DSM in Animal Nutrition & Health
Properties and Metabolism
Vitamin D designates a group of closely related compounds that possess antirachitic activity. It may be supplied through the diet or by irradiation of the body. The two most prominent forms of vitamin D are ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). Ergocalciferol is derived from a common plant steroid, ergosterol, whereas cholecalciferol (Illus. 3-1) is produced from the precursor 7-dehydrocholesterol, exclusively from animal products. The provitamin 7-dehydrocholesterol, derived from cholesterol or squalene, is synthesized in the body and present in large amounts in skin (in most species), the intestinal wall and other tissues. Vitamin D precursors have no antirachitic activity.
Vitamin D, in the pure form, occurs as colorless crystals that are insoluble in water but readily soluble in alcohol and other organic solvents. Vitamin D can be destroyed by over-treatment with ultraviolet (UV) light and by peroxidation in the presence of rancidifying polyunsaturated fatty acids (PUFA). Like vitamins A and E, unless vitamin D3 is stabilized, it is destroyed by oxidation. Its oxidative destruction is increased by heat, moisture and trace minerals. There is less destruction of vitamin D3in freeze-dried fish meals during drying, possibly because of decreased atmospheric oxygen. There is negligible loss of crystalline cholecalciferol during storage for one year or of crystalline ergocalciferol for nine months in amber-evacuated capsules at refrigerator temperatures. Vitamin D from the diet is absorbed from the intestinal tract, and is more likely to be absorbed from the ileal portion in greatest amounts due to the longer retention time of food in the distal portion of the intestine (Norman and DeLuca, 1963). Vitamin D is absorbed from the intestinal tract in association with fats, as are all the fat-soluble vitamins. Like the others, it requires the presence of bile salts for absorption (Braun, 1986), and is absorbed with other neutral lipids via chylomicron into the lymphatic system of animals. It has been reported that only 50% of an oral dose of vitamin D is absorbed. However, considering that sufficient vitamin D is usually produced by daily exposure to sunlight, it is not surprising that the body has not evolved a more efficient mechanism for dietary vitamin D absorption (Collins and Norman, 1991). Effective treatment of rickets by rubbing cod liver oil on the skin, indicates that vitamin D can be absorbed through the skin. There are four important variables that collectively determine the amount of vitamin D3 that will be photochemically produced by an exposure of skin to sunlight (Norman and Henry, 2007). The two principal determinants are the quantity or intensity of ultraviolet light (UV) and the appropriate wavelength of the UV light. The third important variable determining skin vitamin D3synthesis is the actual concentration of 7-dehydrocholesterol present in the skin. The fourth determinant of vitamin D3production is the concentration of melanin in skin (skin color). The darker the skin the longer time required to convert 7-dehydrocholesterol to vitamin D3.Presence of the provitamin 7-dehydrocholesterol in the epidermis of the skin and sebaceous secretions is well recognized. Vitamin D3 is synthesized in the skin of many herbivores and omnivores, including humans, rats, pigs, horses, poultry, sheep and cattle. However, little 7-dehydrocholesterol is found in the skin of cats and dogs (and likely other carnivores), and therefore little vitamin D3 is produced in the skin (How et al., 1995). For poultry, Tian et al. (1994) reported that skin of the legs and feet of chickens contains about 30 times as much 7-dehydrocholesterol (provitamin D3) as the body skin. The cholecalciferol formed by the UV irradiation of 7-dehydrocholesterol in the skin is removed from the skin into the circulatory system by the blood transport protein for vitamin D, the vitamin D-binding protein (DBP) (Norman and Henry, 2007) then becomes immediately available for further metabolism (Imawari et al., 1976). Some of the vitamin D3 formed in and on the skin ends up in the digestive tract as many ruminant animals consume the vitamin as they lick their skin and hair. Vitamin D undergoes a multiple series of transformations and multi-site interactions in the living system (Deluca, 1979; Norman and Henry, 2007). Vitamin D metabolism in ruminants begins prior to absorption in that rumen microbes are capable of degrading vitamin D to inactive metabolites (Sommerfeldt et al., 1983). This may explain the higher vitamin D requirements in ruminants.
Production and metabolism of vitamin D (both D2 and D3) that is necessary to activate the target organs are illustrated in Figure 3-1. By itself vitamin D is biologically inactive and must be converted to a hormonal form in a two-step process before it can function as the hormone 1,25-(OH)2 D3. Once in the liver, the first transformation occurs in which a microsomal system hydroxylates the 25-position carbon in the side chain to produce 25-hydroxy-vitamin D [25-(OH)D3]. The concentration of 25(OH)D3 in the circulation is considered a good indicator of vitamin D status in general, able to indicate an adequacy, deficiency or toxicity of vitamin D (McDowell, 2000; Bar 2003). The 25-(OH)D3 is then transported to the kidney on the vitamin D transport globulin, where it can be converted in the proximal convoluted cells to a variety of compounds, of which the most important appears to be 1,25 dihydroxy-vitamin D3[1,25-(OH)2D3] (DeLuca, 2008). The 1,25-(OH)2D3 is also referred to as calcitriol. Although the kidney is the main site of 1-hydroxylation other organs can also form 1,25-(OH)2D3 including the placenta (Johnston, 2006; DeLuca, 2008). Once formed in the kidney, 1,25-(OH)2D3 is then transported to the intestine, bones or elsewhere in the body, where it is involved in the metabolism of calcium and phosphorus. From studies of vitamin D metabolism, it has been found that the vitamin functions as a hormone. The hormonal form, 1,25-(OH)2D3, is the metabolically active form of the vitamin that functions in intestine and bone, whereas 25-(OH)D and vitamin D do not function at these specific sites under physiological conditions (DeLuca, 2008).
