DSM in Animal Nutrition & Health
Properties and Metabolism
Vitamin A itself does not occur in plant products, but its precursors, carotenes, do occur in several forms. These compounds (carotenoids) are commonly referred to as provitamin A because the body can transform them into active vitamin A. The combined potency in a feed, represented by its vitamin A and carotene content, is referred to as its vitamin A value. Vitamin A occurs in three forms: retinol, retinal, and retinoic acid. Retinol is the alcohol form of vitamin A. Replacement of the alcohol group (-OH) by an aldehyde group (-CHO) yields retinal, and replacement by an acid group (-COOH) gives retinoic acid. Vitamin A products for feed use include the acetate, propionate and palmitate esters. Vitamin A alcohol (retinol) is a nearly colorless, fat-soluble, long-chain, unsaturated compound with five bonds (Illus. 2-1). 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 (McDowell, 2000).
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. As an example, both alpha-carotene and cryptoxanthin are about one-half the conversion rate of beta-carotene (Tanumihardjo and Howe, 2005). 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. 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 milk fat. 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. Although cats cannot convert beta-carotene to vitamin A, they do absorb carotenoids (Schweigert et al., 2002). Although beta-carotene is found in plasma of cats, it is never in tissues (Raila et al., 2001). 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 (Stahlet 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. Different species of animals convert beta-carotene and vitamin A with varying degrees of efficiency. Some factors that influence the rate at which carotenoids are converted to vitamin A are type of carotenoid, class and production level of animal, individual genetic differences in animals and level of carotene intake (McDowell, 2000). Efficiency of vitamin A conversion from beta-carotene is decreased with higher levels of intake (Van Vliet et al., 1996). The conversion rate of the rat has been used as the standard value, with 1 mg of beta-carotene equal to 1,667 IU of vitamin A. Based on this standard, the comparative efficiencies of various species are shown in Table 2-1. Of the species studied, only poultry are equal to the rat in vitamin conversion, with cattle being only 24% as efficient. For the dog, conversion of beta-carotene to vitamin A is 50% as efficient as the rat and chicken. For the chicken, as beta-carotene level is increased, conversion efficiency drops from a ratio of 2:1 to 5:1. For the calf, it decreases from 8:1 to 16:1 (Bauernfeind, 1972).
Stress conditions, such as extremely hot weather, viral infections and altered thyroid function, have also been suggested as causes for reduced carotene to vitamin A conversion. Vitamin A requirements are higher under stressful conditions such as abnormal temperatures or exposure to disease conditions. As an example with poultry, coccidiosis not only causes destruction of vitamin A in the gut but also injures the microvilli of the intestinal wall. 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 (McDowell and Ward, 2008).
The efficiency of beta-carotene in meeting the vitamin A requirements of trout and salmon apparently is dependent on water temperature. Cold-water fish utilize precursors of vitamin A at 12.4 to 14°C, but do not at 9°C (Poston et al., 1977). Activity of beta-carotene-15, 15′-dioxygenase, which oxidizes beta-carotene to retinal in the intestinal mucosa, may be restricted at cold temperatures.
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 overall absorption of vitamin A is of the order of 80% to 90% (Olson, 2001). 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. The primary storage form is retinyl palmitate. 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. The receptor was found to be located in all tissues known to require retinol for their function, particularly the pigment epithelium of the eye (Wolf, 2007).
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, and 9-cis-retinoic acid 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. Dogs differ from other species with respect to the occurrence of a high percentage of retinyl esters in blood plasma and the excretion of substantial amounts of vitamin A in the urine. Contrary to plasma, urine concentrations of retinol and retinyl esters in dogs may possibly serve as indicators of dietary intake of vitamin A in excess of requirements (Schweigert and Bok, 2000).
The liver normally contains about 90% of the total body vitamin A. The remainder is stored in the kidneys, lungs, adrenals and blood, with small amounts also found in other organs and tissues. Several studies have shown that the liver can store enough vitamin A to protect the animal from long periods of dietary scarcity. 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 a biopsy is a useful technique in studies of vitamin A status and requirements.
During periods of low dietary carotene, stored vitamin A can be mobilized and utilized without the animal showing signs of vitamin A deficiency. At birth, the mammal usually does not have sufficient vitamin A reserves to provide for its needs for any substantial time. Accordingly, it is important that young animals receive colostrum, which generally is high in vitamin A, within a few hours after birth. If the dam has received a diet low in vitamin A content, the newborn animal is likely to be susceptible to a vitamin A deficiency because body reserves are low and colostrum will have a subnormal content (Miller et al., 1969).
