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DSM in Animal Nutrition & Health

Vitamin D

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 the skin (in most species), the intestinal wall and other tissues. Vitamin D precursors have no antirachitic activity and must undergo ultraviolet irradiation (sunlight) to be activated to vitamins D2 and D3.

 

Illustration 3-1

3Illustration_3-1_VitaminD

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, 2007). Hymøller and Jensen (2010) showed no degradation of vitamin D in the rumen of high producing dairy cows. Both ergocalciferol and cholecalciferol were added to the rumen contents through a rumen fistula. Later both forms of vitamin D were found at constant levels, indicating no degradation in the rumen. 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 selectively 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 D synthesis is the actual concentration of 7-dehydrocholesterol present in the skin. The fourth determinant of vitamin D3 production 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 absorbed and transported by the blood, primarily bound to gamma-globulin, and 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, 1992, 2008; Dittmer and Thompson, 2011). 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), which may explain the higher vitamin D requirements in ruminants.

Production and metabolism of vitamin D (both D2 and D3) necessary to activate the target organs are illustrated in Figure 3-1. 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 D3 [25-(OH)D3]. This metabolite is the major circulating form of vitamin D under normal conditions and during vitamin D excess (Littledike and Horst, 1982). 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 D [1,25-(OH)2D3] (DeLuca, 2008). The 1,25-(OH)2D3 is also referred to as calcitriol. 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)D3 and vitamin D3 do not function at these specific sites under physiological conditions (DeLuca, 1992).

 

Figure 3-1: The Functional Metabolism of Vitamin D3 Necessary to Activate Target Organs of Intestine, Bone and Kidney

3Figure_3-1_The_Functional_Metabolism_of_Vitamin_D3

Production of 1,25-(OH)2D3 is very carefully regulated by parathyroid hormone (PTH) in response to serum calcium and phosphate (PO43-) concentrations. Under conditions of calcium stress, PTH activates renal mitochondrial 1-alpha-hydroxylases, which convert 25-(OH)D3 to 1,25-(OH)2D3, 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 1alpha-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 in 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 most mammals, vitamin D, 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 D3, 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 in 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).

 

Functions

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. Recently, evidence also suggests 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)2D3is 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 that are 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 through its 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), requires metabolic energy, and 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 Ca+2-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 the 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.

 

Requirements

With adequate direct exposure to sunlight, ruminants do not have an absolute dietary requirement for vitamin D, due to its production in the skin through the action of UV radiation on 7-dehydrocholesterol. Other factors influencing dietary vitamin D requirements include (1) amounts and ratio of dietary calcium and phosphorus; (2) bioavailability of calcium and phosphorus; (3) species; and (4) physiological state of the animal. Vitamin D metabolism differences have been shown between Bos indicus and Bos taurus cattle (Montgomery et al., 2004c). Bos indicus cattle have greater 1,25-(OH)2D3 in tissues, and greater 1,25(OH)2D3 plasma concentrations than Bos taurus cattle. Waters et al. (2009) reported differences in serum 25-(OH)D3 concentrations between white-tailed deer and elk. Thus, the requirement for vitamin D differs between breeds and species. Vitamin D becomes nutritionally important in the absence of sufficient sunlight. Sunlight passing through ordinary window glass is ineffective in producing vitamin D3 in skin because glass blocks penetration of UV radiation. Both time of exposure and intensity of UV radiation affect vitamin D3production in skin. Additionally, coat color, coat thickness and skin pigmentation are important determinants of vitamin D production by irradiation. Irradiation is less effective on dark-pigmented skin. Ruminants housed in confinement depend on their diet for their vitamin D requirements. In the current agricultural economy, confinement livestock production is common. A significant proportion of dairy cattle and calves are housed under conditions with little or no exposure to sunlight. Cattle that are not confined are subject to seasonal variation in sunlight exposure. The vitamin D requirements of ruminants are listed in NRC publications in units of IU per kg (lb) of body weight per day, IU per kg (lb) of diet, or IU per head per day. One IU of vitamin D is defined as 0.025 µg of cholecalciferol (D3) or its equivalent, and therefore 1 mg of cholecalciferol possesses 40,000 IU of vitamin D activity. Typical requirements of cattle, sheep and goats are shown in Table 3-1. The vitamin D requirement of beef cattle (NRC, 1996) is 275 IU per kg (125 IU per lb) of diet on a dry basis. Vitamin D requirements for dairy cattle are from 600 to 864 IU per kg (273 to 393 IU per lb) of diet for lactating cows, 951 to 1,511 IU per kg (432 to 687 IU per lb) of diet for fresh cows, 1,520-2,249 IU per kg (691 to 1022 IU per lb) of diet for dry pregnant cows and 1769 to 1887 (804 to 858 IU per lb) for growing pregnant heifers (NRC, 2001). For the lactating cows allowance was made for varying levels of milk production, which had not been calculated for the previous NRC (1989).