Figure 3-1: The Functional Metabolism of Vitamin D3 Necessary to Activate Target Organs of Intestine, Bone and Kidney
Production of 1,25-(OH)2D3 is very carefully regulated by parathyroid hormone (PTH) in response to serum calcium and phosphate (PO43-) concentrations. It is now known that the most important point of regulation of the vitamin D endocrine system occurs through the stringent control of the activity of the renal 1-hydroxylase. In this way, the production of the hormone 1,25-(OH)2D3can be modulated according to the calcium needs of the organism (Miller and Norman, 1984). Factors known to affect the activity of the 1-hydroxylase include calcium, PTH, and vitamin D status. Besides 1,25-(OH)2D3, the kidney also converts 25-(OH)D3 to other known compounds, including 24,25-(OH)2D3, 25,26-(OH)2D3 and 1,24,25-(OH)3D3. Henry and Norman (1978) have shown that hatchability of eggs from hens is severely depressed in vitamin D3-deficient hens even though they are fed 1,25-(OH)2D3. Evidently, 1,25-(OH)2D3 is effective in maintaining blood calcium levels so that egg production and eggshell thickness remain normal. However, without vitamin D, the upper mandible of the chicks fails to develop, and consequently, the chicks cannot crack the shell, resulting in mortality. The reason for this relates to failure of 1,25-(OH)2D3 from being passed from the hen to the egg, unlike the forms of D3 and 25-(OH)D3. Under conditions of calcium stress, PTH activates renal mitochondrial 1 alpha-hydroxylases, which convert 25-(OH)D to 1,25-(OH)2D, and inactivates renal and extrarenal 24- and 23-hydroxylases, which convert the 25-(OH)D3 [and any 1,25-(OH)2D3 formed] to inactive metabolites (Goff et al., 1991b). Under conditions with little calcium stress (when little PTH is secreted), the 1 alpha-hydroxylase can also be stimulated by low blood calcium or phosphorus concentration directly. High plasma 1,25-(OH)2D3 concentration has an inhibitory effect on renal 1 alpha-hydroxylase and a stimulatory effect on tissue 24- and 23-hydroxylases (Engstrom et al., 1987). Thus, production and catabolism of the hormone 1,25-(OH)2D3 are tightly regulated. It is now known that the most important point of regulation of the vitamin D endocrine system occurs through stringent control of the activity of the renal 1 alpha-hydroxylase. In this way, the production of the hormone 1,25-(OH)2D3 can be modulated according to the calcium needs of the organism (Collins and Norman, 1991).
For mammals, 1,25-(OH)2D3 is a critical factor in the maintenance of sufficient maternal calcium for transport to the fetus and may play a role in normal skeletal development of the neonate (Lester, 1986). Deletion of vitamin D3supplementation in diets fed to sows during gestation and lactation significantly compromised skeletal bone mineral content in offspring at 13 weeks of age and decreased the age at which pigs displayed kyphosis (backward curvature of spinal column) (Crenshaw, 2010). A liberal intake of vitamin D during gestation does provide a sufficient store in newborns to help prevent early rickets. For example, newborn lambs can be provided enough to meet their needs for six weeks. Parenteral cholecalciferol (vitamin D3) treatment of sows before parturition proved an effective means of supplementing young piglets with cholecalciferol (via the sow’s milk) and its more polar metabolites via placental transport (Goff et al. 1984). Experimental evidence suggests that vitamin D may play a role in the maternal-conceptus cross talk (Viganό et al., 2003). However, irrespective of the dietary dose and form of vitamin D (D3 or 25-(OH)D3) provided to sows, very little vitamin D was transferred to the progeny (Lauridsen et al., 2010).
One question that is still unanswered is whether the hormone form 1,25(OH)2 D3 acts alone or if there is some response from a second vitamin D metabolite or hormone (e.g. 24,25(OH)2 vitamin D3) (Feldman et al., 2003). However, this question was probably answered in a study where the 24-position of 25-(OH)D3 was blocked with fluoro groups to prevent 24-hydroxylation (DeLuca, 2008). For two generation all systems were normal, indicating a need for only 1,25-(OH)2 D3. Therefore, research indicates that 1,25-(OH)2 D3 appears to be the only functional form of vitamin D in biology (DeLuca, 2008).
For most mammals, vitamin D3, 25-(OH)D3, and possibly 24,25-(OH)2D3 and 1,25-(OH)2D3 are all transported on the same protein, called transcalciferin, or vitamin D-binding protein (DBP). In contrast to aquatic species, which store significant amounts of vitamin D in the liver, land animals, do not store appreciable amounts of the vitamin. The body has some ability to store vitamin D, although to a much lesser extent than vitamin A. Principal stores of vitamin D occur in blood and liver, but it is also found in lungs, kidneys and elsewhere in the body. During times of deprivation, vitamin D in these tissues is released slowly, thus meeting vitamin D needs of the animal over a longer period of time (Collins and Norman, 1991). Excretion of absorbed vitamin D and its metabolites occurs primarily through the feces with the aid of bile salts.
For mammals, 1,25-(OH)2D3 is a critical factor in the maintenance of sufficient maternal calcium for transport to the fetus and may play a role in normal skeletal development of the neonate (Lester, 1986). A liberal intake of vitamin D during gestation does provide a sufficient store in newborns to help prevent early rickets. For example, newborn lambs can be provided enough to meet their needs for six weeks. Parenteral cholecalciferol treatment of sows before parturition proved an effective means of supplementing young piglets with cholecalciferol (via the sow’s milk) and its more polar metabolites via placental transport (Goff et al., 1984).