A. Vitamin A
Vitamin A is necessary for support of growth, health and life of major animal species. In the absence of vitamin A, animals will cease to grow and eventually die. Vitamin A and its derivatives, the retinoids, have a profound influence on organ development, cell proliferation, and cell differentiation and their deficiency originates or predisposes animals to a number of disabilities (McDowell, 2000; Esteban-Pretel et al., 2010). Even with a mild vitamin A deficiency in pregnant rats, there was reduction of nephrons in the fetuses resulting in kidney malfunction (Márin et al., 2005; Bhat and Manolescu, 2008). Vitamin A deficiency in rats included anatomic and metabolic changes comparable to those associated with neurodegenerative disorders; slowing of cerebral growth was correlated with retinol level (Rahab et al., 2009). During embryogenesis, retinoic acid has been shown to influence processes governing the patterning of neural tissue and cranio-facial, eye and olfactory system development; retinoic acid continues to influence the development, regeneration, and well-being of neurons (Asson-Batres et al., 2009). Retinoic acid also regulates the differentiation of epithelial, connective and hematopoietic tissues (Safonova et al., 1994). It has been suggested that retinoic acid is an effective antidiabetic agent that could be considered in treatment of type 2 diabetes (Wolf, 2009; Manolescu et al., 2010). The nature of the growth and differentiation response elicited by retinoic acid depends upon cell type. Retinoic acid can be an inhibitor of many cell types with a potential to reduce adipose tissues in meat producing animals (Suryawan and Hu, 1997). Although retinol is needed for normal vision and some aspects of reproduction, recent discoveries have revealed that most, if not all, actions of vitamin A in development, differentiation and metabolism are mediated by nuclear receptor proteins that bind retinoic-acid, the active form of vitamin A (Anonymous, 1993). A group of retinoic acid-binding proteins (receptors) function in the nucleus by attaching to promoter regions in a number of specific genes to stimulate their transcription and thus affect growth, development and differentiation. Retinoic acid receptors in cell nuclei are structurally homologous and functionally analogous to the known receptors for steroid hormones, thyroid hormone (triiodothyronine) and vitamin D (1,25-OH2D). Thus, retinoic acid is now recognized to function as a hormone to regulate the transcription activity of a large number of genes (Ross, 1993; Shin and McGrane, 1997; Solomons, 2006). Vitamin A deficiency causes at least four different and probably physiologically distinct lesions: loss of vision due to a failure of rhodopsin formation in the retina; defects in bone growth; defects in reproduction (i.e., failure of spermatogenesis in the male and resorption of the fetus in the female); and defects in growth and differentiation of epithelial tissues, frequently resulting in keratinization. Keratinization of these tissues results in loss of function in the alimentary, genital, reproductive, respiratory and urinary tracts. Such altered characteristics increase the susceptibility of the affected tissue to infection. Thus, diarrhea and pneumonia are typical secondary effects of vitamin A deficiency. More is known about the role of vitamin A in vision than any of its other functions. Retinol is utilized in the aldehyde form (trans form to 11-cis-retinal) in the retina of the eye as the prosthetic group in rhodopsin for dim light vision (rods) and as the prosthetic group in iodopsin for bright light and color vision (cones). Retinoic acid has been found to support growth and tissue differentiation but not vision or reproduction (McDowell, 2000; Solomons, 2006). Vitamin A-deficient rats fed retinoic acid were healthy in every respect, with normal estrus and conception, but failed to give birth and resorbed their fetuses. When retinol was given even at a late stage in pregnancy, fetuses were saved. Male rats on retinoic acid were healthy but produced no sperm, and without vitamin A both sexes were blind (Anonymous, 1977). Disease resistance is a function of vitamin A, with the vitamin needed for maintenance of mucous membranes and normal function of the adrenal gland. Vitamin A deficiency can impair regeneration of normal mucosal epithelium damaged by infection or inflammation (Ahmed et al., 1990; Stephensen et al., 1993) and thus could increase the severity of an infectious episode and/or prolong recovery from that episode. Adequate dietary vitamin A is necessary to help maintain normal resistance to stress and disease. An animal’s ability to resist infectious disease depends on a responsive immune system, with a vitamin A deficiency causing a reduced immune response. In many experiments with laboratory and domestic animals, the effects of both clinical and subclinical deficiencies of vitamin A on the production of antibodies and on the resistance of different tissues against microbial infection or parasitic infestation have frequently been demonstrated (Kelley and Easter, 1987; Lessard et al., 1997). Supplemental vitamin A improved the health of animals infected with roundworms, of hens infected with the genus Capillaria and of rats with hookworms (Herrick, 1972). Vitamin A is a valuable nutritional aid in the management of ringworm (Trichophyton verrucosum) infection in cattle.
Vitamin A deficiency affects immune function, particularly the antibody response to T cell-dependent antigens (Ross, 1992). The RAR-alpha mRNA expression and antigen-specific proliferative responses to T-lymphocytes are influenced by vitamin A status in vivo, and directly modulated by retinoic acid (Halevy et al., 1994). Vitamin A deficiency affects a number of cells of the immune system, and repletion with retinoic acid effectively reestablishes the number of circulating lymphocytes (Zhao and Ross, 1995).
A diminished primary antibody response could also increase the severity and/or duration of an episode of infection, whereas a diminished secondary response could increase the risk of developing a second episode of infection. Vitamin A deficiency causes decreased phagocytic activity in macrophages and neutrophils. The secretory immunoglobulin A (IgA) system is an important first line of defense against infections of mucosal surfaces (McGhee et al., 1992). Several studies in animal models have shown that the intestinal IgA response is impaired by vitamin A deficiency (Wiedermann et al., 1993; Stephensen et al., 1996).
