from mildly deficient to toxic levels affect digestion and uptake. Percentage transfer from the digestive tract from supplemental dietary levels of 0, 100 and 12,000 µg retinol per kg were 91%, 58% and 14%, respectively. Wing (1969) reported that the apparent digestibility of carotene in various forages fed to dairy cattle averaged about 78%. Variables that influenced carotene digestibility included month of forage harvest, type of forage (hay, silage, greenchop or pasture), species of plant and plant dry matter. In general, carotene digestibility was higher during warmer months than during winter.

Several papers indicate that appreciable amounts of carotene or vitamin A may be degraded in the rumen. Various studies with different diets have indicated preintestinal vitamin A disappearance values ranging from 40% to 70% (Ullrey, 1972). Rode et al. (1990) compared microbial degradation of vitamin A (retinol acetate) from steers fed concentrate, hay or straw diets. Estimated effective rumen degradation of biologically active vitamin A was 67% for cattle fed concentrates compared to 16% and 19% for animals fed hay and straw diets, respectively.

In most mammals the product ultimately absorbed from the intestinal tract as a result of feeding carotenoids is mainly vitamin A itself. There is considerable species specificity regarding the ability to absorb dietary carotenoids. In some species, such as the rat, pig, goat, sheep, rabbit, buffalo and dog, almost all the carotene is cleaved in the intestine. In humans, some breeds of cattle, horses and carp, significant amounts of carotene can be absorbed. Absorbed carotene can be stored in the liver and fatty tissues. Hence, these latter animals have yellow body and milk fat, whereas animals that do not absorb carotene have white fat. In the case of cattle, there is a strong breed difference in absorption of carotene. The Holstein is an efficient converter, having white adipose tissue and milk fat. The Guernsey and Jersey breeds, however, readily absorb carotene, resulting in yellow adipose fat and also butterfat.

Beta-carotene present in feed is converted in the intestinal mucosa by an enzyme to retinal, which is then reduced to retinol (vitamin A). However, extensive evidence exists also for random (excentric) cleavage, resulting in retinoic acid and retinal, with a preponderance of apocarotenals formed as intermediates (Wolf, 1995). The cleavage enzyme has been found in many vertebrates but is not present in the cat or mink. Therefore, these species cannot utilize carotene as a source of vitamin A. The main site of vitamin A and carotenoid absorption is the mucosa of the proximal jejunum. Although carotenoids are normally converted to retinol in the intestinal mucosa, they may also be converted in the liver and other organs, especially in yellow fat species such as Guernsey and Jersey breeds of cattle (McGinnis, 1988). Either dietary retinol or retinol resulting from conversion of carotenoids is then esterified with a long-chain fatty acid, usually palmitate.

A number of factors affect absorption of carotenoids. Cis-trans isomerism of the carotenoids is important in determining their absorbability, with the trans forms being more efficiently absorbed (Stahl et al., 1995). Among carotenoids, there is a differential uptake and clearance of specific carotenoids (Bierer et al., 1995; Johnson et al., 1997). Dietary fat is important (Roels et al., 1958; Fichter and Mitchell, 1977). Dietary antioxidants (e.g., vitamin E) also appear to have an important effect on the utilization and perhaps absorption of carotenoids. It is uncertain whether the antioxidants contribute directly to the efficient absorption or whether they protect both carotene and vitamin A from oxidative breakdown. Protein deficiency reduces absorption of carotene from the intestine.

Several studies have shown that liver can store enough vitamin A to protect animals from deficiency for long periods of low vitamin A/carotene intake. Hepatic retinol concentrations above 20 µg/g of fresh tissue provide adequate reserves to maintain serum retinol within normal limits (Herdt and Stowe, 1991). This large storage capacity must be considered in studies of vitamin A requirements to ensure that intakes that appear adequate for a given function are not being supplemented by reserves stored prior to the period of observation. Measurement of the liver store of vitamin A at slaughter or by biopsy ( Illus. 2) is a useful technique in studies of vitamin A status and requirements. Plasma retinol concentration is not a reliable measure of liver stores, because of its tight homeostatic regulation.

Besides being partially converted to retinol, beta-carotene and other carotenoids such as lutein are absorbed in lipid micelles and transferred to the bloodstream, where they are transported primarily in association with lipoproteins (Yang et al., 1992). Sheep, goats and cattle differ in the proportion of carotenoids associated with the VLDL, LDL and HDL lipoprotein fractions of plasma (Yang et al., 1992). Compared to sheep and goats, cattle have tenfold higher concentrations of beta-carotene in the liver, 7 versus 0.7 µg/g of tissue (Yang et al., 1992).