The primary function of vitamin D is to elevate plasma calcium and phosphorus to a level that will support normal mineralization of bone as well as other body functions. It is now known that vitamin D is not only important for mineralization and skeletal growth but plays many other roles. These include regulation of the parathyroid gland, in the immune system, in skin, in cancer prevention, in foreign chemicals metabolism and in cellular development and differentiation. There is a regulatory role of vitamin D (1,25-(OH)2D3) in immune cell functions (Reinhardt and Hustmeyer, 1987), the release of insulin in relation to glucose challenge (DeLuca, 1988), and reproduction in both males and females (DeLuca, 1992). Experiments in mice have illustrated that the autoimmune diseases of multiple sclerosis and rheumatoid arthritis can be successfully treated with the vitamin D hormone and its analogs (DeLuca and Zierold, 1998). Two hormones, thyrocalcitonin (calcitonin) and PTH, function in a delicate relationship with 1,25-(OH)2D3 to control blood calcium and phosphorus levels (Engstrom and Littledike, 1986; McDowell, 2000). Production rate of 1,25-(OH)2D3 is under physiological control as well as dietary control. Calcitonin, contrary to the other two, regulates high serum calcium levels by (1) depressing gut absorption, (2) halting bone demineralization, and (3) depressing reabsorption in the kidney. Vitamin D elevates plasma calcium and phosphorus by stimulating specific ion pump mechanisms in the intestine, bone and kidney. These three sources of calcium and phosphorus provide reservoirs that enable vitamin D to elevate calcium and phosphorus in blood to levels necessary for normal bone mineralization and for other functions ascribed to calcium.In the target tissue, the hormone enters the cell and binds to a cytosolic receptor or a nuclear receptor. 1,25-(OH)2D3 regulates gene expression by binding to tissue-specific receptors and subsequent interaction between the bound receptor and the DNA (Collins and Norman, 1991). The receptor-hormone complex moves to the nucleus where it binds to the chromatin and stimulates the transcription of particular genes to produce specific mRNAs, which code for the synthesis of specific proteins. Evidence for transcription regulation of a specific gene typically includes 1,25-(OH)2D3-induced modulation in mRNA levels. Additionally, evidence may include measurements of transcription and/or the presence of a vitamin D responsive element within the promoter region of the gene (Hannah and Norman, 1994).
Recent studies have identified a heterodimer of the vitamin D receptor (VDR) and a vitamin A receptor (RXR) within the nucleus of the cell as the active complex for mediating positive transcriptional effects of 1,25-(OH)2D3. The two receptors (vitamins D and A) selectively interact with specific hormone response elements composed of direct repeats of specific nucleotides located in the promoter of regulated genes. The complex that binds to these elements actually consists of three distinct elements: the 1,25-(OH)2D3 hormonal ligand, the vitamin D receptor (VDR) and one of the vitamin A (retinoid) X receptors (RXR) (Kliewer et al., 1992; Whitfield et al., 1995).
A. Intestinal Effects
It is well known that vitamin D stimulates active transport of calcium and phosphorus across intestinal epithelium. This stimulation does not involve PTH directly but involves the active form of vitamin D. Parathyroid hormone indirectly stimulates intestinal calcium absorption by stimulating production of 1,25-(OH)2D3 under conditions of hypocalcemia.
The mechanism whereby vitamin D stimulates calcium and phosphorus absorption is still not completely understood. Current evidence (Wasserman, 1981) indicates that 1,25-(OH)2D3 is transferred to the nucleus of the intestinal cell, where it interacts with the chromatin material. In response to the 1,25-(OH)2D3, specific RNAs are elaborated by the nucleus, and when these are translated into specific proteins by ribosomes, the events leading to enhancement of calcium and phosphorus absorption occur (Scott et al., 1982).
In the intestine, 1,25-(OH)2D3 promotes synthesis of calbindin (calcium-binding protein, CaBP) and other proteins and stimulates calcium and phosphorus absorption. Vitamin D has also been reported to influence magnesium (Mg) absorption as well as calcium and phosphorus balance (Miller et al., 1965). This calbindin is not present in the intestine of rachitic chicks but appears following vitamin D supplementation. Originally, it was felt that vitamin D did not regulate phosphorus absorption and transport, but in 1963 it was demonstrated, through the use of an in vitro inverted sac technique, that vitamin D does in fact play such a role (Harrison and Harrison, 1963).
B. Bone Effects
During bone formation in young animals, minerals are deposited on the protein matrix. This is accompanied by an invasion of blood vessels that gives rise to trabecular bone. This process causes bones to elongate. During a vitamin D deficiency, this organic matrix fails to mineralize, causing rickets in the young and osteomalacia in adults. The active metabolite, 1,25-(OH)2D3, brings about mineralization of the bone matrix.
Vitamin D has another function in bone, namely, in mobilization of calcium from bone to the extracellular fluid compartment. This function is shared by PTH (Garabedian et al., 1974), and requires metabolic energy. It presumably transports calcium and phosphorus across the bone membrane by acting on osteocytes and osteoclasts. Rapid, acute plasma calcium regulation is due to the interaction of plasma calcium with calcium-binding sites in bone material as blood comes in contact with bone. Changes in plasma calcium are brought about by a change in the proportion of high- and low-affinity calcium-binding sites, access to which is regulated by osteoclasts and osteoblasts, respectively (Bronner and Stein, 1995). Another role of vitamin D has been proposed in addition to its involvement in bone, namely, in the biosynthesis of collagen in preparation for mineralization (Gonnerman et al., 1976).