B. Beta-Carotene Function Independent of Vitamin A
Recent animal studies indicate that certain carotenoids have antioxidant capacities, but without vitamin A activity. Carotenoids can enhance many aspects of immune functions, act directly as antimutagens and anticarcinogens, protect against radiation damage and block the damaging effects of photosensitizers. (Chew, 1995; Koutsos, 2003; Lindshield and Erdman, Jr., 2006). Beta-carotene can function as a chain-breaking antioxidant, it deactivates reactive chemical species such as singlet oxygen, triplet photochemical sensitizers and free radicals which would otherwise induce potentially harmful processes (e.g., lipid peroxidations) (McDowell, 2004). In humans, carotenoids had a preventative role in bladder cancer and implications for prevention of other types of cancer (Schabath et al., 2004). Injectable beta-carotene has increased conception rates in swine and improved live births and live weights in pigs (Chew, 1993). Beta-carotene supplementation had a positive effect on the pregnancy rate of mares in some studies (Ahlswede and Konermann, 1980; Peltier et al., 1997). Vitamin A and beta-carotene have important roles in protecting animals against numerous infections, including mastitis. Potential pathogens are regularly present in the teat orifice, and under suitable circumstances can invade the mammary gland and initiate clinical mastitis. Any unhealthy state of the epithelium would increase susceptibility of the mammary gland to invasion by pathogens. There are reports of improved mammary health in dairy cows supplemented with beta-carotene and vitamin A during the dry (Dahlquist and Chew, 1985) and lactating (Chew and Johnston, 1985) periods. Polymorphonuclear neutrophils (PMN) are the major line of defense against bacteria in the mammary gland. Beta-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. (1991a, b) reported that beta-carotene enhanced the bactericidal activity of blood and milk PMN, against S. auereusbut did not affect phagocytosis. Vitamin A either had no effect or supressed 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. Beta-carotene, on the other hand, does have significant antioxidant properties and effectively quenches singlet oxygen free radicals (Di Mascio et al., 1991; Zamora et al., 1991). Currently, there are significantly new views on the health benefits of megavitamin doses, particularly the antioxidant vitamins (C, E, and beta-carotene). Because free radical-induced damage to mammalian tissues is believed to contribute to the aging process and to the development of some degenerative diseases (Canfield et al., 1992), it has been proposed that dietary carotenoids serve as antioxidants in tissues (Thurnham, 1994). In the lipid environment of biological membranes a combination of carotenoids and other antioxidants, especially tocopherols, may provide better protection than tocopherols alone. Antioxidant properties of carotenoids include scavenging singlet oxygen and peroxyl radicals, sulfur, sulfonyl and NO2 radicals and providing protection of lipids from superoxide and hydroxyl radical attack (Surai, 2002). Therefore, carotenoids can actively quench singlet oxygen (O2) and prevent lipid peroxidation. One molecule of beta-carotene is able to quench 1000 molecules of O2 before it reacts chemically and forms products. Although the principal antioxidant carotenoid is beta-carotene, other carotenoids (e.g., lutein, lycopene and zeaxanthin) have strong antioxidant activities (McDowell, 2006).
Practical factors influencing vitamin A requirements for various species are listed in Table 2-2. Minimum requirements, determined by various methods, include amounts required to prevent night blindness, amounts required for storage and reproduction, and amounts required for maintenance of normal pressure in the cerebrospinal fluid (CSF). The minimum vitamin A requirement for normal growth may be lower than that required for higher rates of gain, resistance to various diseases and normal bone development.
Table 2-2: Factors Influencing Vitamin A Requirements
- Type and level of production (growth, pregnancy, lactaction).
- Genetic differences (species, breed, strain).
- Carryover effect of stored vitamin A (principally in the liver, but kidney also important for cats).
- Conversion efficiency of carotenes to vitamin A (not a factor for cats).
- Variations in level, type and isomerization of carotenoid vitamin A precursors in feedstuffs (not a factor for cats).
- Presences of adequate bile in vivo.
- Destruction of vitamin A in feeds through oxidation, long length of storage, high temperatures of pelleting, catalytic effects of trace minerals and peroxidizing effects of rancid fats.
- Presence of disease and/or parasites.
- Environmental stress and temperature
- Adequacy of dietary fat, protein, zinc, phosphorus and antioxidants (including vitamin E, vitamin C and selenium.
- Pelleting and subsequent storage of feed.
Adapted from McDowell (2000)
The most common carotenoids that have vitamin A activity are beta-carotene, alpha-carotene and cyptoxanthin. The provitamin A value of alpha-carotene and cryptoxanthin is about one-half that of beta-carotene (Tanumilhardjo and Howe, 2005). Different species of animals convert beta-carotene to vitamin A with varying degrees of efficiency (Table 2-1). Some factors that influence the rate at which carotenoids are converted to vitamin A are type of carotenoid, class and production level of animal, individual genetic differences in animals and level of carotene intake. Efficiency of vitamin A conversion from beta-carotene is decreased with higher levels of intake (Van Vliet et al., 1996). The conversion rate of the rat has been used as the standard value, with 1 mg of beta-carotene equal to 1,667 IU of vitamin A. Dogs are only 50% as efficient in this conversion, while the cat cannot utilize vitamin A precursors. Stress conditions, such as extremely hot weather, viral infections, and altered thyroid function, have also been suggested as cause for reduced carotene to vitamin A conversion. Vitamin A requirements are higher under stressful conditions such as abnormal temperatures or exposure to disease conditions. As an example with poultry, coccidiosis not only causes destruction of vitamin A in the gut but also injures the microvilli of the intestinal wall, thereby decreasing absorption of vitamin A and at the same time causing anorexia for several days (Scott et al., 1982). Vitamin A deficiency is often seen in heavily parasitized animals that supposedly were receiving an adequate amount of the vitamin (McDowell, 2004). Vitamin A requirements should be sufficient to provide for satisfactory growth, reproduction and lactation and prevention of disease deficiency signs. Present requirements should be designed to be adequate for these purposes under practical conditions of feeding and management as well as allow for a certain amount of storage. Storage of vitamin A in the liver certainly appears to be desirable since under conditions of little or no liver storage, stresses and diseases may precipitate vitamin A deficiency. The minimum vitamin A requirement for normal growth may be lower than those required for higher rates of gain, resistance to various diseases, and normal bone development. Friedman and Sklan (1997) report that optimum immune responses in growing chicks and turkeys were obtained with vitamin A intakes that were three- to ten-fold higher than NRC recommended levels. In humans vitamin A stores were positively associated with several measures of innate immune activity across a broad range of stores, suggesting that vitamin A enhances protection against diverse pathogens even at concentrations above those needed to maintain normal vision (Ahmad et al., 2009).
A. Requirements for Dogs
Frohring (1935; 1937) fed vitamin A-deficient diets to puppies and determined that for growth 100 IU vitamin A per kg (45.5 IU per lb) body weight was lost from the liver daily. Crimm and Short (1937), using a similar vitamin A depletion technique, estimated that the minimal daily vitamin A requirement of adult dogs was 22 to 47 IU per kg (10 to 21.4 IU per lb) of body weight. The NRC (2006) suggests that the daily vitamin A requirement would be met by 1,515 IU per kg (689 RE per lb) of diet for all classes of dogs. The Association of American Feed Control Officials (AAFCO, 2007) recommendations, which suggest 5,000 IU of vitamin A per kg (2,272.7 IU per lb) of diet.