Ruminant animals are born with very low liver reserves of vitamin A. Therefore, it is critical that calves receive adequate amounts of colostrum, which contains high levels of vitamin A activity, within a few hours after birth. Colostrum deprivation during the first 24 hours of life impairs overall absorption of vitamins A, D and E through seven days of age (Blum et al., 1997). Adequate fat intake is required for fat-soluble vitamin absorption in calves (Rajaraman et al., 1997) and is a consideration in the formulation of milk replacers.

Low vitamin A intake by the cow during pregnancy increases the likelihood of vitamin A deficiency in the calf, because the cow’s body reserves are low and colostrum will have a subnormal vitamin A content (Miller et al., 1969). Deficiencies of dietary protein, phosphorus, zinc and iodine during gestation can also impair vitamin A metabolism in the cow and reduce colostral vitamin A supply to the calf. Addition of 200 g/day supplemental fat to the diet of dry dairy cows increased plasma beta-carotene (as well as vitamin E) concentrations in the peripartum period (Weiss et al., 1994).

Grazing livestock that have access to high quality, green pasture can store sufficient vitamin A in the liver to withstand periods of low carotene intake during the winter or dry season, perhaps for as long as four to six months. Cattle grazing good pasture can attain 30 to 80 mg per kg (13.6 to 36.4 mg per lb) of liver vitamin A (Rumsey, 1975). Beef cattle entering the feedlot with 20 to 40 mg per kg (9.1 to 18.2 mg per lb) of liver vitamin A will have adequate stores for three to four months (Perry et al., 1968). Intramuscular injection of 1 million IU of emulsified vitamin A apparently provides sufficient vitamin A to prevent deficiency signs for two to four months in growing or breeding beef cattle (NRC, 1996).

Sheep grazing green forage during the normal growing season can tolerate a low-carotene diet for four to six months (NRC, 1985). Goats that have had access to good quality, green forage can function with low carotene diets for at least three months without detrimental effects (NRC, 1981). The browsing nature of the goat allows it to select palatable green plant leaves, buds, etc., and may impart to it an advantage over other ruminant species in obtaining carotene under conditions of sparse vegetation.

Dietary retinyl esters are hydrolyzed to retinol in the intestine; they are absorbed as the free alcohol and then re-esterified in the mucosa. The retinyl esters are transported mainly in association with lymph chylomicrons to the liver where they are hydrolyzed to retinol and re-esterified for storage. Hydrolysis of the ester storage form mobilizes vitamin A from the liver as free retinol.

Retinol is released from the hepatocyte as a complex with retinol-binding protein (RBP). Retinol in association with RBP circulates to peripheral tissues complexed to a thyroxine-binding protein, transthyretin (Blomhoff et al., 1991; Ross, 1993). The retinol-transthyretin complex is transported to target tissues, where the complex binds to a cell-surface receptor and the retinol is transported into cells of target tissue.

Once the retinoids are transferred into the cell they are quickly bound by specific binding proteins in the cell cytosol. The intracellular retinoid-binding proteins bind retinol, retinal and retinoic acid for purposes of protection against decomposition, solubilize them in aqueous medium, render them nontoxic and transport them within cells to their site of action. These binding proteins also function by presenting the retinoids to the appropriate enzymes for metabolism (Wolf, 1991). Some of the principal forms of intracellular (cytoplasmic) retinoid-binding proteins are cellular retinol-binding proteins (CRBP, I and II), cellular retinoic acid-binding proteins (CRABP, I and II), cellular retinaldehyde binding protein (CRALBP) and six nuclear retinoic acid receptors (RAR and RXR, with alpha, beta and gamma forms). There are two classes of nuclear receptors with all-trans retinoic acid the ligand for RAR; 9-cis-retinoic acid is the ligand for RXR (Kasner et al., 1994; Kliewer et al., 1994).

Retinol is readily transferred to the egg in birds, but the transfer of retinol across the placenta is marginal and mammals are born with very low liver stores of vitamin A. In the pig, uterine RBP has been identified in the uterus, with the function of delivering retinol to the fetus (Clawitter et al., 1990). The main excretory pathway for vitamin A is by elimination as glucuronide conjugates in the bile prior to fecal excretion.

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