C. Kidney Effects
There is evidence that vitamin D functions in the distal renal tubules to improve calcium reabsorption and is mediated by the calcium-binding protein, calbindin (Bronner and Stein, 1995). It is known that 99% of the renal-filtered calcium is reabsorbed in the absence of vitamin D and PTH. The remaining 1% is under control of these two hormonal agents, although it is not known whether they work in concert. It has been shown that 1,25-(OH)2D3 functions in improving renal reabsorption of calcium (Sutton and Dirks, 1978).
D. Functions of vitamin D Beyond Bone Mineralization
The well-known effects of vitamin D relate to biochemical changes occurring in the intestine, bone and kidney. Vitamin D is important for more than just its traditional role in calcium metabolism. A receptor for the active metabolite 1,25-(OH)2D3 has been isolated in the pancreas, parathyroid glands, bone marrow, certain cells of the ovary and brain, endocrine cells of the stomach, breast epithelial cells, skin fibroblasts and keratinocytes, suggesting that 1,25-(OH)2D3has additional functions in a wide variety of cells, glands and organs (Machlin and Sauberlich, 1994). To date, more than 50 genes have been reported to be transcriptionally regulated by 1,25-(OH)2D3 (Hannah and Norman, 1994).
Vitamin D has also been shown to be required for embryonic development of the chick. Vitamin D treatment stimulated yolk calcium mobilization and the vitamin D-dependent Ca2+-binding protein, calbindin, is present in the yolk sac (Tuan and Suyama, 1996). These findings strongly suggest that the hormonal action of 1,25-(OH)2D3 on yolk sac calcium transport is mediated by the regulated expression and activity of calbindin, analogous to the response of the adult intestine. 1,25-(OH)2D3 is also essential for the transport of eggshell calcium to the embryo across the chorioallantoic membrane (Elaroussi et al., 1994).
The actions of 1,25-(OH)2D3 are recognized as being involved in regulation of the growth and differentiation of a variety of cell types, including those of hematopoietic and immune systems (Lemire, 1992). Recent studies have suggested 1,25-(OH)2D3 as an immunoregulatory hormone. Elevated 1,25-(OH)2D3 also was associated with a significant 70% enhancement of lymphocyte proliferation in cells treated with pokeweed mitogen (Hustmeyer et al.,1994). 1,25-(OH)2D3 also inhibits growth of certain malignant cell types and promotes their differentiation (Colston et al., 1981; DeLuca, 1992). 1,25-(OH)2D3 has been reported to inhibit proliferation of leukemic cells (Pakkla et al.,1995), breast cancer cells (Vink van Wijngaarden et al., 1995) and colorectal cells (Cross et al., 1995). A deficiency of vitamin D may promote prostate cancer (Skowronski et al., 1995). Also, 1,25-(OH)2D3 and its analogs may be effective in treating some forms of psoriasis (Kragballe et al., 1991). The therapeutic value of vitamin D and its analogs has been under rigorous evaluation in numerous laboratories around the world.
Swine do not have a nutritional requirement for vitamin D when sufficient sunlight is available, since vitamin D3is produced in skin through action of UV irradiation on 7-dehydrocholesterol. In addition to sunlight, other factors influencing dietary vitamin D requirements include (1) amount and ratio of dietary calcium and phosphorus, (2) availability of calcium and phosphorus, (3) species, and (4) physiologic factors. Vitamin D becomes a nutritionally important factor in the absence of sufficient sunlight. Sunlight that comes through ordinary window glass is ineffective in producing vitamin D3in the skin, since glass does not allow penetration of UV rays, and its effectiveness is dependent on length and intensity of UV rays that reach the body. Animals housed in confinement must depend on their feed for the vitamin D they need. In a modern agricultural economy this applies particularly to intensive swine and poultry production. The colors of the skin are important in determining response to irradiation. Irradiation is less effective on dark-pigmented skin. This has been shown to be true for white and black breeds of hogs. White pigs have been shown to resist vitamin D deficiency signs about twice as long as colored pigs; an average of 45 minutes of daily exposure to January sunshine for two weeks was sufficient to cure rickets in white pigs in Minnesota (Cunha, 1977). The vitamin D requirements of swine as listed in NRC (1998) range from 150 to 220 IU per kg (68 to 100 IU per lb) of diet. The estimated requirement for breeding and lactating animals is 200 IU per kg (91 IU per lb) of diet. The British Society of Animal Science (2003) suggests up to 1,000 IU vitamin D per kg (455 IU per lb) of feed for some classes of swine. In the NRC (1998) there is a notation that no studies of the vitamin D requirements of sows during gestation or lactation have been reported. Now, however, Lauridsen et al. (2010) carried out an experiment with gilts and lactating sows to evaluate reproductive performance and bone status. They concluded that a dietary dose of approximately 1,400 IU of vitamin D per kg (636 IU per lb) is recommended for reproducting swine. In the gilt trial they found that the ultimate strength of bones and their ash content were greater when vitamin D3 was supplemented in doses larger than 800 IU per kg (364 IU per 1b) of feed. Miller et al. (1964) reported that the vitamin D2 requirement of the baby pig fed a casein-glucose diet was 100 IU per kg (46 IU per lb) of diet. However, if isolated soy protein is fed, the requirement is higher (Miller et al., 1965b).