B. Requirements for Cats
The cat, compared to other species, has a high vitamin A requirement relative to its body size. Controlled studies designed to define the vitamin A requirements have not been published. However, the requirement has been estimated to range from 1,600 to 2,000 IU of preformed vitamin A per head per day (Scott, 1965; Gershoff et al., 1957a). In long-term studies, 4,000 IU retinol per kg (1,818.2 IU per lb) of diet was not adequate for pregnancy, but 6,000 IU per kg (2,727 IU per lb) prevented deformities and provided for normal kitten development during lactation (NRC, 1985). The NRC (2006) vitamin A recommendations for cats is 1,000 µg retinol per kg (455 µg retinol per lb) for kittens and adults at maintenance and double this level for cats in late gestation and at peak lactation. The AAFCO (2007) recommendation for cats is 5,000 IU per kg (2,273 IU per lb) of diet for maintenance and 9,000 IU per kg (4,091 IU per lb) for both growth and reproduction functions.
Concentrations of vitamin A in feedstuffs are highly variable. The naturally occurring sources with the highest vitamin A activity are fish oils and liver. Among the common foods of animal origin, milk fat, egg yolk and liver are sources of high vitamin A activity, provided the animal from which they were obtained received adequate vitamin A for an extended period.
Sources of supplemental vitamin A are derived primarily from fish liver oils, in which the vitamin occurs largely in esterified form, and from industrial chemical synthesis. Before the era of the chemical production of vitamin A, the principal source of vitamin A concentrates was the liver and/or body oils of marine fish. Since industrial synthesis was developed in 1949, the synthetic form has become the major source of the vitamin to meet the requirements of domestic animals and humans. The synthetic vitamin usually is produced as the all-trans retinyl palmitate or acetate.
Provitamin A carotenoids, mainly beta-carotene in green feeds, are the principal source of vitamin A for grazing livestock. Although the yellow color of carotenoids is masked by chlorophyll, all green parts of growing plants have high carotene content, and thus, have high vitamin A value. In fact, the degree of green color in a roughage is a good index of its carotene content. Good pasture always provides a liberal supply, and type of pasture plant, whether grass or legume, appears to have minor importance. At maturity, however, leaves contain much more carotene than stems, and, therefore, legume hay is higher in vitamin content than grass hay (Maynard et al., 1979). In all hays and other forage, vitamin A value decreases after the bloom stage. Plants at maturity can have 50% or less of the maximum carotenoid value of immature plants. Wing (1969) reported carotene digestibility in plants to be greater during the warmer months. Variations were found in the digestibility of carotenes in plants according to year, species of plant, dry matter content, and form of forage. Average published values of carotene content can serve only as approximate guides in feeding practice because of many factors affecting actual potency of individual samples as fed (NRC, 1982).
Cooking processes commonly used in human food preparation do not cause much destruction to the vitamin potency. The blanching and freezing process generally causes little loss of carotenoid to cis-forms. In a report from Indonesia, isomerization during traditional cooking caused a loss of up to 9% of vitamin A potency (Van Der Pol et al., 1998). Hydrogenation of fats lessens their vitamin A value, while saponification does not destroy the vitamin if oxidation is avoided.
Gamma radiation has been used to sterilize pet diets. Caulfield et al. (2008) note that gamma irradiation can have profound, selective effects on the vitamin A and peroxide contents of dry diets, and caution is advised when feeding such diets long-term, particularly for cats. Furthermore, pasteurization (with its fewer deleterious effects) may represent an alternative method of decontaminating diets for rodents, dogs, and cats.
Both carotene and vitamin A are destroyed by oxidation, which is the most common cause of any depreciation that may occur in the activity of sources (Table 2-3). The process is accelerated at high temperatures, but heat without oxygen has a minor effect. Butter exposed in thin layers in air at 50°C loses all its vitamin A potency in 6 hours, but in the absence of air there is little destruction at 120°C over the same period. Cod liver oil in a tightly corked bottle had shown activity after 31 years, but it may lose all of its potency in a few weeks when incorporated in a feed mixture stored under usual conditions (Maynard et al., 1979). Much of the carotene content is destroyed by oxidation in the process of field curing. Russell (1929) found that there may be a loss of more than 80% of the carotene of alfalfa during the first 24 hours of the curing process. It occurs chiefly during the hours of daylight, due in part to photochemical activation of the destructive process. In alfalfa leaves, sunlight-sensitized destruction is 7% to 8% of the total pigment present, while enzymatic destruction amounts to 27% to 28% (Bauernfeind, 1972). Enzymatic destruction requires oxygen, is greatest at high temperatures, and ceases after complete dehydration.
All-trans beta-carotene is the predominant isomer in feeds, thermal processing can substantially increase the proportions of cis isomers (9-c and 13-c); beta-carotene is the more bioavailable isomer (Deming et al., 2002). It would seem likely that the isomers of beta-carotene would increase in the fermentation process of silage, resulting in a lower bioavailable beta-carotene.
Hay crops cut in the bloom stage or earlier and cured without exposure to rain or too much sun retain a considerable proportion of their carotene content. Hay crops cut in the seed stage and exposed to rain and sun for extended periods lose most of their carotene. Green hay curing in the swath may lose one-half of its vitamin A activity in one day’s exposure to sunlight. Thus, hay usually has only a small proportion of the carotene content of fresh grass. Under similar conditions of curing, alfalfa and other legume hays are much higher in carotene content than grass hays because of their leafy nature, but a poor grade of alfalfa may have less carotene than a good grade of grass hay (Maynard et al., 1979).