Vitamin D requirements of swine are suggested to be sufficiently high to produce normal growth, calcification, production and reproduction in the absence of sunlight, provided that diets contain recommended levels of calcium and available phosphorus. Wahlstrom and Stolte (1958) found that there is no need for supplemental vitamin D in highly fortified rations for growing pigs, in confinement housing with regard to growth. However, Wahlstrom and Stolte (1958) suggested that they did not use an adequate depletion period to induce true vitamin deficiency, as endogenous stored vitamin D was apparently available. Species differences can be illustrated by the fact that adequate intakes of calcium and phosphorus in a diet containing enough vitamin D to produce normal bone in the rat or pig will quickly cause the development of rickets in chicks.
The need for vitamin D depends to a large extent on the ratio of calcium to phosphorus. As this ratio becomes either wider or narrower than the optimum, the requirement for vitamin D increases, but no amount will compensate for severe deficiencies of either calcium or phosphorus. It is recommended (NRC, 1998) that the dietary dry matter of rapidly growing young pigs should have a calcium:phosphorus ratio in the range of about 1:1 to 1.5:1. For adult maintenance, wider calcium:phosphorus ratios are possible. In these situations vitamin D requirements seem to be at a minimum and risks of vitamin D deficiency are less. Combs et al. (1966b) indicated the need for vitamin D supplementation may be reduced or eliminated when accompanied by “near optimum” dietary calcium and phosphorus concentrations. Their findings appear to be supported by those of Wahlstrom and Stolte (1958). However, Bethke et al. (1946) concluded that swine have a fundamental requirement for vitamin D even when the feed supplies a satisfactory ration and adequate amounts of calcium and phosphorus. Differences in the length of vitamin D depletion before initiation of the experiments or the inclusion of B-vitamins and antibiotics in the basal ration in some of the experiments, but not others, may partially explain the conflicting findings.
Amounts of dietary calcium and phosphorus and the physical and chemical forms in which they are presented must be considered when determining requirements for vitamin D. High dietary calcium concentrations can precipitate phosphates as insoluble calcium phosphate. Soluble calcium salts are more readily absorbed, and oxalates tend to interfere with absorption, but some of this interference can be overcome by dietary vitamin D or irradiation. Correspondingly, while the phosphorus of inorganic orthophosphate tends to be well absorbed, other factors being favorable, that of phytic acid, which is the predominant phosphorus compound of unprocessed cereal grains and oilseeds, seem to be poorly available to swine. Phosphorus absorption is mostly independent of vitamin D intake, with the inefficient absorption in rickets being secondary to failure of calcium absorption. The improvement upon vitamin D administration is a result of improving calcium absorption.
In light of the cost of phosphorus and environmental concerns over its abundance in pig manure, the interrelationships and possible additive benefits of adding phytase and 1-alpha-hydroxylated vitamin D3 compounds are being investigated. In poultry, both phytase and 1-alpha-hydroxy D3 have been demonstrated to be efficacious for releasing phosphorus and trace minerals from phytase complexes (Edwards, 1993; Biehl et al., 1995). However, Biehl and Baker (1996) reported that 1-alpha-hydroxycholecalciferol was either significantly less effective in pigs than in chicks or not at all effective in improving phytase-phosphorus utilization in young pigs. Baker and Biehl (1996) indicated that 1-alpha-(OH)D3 seems to be much less efficacious in pigs than in chickens. Cromwell et al. (1996) indicated that addition of 1,25-dihydroxycholecalciferol at the rate of 10 mg per kg diet did not improve utilization of phytate phosphorus in pigs. Similarly, Cromwell et al. (1997) determined that addition of 5 to 200 mg per kg of D3 to low-calcium, low-phosphorus diets did not improve phytate phosphorus utilization in growing pigs. The 200 mg per kg level of D3depressed growth rate, bone strength and plasma P in trials conducted with pigs 14 to 21 kg (30.9 to 46.3 lb) initial weight. Adeola et al. (1998) determined that phytase or cholecalciferol supplementation of a low-calcium, low-phosphorus diet produces similar growth performance as a diet with adequate calcium and phosphorus when fed to 20 kg pigs. Li et al. (1998) found that the addition of 2,000 IU per kg Vitamin D3 to a diet containing phytase tended to increase pig performance and tended to further increase digestibility of dry matter, phosphorus and calcium. However, the addition of vitamin D3 did not significantly increase the effectiveness of phytase. Whether or not vitamin D will be found to improve the effectiveness of phytase, both phytase and 1-alpha-hydroxy D3, alone or in combination, have been suggested to offer potential for reducing feed costs while simultaneously reducing phosphorus in manure (Baker and Biehl, 1996).
It was generally assumed that for all but a few species, vitamin D2 and vitamin D3 are equally potent. For poultry and other birds and a few of the rarer mammals that have been studied, including some New World monkeys, vitamin D3 is many times more potent than vitamin D2 on a weight basis. Vitamin D3 may be 30 to 40 times more effective than the D2 form for poultry. Therefore, plant sources of the vitamin (vitamin D2) should not be provided to these species. More recently, studies with swine indicate that D3 is likewise more potent for swine than D2, but the difference between forms is much less dramatic than for poultry. Horst et al. (1981) demonstrated that pigs discriminate in their metabolism of the two forms. Horst et al. (1982) indicated that the rat and pig as well as the chick discriminate between ergocalciferol and cholecalciferol when these vitamins are administered together orally. Pigs given oral doses of a mixture of vitamin D2 and D3 had significantly higher concentrations of plasma vitamin D3, 25-(OH)D3, 24,25-(OH)2D3, 25,26-(OH)2D3 and 1,25-(OH)2D3 than corresponding vitamin D2 counterparts.