The Vitamin A potency of yellow corn is only about one-eighth that of good roughage. There is evidence that yellow corn may lose carotene rapidly during storage. For example, a hybrid corn high in carotene lost about 50% of its carotene content in 8 months of storage at 25°C and about 75% in 3 years at 25°C. Less carotene was lost during storage at 7°C (Quackenbush, 1963). Bioavailability of natural beta-carotene is less than chemically synthesized forms (White et al., 1993). In ferrets, all-trans-beta-carotene was less available from carrot juice than from beta-carotene beadlet-fortified beverages (White et al., 1993). For calves, a small enhancing effect of mild heat treatment of carrots on serum and tissue accumulation of carotenoids has been reported (Poor et al., 1993).
The major source of supplemental vitamin A in animal diets is trans-retinyl acetate (McGinnis, 1988). The acetate, propionate and palmitate esters are chemically synthesized by vitamin A manufacturers. These are available in gelatin beadlet product forms for protection against oxidative destruction in premixes, mash, and cubed and pelleted feeds. Carbohydrates, gelatin and antioxidants also are generally included inside the beadlets to stabilize the vitamin A. These provide physical and chemical protection against factors normally present in feed that are destructive to vitamin A. The vitamin A acetate products most frequently used in ruminant feeds contain 650,000 IU or United States Pharmacopeia Units (USP) per gram of product. (Note that IU and USP units are equal in value and one unit equals the activity of 0.3 µg of all-trans retinol or 0.344 µg of all-trans retinyl acetate.)
Table 2-3: Practical Factors That Affect Vitamin A Stability
Factors Detrimental to Stability
- Prolonged storage of the vitamin products, either in premixes prior to mixing or in the final feed product.
- Vitamin premixes containing trace minerals.
- High environmental temperature and humidity.
- Pelleting, blocking and extrusion.
- Hot feed bins that sweat inside upon cooling.
- Rancid fat in the feed.
- Moisture leakage into feed bins or storage facilities.
Adapted from Hoffman-La Roche (1989)
Vitamin A is necessary for normal vision in animals, maintenance of healthy epithelial or surface tissues and normal bone development. The vitamin A deficiency signs observed in various species vary somewhat, but most relate to changes in these tissues. Numerous studies have also demonstrated increased frequency and severity of infection in vitamin A-deficient animals. Lack of vitamin A results in decreased antibody production and impaired cell-mediated immune processes against infective agents (Davis and Sell, 1989). Clinical signs can be specific for vitamin A deficiency, or only general indications, including loss of appetite, loss of weight, unthrifty appearance, thick nasal discharge and reduced fertility. The normal epithelium in various locations throughout the body becomes replaced by a stratified, keratinized epithelium when vitamin A is deficient. This effect has been noted in the respiratory, alimentary, reproductive and genitourinary tracts, as well as in the eye. Dogs lacking vitamin A had increased infection with associated changes in the blood leukocyte differential count (Scott et al., 1995; NRC, 2006). Vitamin A-deficient cats had extensive infectious sequelae in the lung and occasionally in the conjunctiva and salivary glands (Gershoff et al., 1957a). For many species, keratinization lowers the resistance of the epithelial tissues to the entrance of infectious organisms. Thus, respiratory troubles, such as colds and sinus infections, tend to be more severe in vitamin A deficiency. In fact, death is often a consequence of pneumonia. A number of criteria are available to evaluate the vitamin A status of animals, including production response, liver vitamin A stores, plasma vitamin A, and cerebrospinal fluid pressure. The blood maintains its level of vitamin A at the expense of liver stores. Thus, the blood level is not a reliable indication of vitamin A adequacy or deficiency. For dogs urinary retinol and retinyl esters can serve as an indicator of dietary vitamin A intake (Schweigert and Bok, 2000). The vitamin A level in the liver can be a reliable indication of vitamin A adequacy or deficiency. Nevertheless, with a level of less than 40 µg of vitamin A per 100 ml of blood serum, a borderline deficiency of the vitamin would be expected. Nondetectable levels are the rule in severe deficiency. In severely vitamin A-deficient dogs, blood and liver concentrations of vitamin A were negligible (Ralston Purina, 1987). In histological exams of dogs, changes of squamous metaplasia in epithelial tissue are present, especially in the respiratory tract (Mellanby, 1950; Stewart, 1965) with liver depletion of the vitamin (< 10 µ/g) as conclusive evidence in support of the diagnosis. For male dogs with progressive glomerular disease retinol-binding protein (RBP) was detected in the urine and the RBP increased with the progression of the renal disease (Nabity et al., 2011). A simple test for night blindness evaluation in dogs and cats can be made by darkening an unfamiliar room and observing the animals’ movements in dim red light (Ralston Purina, 1987). More detailed analysis is possible with the electroretinogram in the anesthetized animal. Retinoid treatment of vitamin A deficiency in companion animals is presented in Illus. 2-2.
Illustration 2-2: Retinol Treatment of Vitamin A Deficiency
A. Deficiency in Dogs
Experimental vitamin A deficiency has been extensively studied in dogs, with the initial studies of this fat-soluble vitamin deficiency being made in dogs in the 1920s (Mellanby, 1950). Vitamin A deficiency is usually confined to young animals, and advanced deficiency is always associated with cessation of growth or even weight loss. Steenbock et al. (1921) reported that dogs deprived of fat-soluble vitamins developed an “ophthalmia.” Signs of xeropthalmia (excessive dryness of the eye), night blindness, conjunctivitis, corneal opacity and ulceration, skin lesions and metaplasia of the bronchiolar were associated with rough haircoat, anorexia, growth depression and muscle weakness (Stimson and Hedley, 1933; Crimm and Short, 1937; Russell and Morris, 1939; Singh et al., 1965; Tighe and Brown, 1998). Respiratory infection in prolonged deficiency often resulted in death. Deafness and facial paralysis occurred in puppies with severe chronic deficiency when bone growth of the skull was altered by inadequate remodeling and excessive periosteal growth to constrict cranial nerves. Blood and liver concentrations of vitamin A are negligible in these cases. Mellanby (1938) established that such damage to the cochlear and vestibular divisions of the eighth cranial nerve, plus a serious labyrinthitis, may induce deafness. Similar damage may also affect function of the optic nerve, although this only has been observed after prolonged experimental deficiency (NRC, 2006). Vitamin A deficiency is generally a disease in growing pups, as adults seldom develop signs due to their extremely slow depletion of liver stores. In fact, several litters of pups have been produced while the dam was maintained on a deficient diet (Ralston Purina, 1987).
B. Deficiency in Cats
Vitamin A deficiency signs in cats are characterized by lack of growth, loss of weight and poor appetite after two to three months of feeding a vitamin A-deficient diet. Muscle wasting was severe, while stored body fat may be nearly normal. Epithelial tissues and mucous glands forming the membrane lining of the alimentary, respiratory, urinary and reproductive tracts, as well as the membrane covering the eye and eyelids, were markedly impaired by vitamin A deficiency (Gershoff et al., 1957a). Follicular hyperkeratosis can be recognized as small reddish eruptions around the nose and eyebrows and is coupled with microscopic evidence of atrophy of epidermal adnexae. Acinar dysplasia of the pancreas has been described and marked hypoplasia of the seminiferous tubules, depletion of adrenal cortical lipid, and focal atrophy of the skin were also observed. The reported early clinical signs of vitamin A deficiency were weight loss, an exudate about the eyes, and muscle and weakness (NRC, 2006). Night blindness can be detected early in vitamin A-deficient kittens. As the deficiency progresses, the pupillary reflex to light is delayed from 1 to 2 seconds in the normal state to 5 to 10 seconds in the deficient state. Irreversible degeneration occurs in the rods and cones (visual cells) of the retina. Unfortunately, assessment of night blindness in cats has not been adequately distinguished from retinal degeneration due to taurine deficiency (Knopf et al., 1978), since all experimental vitamin A-deficient diets to date have used casein as the source of protein, a procedure which depletes taurine in cats. With vitamin A deficiency, bones become thickened and constrict the central nervous system. Ataxia, stiff gait and hydrocephalus in newborn kittens from deficient dams suggest an increased cerebrospinal fluid pressure, whereas the description of cleft palate suggests deranged intrauterine mitosis and cell differentiation (Ralston Purina, 1987). Male cats become sterile from testicular hypoplasia; females in severe vitamin A deficiency do not ovulate. With a lesser degree of deficiency, queens conceive and implantation occurs, but resorption or abortion of the fetus results at approximately the 49th day of pregnancy (Gershoff et al., 1957a; Scott, 1965). Bartsch et al. (1975) described ataxia, “star gazing,” blindness, and intermittent convulsions in African lion cubs presumed to be vitamin A deficient. Severe thickening of the cranium, compression of the brain and partial herniation of the cerebellum also occurred.
Vitamin A deficiency would not be expected for dogs or cats in the wild because they are carnivorous and usually consume sufficiently large quantities of the vitamin from organ meats (particularly liver), which can be stored during periods of inadequate consumption. Generally, domesticated cats and dogs are not deficient in vitamin A. However, storage or reserves may be low in cases such as steatorrhea, chronic liver disease or chronic enteritis, where absorption is depressed while turnover of intestinal epithelium is increased. On the other hand, they must start as pups or kittens without any stores, since placental transport is low, and steadily accumulate these reserves without appreciable interruption to have an adequate supply for brief episodes of stress or for pregnancy and lactation (Ralston Purina, 1987). An early, rich source of vitamin A is colostrum. Thus, orphaned kittens and pups should be supplemented until they can consume an adequate supply in their diet. Vitamin A assimilation is decreased, and its utilization is increased, by infection (Nockels, 1988). Aflatoxin ingestion has also been reported to reduce vitamin A stores in the liver and induce its deficiency. Chickens evidenced decreased tissue accumulation of oxycarotenoid pigments when fed aflatoxin. It is therefore important that pets that are sick or are receiving vitamin A antagonists receive diets that are fortified with the vitamin. Supplementation of pet foods would be particularly essential if vitamin A-rich organ meats (particularly liver and kidney) were not part of the diets. For cats, fish by-products (e.g., fish liver oils) could be an important source of vitamin A. Lacking the capability to convert carotene to vitamin A, cats need preformed vitamin A (vitamin A from commercial synthesis or animal origin) to meet their requirement (Gershoff et al., 1957a). Several factors can influence the loss of vitamin A from feedstuffs during storage. Stabilized vitamin A (e.g., beadlets) is not easily destroyed in a vitamin mix, but there is only a 58% retention when the vitamin premix also contains choline and trace minerals (Coelho, 1991). The trace minerals in feeds and supplements, particularly copper, are detrimental to vitamin A stability. Dash and Mitchell (1976) reported the vitamin A content of 1,293 commercial feeds over a three-year period. The loss of vitamin A was over 50% in one year’s time. Vitamin A loss in commercial feeds was evident even if the commercial feeds contained stabilized vitamin A supplements. Vitamin A and carotene destruction also occurs from processing of feeds with steam and pressure. Pelleting effects on vitamin A in feed are caused by die thickness and hole size, which produce frictional heat and a shearing effect that can break supplemental vitamin A beadlets and expose the vitamin. In addition, steam application exposes feed to heat and moisture. Running fines back through the pellet mill exposes vitamin A to the same factors a second time. Between 30% and 40% of vitamin A present at mixing may be destroyed during pelleting (Shields et al., 1982). Most supplemental vitamin A used today for animals is administered orally in a product form that blends uniformly in dry feed. However, in a time of need, an intramuscular injection of the vitamin is probably the surest route of administration, especially if food consumption is depressed or the animal suffers from malabsorption. Because of the poor stability of vitamin A, particularly when exposed to oxygen, trace minerals, pelleting, feed storage and other factors, the feed industry has readily accepted the dry, stabilized forms of the vitamin. The stability of vitamin A in pet foods has been improved in recent years by chemical stabilization as an ester and by physical protection using antioxidants, emulsifying agents and stabilized materials, including gelatin, crosslinked gelatin and sugar in spray-dried beadlet, or prilled products (Bauernfeind and DeRitter, 1972; Shields et al., 1982). Stabilized and protectively coated (or beaded) forms of vitamin A slow destruction of the vitamin, but for highest potency, fresh supplies of the mixture should be available on a regular basis. Practical considerations that affect vitamin A stability are listed in Table 2-4. The gelatin beadlet in which the vitamin A ester (palmitate or acetate) is emulsified into a gelatin-plasticizer-antioxidant viscous liquid formulation and spray-dried onto discrete dry particles results in products with good chemical stability, good physical stability and excellent biological availability (Bauernfeind and DeRitter, 1972).