Sources of vitamin D are natural foods, irradiated sebaceous material licked from skin or hair, and directly absorbed products or irradiation formed on or in the skin. The distribution of vitamin D is very limited in nature, although provitamins occur widely. Of feeds for livestock, grains, roots and oilseeds, as well as their numerous by-products, contain insignificant amounts of vitamin D. Green fodders are equally poor sources. Ergocalciferol occurs naturally in some mushrooms and cholecalciferol occurs naturally in fish (Johnson and Kimlin, 2006). The principal source of the antirachitic factor in the diets of farm animals is provided by the action of radiant energy upon ergosterol in forages. Legume hay that is cured to preserve most of its leaves and green color contains considerable amounts of vitamin D activity. Alfalfa, for example, will range from 650 to 2,200 IU per kg (295 to 1,000 IU per lb) (Maynard et al., 1979). Swine that received sun-cured alfalfa leaf meal in their diet would receive a limited amount of vitamin D from this source. However, fewer commercial swine operations use alfalfa leaf meal than in the past. For non-forage-consuming species, the vitamin D that occurs naturally in unfortified food is generally derived from animal products. Saltwater fish, such as herring, salmon and sardines, contain substantial amounts of vitamin D, and fish liver oils are extremely rich sources. Bethke et al. (1946) reported that either irradiated yeast or cod-liver oil can be an effective source of vitamin D.
Due to lack of vitamin D in feeds and management systems without direct sunlight, modern swine operations must provide a supplemental source of the vitamin. Vitamin D3 is the principal source of supplemental vitamin D for livestock and poultry. Vitamin D3 is commercially available as a resin or crystal, usually containing 24 to 40 million IU per gram, for use as the vitamin D source in various vitamin products. Vitamin D3 products for feed include gelatin beadlets (with vitamin A), oil dilutions, oil absorbates, emulsion, and spray- and drum-dried powders. Test results have shown that the gelatin beadlet offers the best vitamin D3 stability. Incorporating vitamins D3 and A in the beadlet form provides physical protection from oxidation, and the antioxidants included in the beadlets afford chemical protection (Hoffmann La-Roche, 1988).
The 25-(OH)D3 form of vitamin D (commercial product referred to as Rovimix Hy·D®) is now also a potential source of supplementation. It is absorbed in the intestine better than vitamin D3 (Ward, 2004; Chung, 2006). The more rapid uptake of 25-(OH)D3 may be due, at least in part to the intestinal binding proteins. This protein has affinity for 25-(OH)D3 that is at least 1,000 times greater than for other D3 metabolites (Teegarden et al., 2000). Also, studies indicate that the intestinal uptake of 25-(OH)D3 occurs irrespective of bile acid secretion and micelle formation/fat absorption.
The primary disease of vitamin D deficiency is rickets, generally characterized by a decreased concentration of calcium and phosphorus in the organic matrices of cartilage and bone. A deficiency of vitamin D results in clinical signs similar to those of a lack of calcium or phosphorus or both, as all three are concerned with proper bone formation. In the adult, osteomalacia is the counterpart of rickets, and since cartilage growth has ceased, it is characterized by a decreased concentration of calcium and phosphorus in the bone matrix. Outward signs of rickets include the following skeletal changes, varying somewhat with species depending on anatomy and severity: (a) weak bones cause curving and bending of bones, (b) enlarged hock and knee joints, (c) tendency to drag hind legs, and (d) beaded ribs and deformed thorax. Although there appear to be differences among species in the susceptibility of different bones to such degenerative changes, as well as differences that probably reflect bodily conformation (e.g., pig compared with sheep), there is nevertheless an apparent common pattern (Abrams, 1978). Spongy parts of individual bones and bones relatively rich in such tissue are generally the first and most severely affected. As in simple calcium deficiency, the vertebrae and the bones of the head suffer the greatest degree of resorption. Next come the scapula, sternum and ribs. The most resistant bones are the metatarsals and the shafts of long bones. For swine specifically, a deficiency of vitamin D causes poor growth, stiffness, lameness (Illus. 3-2), stilted gait, a general tendency to “go down” or lose the use of the limbs (posterior paralysis), frequent cases of fractures, softness of bones, bone deformities, beading of the ribs, enlargement and erosion of joints and unthriftiness (Cunha, 1977). Bones may also be deformed by the weight of the animal and the pull of body muscles.
Illustration 3-2: Vitamin D Deficiency, Rickets
In severe vitamin D deficiency, pigs may exhibit signs of calcium and magnesium deficiency, including tetany (NRC, 1998). It takes four to six months for pigs fed a vitamin D-deficient diet to develop signs of a deficiency (Johnson and Palmer, 1939; Quarterman et al., 1964). Reproductive performance in relation to stillborn pigs was influenced by vitamin D supplementation (Lauridsen et al., 2010). There was a decreased number of stillborn piglets with larger doses of vitamin D (1,400 and 2,000 IU per kg (635 and 907 IU/lb) of vitamin D in feed resulting in 1.17 and 1.13 stillborn piglets per litter, respectively) compared with the smaller doses of vitamin D per kg (90.7 and 3631 IU/lb) of feed (200 and 800 I IU of vitamin D, resulting in 1.98 and 1.99 stillborn piglets per litter, respectively). The trend toward confinement of swine in completely enclosed houses through their life cycle increases the importance of adequate dietary vitamin D fortification. Goff et al.(1984) concluded that subclinical rickets might become more of a problem as swine producers convert to confinement operations, which deprive sows and piglets of the UV irradiation needed for the endogenous production of cholecalciferol. Research has shown that sunshine cannot always be depended on to meet vitamin D requirements of growing or finishing pigs during winter in northern latitudes. Of all vitamins provided in swine feeds, vitamin D is one of two (the other being vitamin B12) that is most likely to be deficient. Typical grain- and soybean-based diets contain virtually no vitamin D. Also the trend towards complete confinement eliminates UV light as a source of the vitamin. Therefore, supplemental vitamin D must be provided for all swine operations in which growing and breeding animals remain in confinement.