Table 2-4: Practical Factors that Affect Vitamin A Stability
Factors Detrimental to Stability
- Prolonged storage of the vitamin products, either in premixes prior to mixing or in the final feed product.
- Vitamin premixes containing trace minerals.
- High environmental temperature and humidity.
- Pelleting, blocking and extrusion.
- Hot feed bins that sweat inside upon cooling.
- Rancid fat in the feed.
- Moisture leakage into feedbins or storage facilities.
Factors Promoting Stability
- Minimize time between manufacture of the vitamin product and consumption by the animal.
- Store vitamins in a cool, dark, dry area in closed containers.
- Don’t mix vitamins and trace minerals in the same premix until ready to mix the feed.
- Control the premix to maintain pH within a range of 4.5 to 6.5; avoiding extremely acidic or alkaline pH.
- Use good quality feed ingredients and vitamins.
- Use appropriate antioxidant systems in the vitamin A product forms.
- Proper maintenance of storage bins and other equipment.
- Minimize time between purchase and use.
Adapted from Hoffmann-La Roche (1989)
The level of vitamin A supplementation should be based primarily on the degree of the deficiency that could potentially be problematic. As with most nutrients, a borderline deficiency is much more likely than a severe deficiency. A marginal deficiency adversely affecting performance is not easily detected. Based on the positive results that may be derived, and considering that vitamin A supplementation is inexpensive and (reportedly) nontoxic at recommended levels, it is beneficial to include supplemental vitamin A in pet foods. Some commercial foods for dogs and cats contain insufficient vitamin A. In 89 Australian brands, 8% of the dog foods and 14% of the cat foods had vitamin A concentrations below the minimum recommended for dogs and pregnant or lactating cats (Heanes, 1990). In contrast, canned pet foods stated to contain liver or kidney showed favorable vitamin A concentrations. The importance of synthetic retinoids in veterinary dermatology is now being explored. In dogs, the retinoids have been used primarily to treat skin diseases purported to be keratinization disorders. Many synthetic retinoids have been developed to offer better therapeutic response and less toxicity than naturally occurring vitamin A compounds. The most commonly used synthetic retinoids include tretinoin, isotretinoin and etretinate. Synthetic retinoids have been used in cats for cutaneous neoplastic diseases. In both species, current data suggest that isotretinoin and etretinate are well tolerated clinically and the incidence of clinical side effects appears to be lower than in humans. Many canine skin diseases are proposed to be disorders of the keratinization process, based on clinical and histological features (Illus. 2-3). Seborrheic dermatosis is the term commonly used to refer to these disorders. Their occurrence in certain breeds of dogs suggests that these disorders are genetically determined. Vitamin A-responsive dermatosis is a rare condition that is seen almost exclusively in cocker spaniels (Watson, 1998). More recently it has also been recognized in a Labrador retriever and a miniature schnauzer (Scott et al., 2001). The condition is characterized by adult-onset, medically refractory seborrheic skin disease with marked follicular plugging and hyperkeratotic plaques, primarily on the ventral and lateral thorax and abdomen (Scott et al., 2001). Response to etretinate has been reported to be good to excellent for idiopathic seborrhea in some breeds of dogs, but ineffective for others (Fadok, 1986; Scott, 1986; Miller, 1989). Beneficial response was apparent after two months of therapy, and improvement continued in the subsequent two months of the trial. Clinical signs recurred three weeks to three months after therapy was discontinued, indicating a continual need for the retinoid medication. Dogs with sebaceousadentis were administered 380 to 2667 IU per kg of diet (173 to 1213 IU per lb) (Lam et al., 2011). The majority of dog owners were satisfied with the overall appearance of their dogs reporting ≤ 25% improvement in clinical signs, including level of pruritus, amount of scale, alopecia and overall coat quality, compared with pretreatment appearance.
Various skin disorders, including sebaceous adenitis, canine ichthyosis, solar-induced precancerous lesions and squamous cell carcinoma have all responded to retinoid therapy (Parker et al., 1983; Ihrke and Goldschmidt, 1983; Power and Ihrke, 1990; Power et al., 1992; Lam et al., 2011). In both dogs and cats with mycosis fungoides, isotretinoin seems to be a well-tolerated, relieving type of nutritional therapy. It is estimated that three out of four non-accidental causes of death in dogs are cancer, renal failure and heart disease. Recent evidence suggests that many “age- related” diseases, such as cancer and heart disease, are caused in part by free radical damage (Parr, 1996; Dove, 2001; Freeman et al., 2006). Dogs with chronic renal disease affected the concentration of retinol in both plasma and urine (Raila et al., 2003). Retinoic acid was found as a novel therapy in treatment of ACTH-secreting tumors for dogs with Cushing’s disease (Castillo et al., 2006). Supplementing the pet diet with antioxidants such as vitamin E, vitamin C and beta-carotene can prevent or reduce the negative impact of free radical damage (Stowe et al., 2006). Recommendations for daily antioxidant fortification rates of vitamin E, vitamin C and beta-carotene have been suggested for dogs and cats by Parr (1996). For example, the daily beta-carotene supplemental level for a 13.6-kg (30-lb) dog is 4.5 mg and for a 2.7-kg (6-lb) cat is 0.90 mg.
In general, the possibility of vitamin A toxicity for dogs and cats is remote. However, of all vitamins, vitamin A is most likely to be provided in toxic concentrations to dogs and cats. Excess vitamin A has been demonstrated to have toxic effects in most species studied. Presumed upper safe levels are four to 10 times the nutritional requirements for nonruminant animals, including dogs and cats (NRC, 1987). Most of the harmful effects have been obtained by feeding over 100 times the daily requirements for an extended period of time. Thus, small excesses of vitamin A for short periods of time should not exert any harmful effects. Recommended upper safe levels of vitamin A for dogs are 33,330 IU per kg (15,150 IU per lb) of diet and 100,000 IU per kg (45,455 IU per lb) of diet for cats. Excessive consumption of foods or supplements high in vitamin A can lead to hypervitaminosis A. This metabolic disorder in cats is most frequently associated with diets having a high liver or fish liver oil content (Lucke et al., 1968; Goldman, 1992; Goldston and Hoskins, 1995). Special care should be taken to avoid long-term feeding of such diets when trying to stimulate appetite in anorexic cats. When a cat is found to be sick and its diet consists wholly or partly of raw liver, vitamin A toxicity should be suspected. The feeding of raw liver should be stopped and a balanced feline diet instituted (Goldman, 1992). The most characteristic signs of hypervitaminosis A are skeletal malformation, spontaneous fractures and internal hemorrhage. Other signs include anorexia, slow growth, weight loss, skin thickening, suppressed keratinization, increased blood clotting time, enteritis, congenital abnormalities, conjunctivitis, fatty liver and reduced function of the liver and kidney. Vitamin A toxicosis in cats results in a disorder called deforming cervical spondylosis (Illus. 2-4) (Case et al., 1995). The clinical signs are pain, difficult movement, lameness, and crippling in severe cases. In a crippling bone disease there is tenderness of the extremities associated with gingivitis and tooth loss that has been described in cats given prolonged excessive doses of this vitamin either as vitamin A itself or by feeding large quantities of raw liver (Seawright et al., 1965; Seawright and Hrdlicka, 1974). Cats consuming raw beef liver containing 20,000 to 40,000 µg per kg (9,091 to 18,182 µg per lb) for two to five years produced the main clinical findings of cutaneous hyperesthesia of neck and forelegs, forelimb lameness, pain and skeletal immobility due to bony exostoses of the cervical vertebrae and tendinous tuberosities of long bones (Illus. 2-5) (Seawright et al., 1965). Feeding liver to kittens for five to 12 months with dietary intakes of vitamin A exceeding 150,000 µg per kg (68,182 µg per lb) body weight per day caused weight loss, cachexia, inanition, exophthalmos, hindleg weakness with abnormal gait and death. Bone changes reflected the level of toxicity, with the proliferative effect of vitamin A stimulation manifested by bony exostoses at moderately high doses followed by thinning or lysis of bone in more advanced stages of toxicity (Seawright et al., 1967).
Illustration 2-4: Vitamin A Toxicosis Deforming Cervical Spine
Illustration 2-5: Vitamin Toxicosis in Cats, Joint Calcification
Feeding kittens high vitamin A for four to five weeks depressed appetite and resulted in damage to the epiphyseal plate, altering growth and development of long bones. Peeling of the footpad epithelium and frequent blinking were also observed. A gait abnormality was evidenced by restricted movement of the stifle joint (Clark, 1973). Freytag et al., (2003) demonstrated that a concentration of 30,600 IU per kg (13,909 RE per lb) diet has a potential for causing birth defects in kittens. Historically, it has been known for centuries among Eskimos and Arctic travelers that the ingestion of polar bear liver by men and dogs causes severe illness (Rodahl, 1949). There are stories of polar bear liver having been given to dogs, who later became sick and eventually suffered from loss of hair. It is also said that the Eskimos used to throw the livers of polar bears into the sea so the dogs would not get them. On the other hand, it is known that dogs are reluctant to eat the liver of polar bear, as is generally experienced by the European trappers in North East Greenland with their sled dogs. On one occasion a liver was given to a dog; because the dog was very hungry, he ate a small portion of the liver and subsequently suffered from diarrhea (Rodahl, 1949). Dosed with 100,000 µg per kg (45,455 µg per lb) body weight per day, greyhound pups had poor appetite after 30 days and lost weight with rapid deterioration in condition after seven weeks (Maddock et al., 1949). After 53 days, a variety of clinical signs rapidly appeared, including continuous shivering. Hyperesthesia of the skin and extreme tenderness of the extremities were evident. The puppies were unwilling to stand, although no fractures were noted. The long bone epiphyseal cartilage was markedly narrower; cortices of the femur, tibia, radius, and ulna were less dense and thinner. Remodeling processes were greatly accelerated, and hemorrhage was common in these areas. Ralston Purina (1987) recorded an incident in which a dog breeder initiated routine supplementation of a concentrated “vitamin mix” for all dogs in the kennel. Within two years, fertility was severely depressed and numerous stillbirths, neonatal deaths of puppies and the deaths of two bitches were associated with extraordinary high liver vitamin A concentrations, suggesting that vitamin A toxicity was the principal cause of the problems.