Synthesis of vitamin D3 in the skin of swine through exposure to UV irradiation is limited because of the commercial production trend of complete confinement. In addition, feedstuffs supply negligible amounts of vitamin D activity in the diet. Consequently, all swine diets should be supplemented with vitamin D. Vitamin D3is the primary source of supplemental vitamin D in foods, feeds and pharmaceuticals (Adams, 1978). Some irradiated sterol resins used as the source of vitamin D3 in various vitamin D3 products for feed use may contain excessive amounts of biologically inactive impurities, such as tachysterol and isotachysterol. The vitamin D3 activity in low-purity vitamin D3resins and supplements (Baker, 1978) has been overestimated by the United States Pharmacopeia (USP) Chemical Assay, which does not correct for these biologically inactive compounds. A commercially available vitamin product containing stabilized, high-purity vitamin D for feed or drinking water should be used to ensure the levels needed to prevent deficiency and allow optimum performance in swine. Pure vitamin D3 crystals or vitamin D3 resin is very susceptible to degradation on exposure to heat or contact with mineral elements. In fact, the resin is stored under refrigeration with nitrogen gas. Dry, stabilized supplements retain potency much longer and can be used in high mineral supplements. It has been shown that vitamin D3 is much more stable than D2in feeds containing minerals. Stabilization of the vitamin can be achieved by (1) rapid compression of the mixed feed, for example into pellets so that air is excluded; (2) storing feed in cool, dry, dark conditions; (3) preventing close contact between the vitamin and potent metallic oxidation catalysts, for example, manganese; and (4) including natural or synthetic antioxidants in the mix. The vitamin can also be protected by enclosing it in durable gelatin beadlets. Stability of dry vitamin D supplements is affected most by high temperature, high moisture content and contact with trace minerals, such as ferrous sulfate, manganese oxide and others. Hirsch (1982) reports the results of a “conventional,” or nonstabilized, vitamin D3 product being mixed into a trace mineral premix or into animal feed and stored at ambient room temperature (20 to 25°C) for up to 12 weeks. The mash feed had lost 31% of its vitamin D activity after 12 weeks, and the trace mineral premix had lost 66% of its activity after only six weeks in storage.
In addition to providing supplemental vitamin D in feed and water, injectable sources are available. Parenteral vitamin D3 treatment of sows before parturition provided an effective means of supplementing piglets with vitamin D3 (via the sow’s milk) and its dehydroxy metabolites by placental transport (Goff et al., 1984).
Supplemental vitamin D3 is usually added to animal feed in the form of cholecalciferol (vitamin D3). However, recently, 25(OH)D3 has been commercially available (as Rovimix Hy·D®) and has been approved for use in poultry nutrition (Bar et al., (2003). The use of 25(OH)D3 has resulted in dramatic performance parameters for poultry in relation to growth, egg production and quality of eggs and disease prevention (e.g. tibial dyschondroplasia) (Soto-Salanova and Hernandez, 2004; Ward, 2004; Larroudé et al, 2005, Chung, 2006).
For growing-finishing swine, vitamin D3 and 25-OHD3 were compared (Jakobsen et al 2007). A similar vitamin D status was found for pigs receiving approximately 55 µg of vitamin D per day as vitamin D3 in a mixture of vitamin D3and 25-(OH)D3 or solely as 25-(OH)D3. In a study looking at the performance of gilts and lactating sows, at doses greater than 200 IU, 25(OH)D3 was more bioavailable than vitamin D3 and, as such, could be considered an equivalent or even more advantageous source of vitamin D.
Cost of vitamin D supplementation in livestock diets is nominal (Rowland, 1982). In contrast, the cost of not adding enough vitamin D to prevent deficiencies is very high. Supplemental levels of vitamin D3 administered to pigs through the feed should be adjusted to provide the margin of safety needed to offset the factors influencing the vitamin D needs of swine. This is important to prevent deficiency and allow optimal performance. Factors that increase the amount of vitamin D needed to maximize productive and reproductive responses often may not be reflected in NRC minimum requirements. Consequently, nutritionists often use more than the minimum levels of vitamin D in feeds. Successful nutrition programs may greatly exceed the NRC minimum of vitamin D. However, no amount of vitamin D can make up for lack of enough calcium or phosphorus in the diet.
Besides inadequate quantities of dietary vitamin D, deficiencies may result from (1) errors in vitamin addition to diets, (2) inadequate mixing and distribution in feed, (3) separation of vitamin D particles after mixing, (4) instability of the vitamin content of the supplement, or (5) excessive length of storage of diets in environmental conditions causing vitamin D loss (Hirsch, 1982).
Supplementation considerations are dependent on other dietary ingredients. The requirements for vitamin D are increased several fold by inadequate levels of calcium and (or) phosphorus or by improper ratios of these two elements in the diet. A number of reports have indicated that molds in feeds interfere with vitamin D (Cunha, 1977), for example, when corn contains the mold Fusarium roseum, a metabolite of this mold prevents vitamin D3 in the intestinal tract from being absorbed by the chick. A similar deleterious effect on vitamin D metabolism would be expected in the pig.
Phytic acid (phytate) is a major storage form of phosphorus in almost all seeds; this form of organic phosphorus is considerably less available than inorganic phosphorus. The use of exogenous supplement phytase in swine and poultry to enhance phytate-phosphorus utilization and reduce the flow of phosphorus into the environment is now common (Nasi et al., 1990; Simons et al., 1990; Jongbled et al., 1992; Onyango et al., 2006). The efficiency of supplemental microbial phytase depends on its rate of inclusion, dietary calcium and phosphorus concentrations and the ratio of these minerals, vitamin D, nature of diet, age and physiological status of swine. Adequate vitamin D is important to best utilize phytin phosphorus (McDowell, 2003). The hydroxylated form of vitamin D3(25-(OH)D3) was found to increase phytate phosphorus retention for swine (Jongbloed et al., 1993; Lei et al., 1994; Kornegay, 1996).
Other factors that influence vitamin D status are diseases of the endocrine system, intestinal disorders, liver malfunction, kidney disorders and drugs. Liver malfunction limits production of the active forms of the vitamin, while intestinal disorders reduce absorption. It is possible that swine with certain diseases or heavy infestations of internal parasites may be unable to synthesize the metabolically active forms of vitamin D as a result of liver or kidney damage. Unknown factors in feeds may increase vitamin D requirements. For example, there is evidence of a factor in rye and in soybean fractions that can produce malabsorption of this vitamin in the intestine (MacAuliffe and McGinnis, 1976).
Hendricks et al. (1967) investigated whether antagonism occurs between the source of vitamin A activity and vitamin D. The authors reported that feeding fermentation beta-carotene at 8 mg per kg (3.6 mg per lb) of diet did not increase the need for ergocalciferol above the level normally required by baby pigs fed a purified isolated-soy diet containing retinyl acetate.
Besides the toxicity resulting from excessive vitamin A, vitamin D is the vitamin next most likely to be consumed in concentrations toxic to livestock. Although vitamin D is toxic at high concentrations, short-term administration of as much as 100 times the requirement level may be tolerated. For most species, including swine, the presumed maximal safe level of vitamin D3for long-term feeding conditions (more than 60 days) is four to ten times the dietary requirement. For swine, the upper safe dietary level for short-time exposure is 33,000 IU per kg (15,000 IU per lb) and for over 60 days, 2,200 IU per kg (1000 IU per lb) of diet (NRC, 1998). In practical conditions, vitamin D toxicosis for swine would only be expected as a result of feed mixing errors and then over a relatively long period. Excessive intake of vitamin D produces a variety of effects, all associated with abnormal elevation of blood calcium. Elevated blood calcium is caused by greatly stimulated bone resorption, as well as increased intestinal calcium absorption. The main pathologic effect of ingestion of massive doses of vitamin D is widespread calcification of soft tissues. In pigs, signs of toxicity are anorexia, stiffness, lameness, arching of the back, polyuria, and aphonia. Death was reported in four days when young pigs received 473,000 IU per kg (215,000 IU per lb) in the diet (Long, 1984). Whiting and Bezeau (1958) reported that a high vitamin D content (800 IU per lb of ration) may be involved in parakeratosis by reducing absorption and retention of zinc. Hancock et al. (1986) suggested reduced gains and feed efficiency when young pigs received 22,000 to 44,000 IU per kg (10,000 to 20,000 IU per lb) of diet. Studies in a number of species, including swine, indicate that vitamin D3is 10 to 20 times more toxic than vitamin D2when provided in excessive amounts. The potential for vitamin D toxicosis, that is hypercalcemia and soft tissue calcification, is much higher when animals have access to formulations of 1,25(OH)2D3, since this hormone form of vitamin D has bypassed the stringent physiological point of the vitamin D endocrine system, namely the 25-(OH)D3-1- hydroxylase of the kidney (Norman and Henry, 2007).A possible new role for vitamin D3 is being evaluated: extremely high levels of vitamin D3 are fed to cattle for short periods of time prior to slaughter to increase tenderness of the meat. Several research studies (Swanek et al., 1997, 1998) have been designed to evaluate the effects of feeding cattle supplemental vitamin D3 at 0, 2.5, 5, or 7.5 million IU per head per day for five or 10 days immediately prior to slaughter. The researchers determined that feeding the high vitamin D3 levels reduced the Warner-Bratzler shear force values of tough beefsteaks aged for short periods. The possible mechanism by which beef tenderization occurs is that high levels of vitamin D3 cause increases in calcium concentrations in blood and muscle; at higher concentrations of calcium, calcium-activated proteases associated with the calpain system are more active, increasing meat tenderness. Using a similar approach, Enright et al. (1998) fed swine diets containing three levels of vitamin D3 (331, 55,031, or 176,000 IU/kg) (150, 24,362 or 79,833 IU/lb) to finishing pigs for the last 10 days prior to slaughter. The researchers reported that feeding the higher levels of vitamin D3 reduced feed intake and average daily gains while increasing serum calcium concentration. Reduced drip loss and color values (Hunter L* measurement) and increased subjective color and firmness scores were exhibited in the carcass as a result of increasing dietary vitamin D3. Tenderness was not evaluated in this study. Sparks et al. (1998) determined that the optimal dosage of vitamin D3 was 500,000 IU daily for three days prior to slaughter as based on blood calcium concentration. The researchers stated that the results of feeding high levels of vitamin D3in this initial study did not improve pork tenderness and other measures of pork quality. Wiegand et al. (2002) fed finishing pigs 500,000 IU per day of vitamin D3 for 3 days prior to slaughter. Vitamin D supplementation did not affect quality characteristics (measured by subjective scores) or tenderness (i.e., Warner-Bratzler shear force). However, the treatment did improve Hunter color values at 14 days of storage.