3Table_3-1_Representative_Vitamin_A_D_and_E_Requirements_for_Ruminants_per_Unit_of_Feed

For sheep on a feed basis, vitamin D requirement ranged from 148 to 216 IU per kg (67 to 98 IU per lb) of diet for all sheep (NRC, 1985). Vitamin D requirements for all classes of goats are 300 IU per kg (136 IU per lb) of diet (NRC, 1981). A newer NRC (2007b), which is for small ruminants, noted that the requirements for all classifications of domesticated small ruminants were comparable to previous recommendations for goats (NRC, 1981) and sheep (NRC, 1985). The vitamin D requirement for maintenance and early pregnancy is 5.6 IU per kg (2.5 IU per lb) bodyweight for small ruminants (NRC 2007b). Additional vitamin D requirements are 213 IU per day for late pregnancy and 760 IU per kg (345 IU per lb) for milk production. For growing and developing small ruminants, an additional 54 IU per day vitamin D is required for every 50 g daily weight gain. Vitamin D requirements are 20 percent greater for animals less than 4 months of age that have been weaned early.

It was once assumed that dietary vitamin D requirements of ruminants exceeded that of nonruminants in part due to ruminal degradation (Parakkasi et al., 1970; Sommerfeldt et al., 1979). However, that view is now challenged as Hymøller and Jensen (2010) showed no degradation of vitamin D (ergocalciferol and cholecalciferol) when added to the rumen contents of high producing dairy cows.

Vitamin D3 (cholecalciferol) is more biologically active than vitamin D2 (ergocalciferol) in dairy calves (Sommerfeldt et al., 1983).

The vitamin D requirement is influenced by the ratio of calcium to phosphorus in the diet (Scott and McLean, 1981). As the calcium:phosphorus ratio becomes either wider or narrower than the optimum, the requirement for vitamin D increases. However, vitamin D cannot compensate for severe deficiencies of either calcium or phosphorus. The apparent optimum range of calcium:phosphorus ratios in the diet of rapidly growing young stock is 1.2:1 to 1.5:1. Mature animals at maintenance tolerate lower dietary calcium levels and wider calcium:phosphorus ratios than growing or lactating animals. Wise et al. (1963) tested nine calcium:phosphorus ratios ranging from 0.41:1 to 14.3:1 in cattle. They reported that dietary calcium:phosphorus ratios below 1:1 and above 7:1 adversely affected growth and feed efficiency. Metacarpal bone growth and histology of ewes was not affected by dietary calcium:phosphorus ratios ranging from 0.88:1 to 4.3:1, in diets containing 0.42% phosphorus (Oberbauer et al., 1988). Therefore, in the presence of adequate phosphorus, excess dietary calcium within these ranges was not detrimental to bone growth and maturation. Intakes of dietary calcium and phosphorus and the physical and chemical forms in which they are presented must be considered when determining vitamin D requirements and optimal fortification levels. Excess dietary calcium concentrations can precipitate phosphates as insoluble calcium phosphate. Soluble calcium salts are more readily absorbed than insoluble compounds and oxalates can interfere with calcium absorption. Dietary vitamin D or irradiation can overcome part of this interference. Correspondingly, the phosphorus of inorganic orthophosphate tends to be well absorbed while other sources are less available. The phosphorus of phytic acid, which is the predominant phosphorus compound of unprocessed cereal grains and oilseeds, is poorly available to nonruminants but is available to ruminants due to the rumen microbial production of phytase enzymes (Abrams, 1978). Phosphorus absorption is largely independent of vitamin D intake. The inefficient phosphorus absorption in rickets is secondary to the failure of calcium absorption, and its improvement upon vitamin D administration is a result of increasing calcium absorption.

 

Sources

The distribution of vitamin D2 and D3 (ergocalciferol and cholecalciferol) in nature is limited, although their precursors, ergosterol in plants and 7-dehydrocholesterol in animals, occur widely. Grains, roots and oilseeds, and their numerous by-products used in livestock feeds, contain insignificant amounts of vitamin D. Ergocalciferol occurs naturally in some mushrooms and cholecalciferol occurs naturally in fish (Johnson and Kimlin, 2006). The principal source of vitamin D in the diets of farm animals is vitamin D2 (ergocalciferol) produced by the action of UV light on the ergosterol in forages.  Green, uncured forages are poor sources of vitamin D, but vitamin D2 is formed by UV radiation during field curing of hay or silage. Potency depends on local climatic conditions. Hay or silage produced under cloudy conditions will have less vitamin D activity than when cured under bright sunlight. Artificially dried forages that have been removed directly after harvest to drying facilities will have less vitamin D content than field cured forage (Abrams, 1952). Even hay dried in the dark immediately after cutting has some vitamin D activity because the dead or injured leaves on the growing plant are responsive to UV irradiation even though the living tissues are not. This phenomenon is also largely responsible for the vitamin D activity found in corn silage (Maynard et al,, 1979). Legume hay that is properly 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). 

3Table_3-2_Vitamin_D2_Concentration_in_Representative_Feedstuffs

The naturally occurring vitamin D activity in concentrate feeds is generally derived from animal products. Saltwater fish and their oils in particular are extremely rich sources of vitamin D. Milk fat contains a variable amount of vitamin D activity (5 to 40 IU in cow’s milk per quart) depending on fat content, but neither cow’s milk nor human milk contains sufficient vitamin D to protect the nursing young against rickets in the absence of sunlight (Maynard et al., 1979). Vitamin D in colostrum provides come reserve. Cow’s milk is reportedly higher in vitamin D when produced in the summer than in the winter.

In general, it has been assumed that for all but a few species, vitamin D2 and D3, are equally active as sources of 1,25-(OH)2D. For poultry and other birds and a few of the rare mammalian species that have been studied, including some New World monkeys, vitamin D3 is many times more potent than D2 on a weight basis. Recently, the belief that mammals (other than the New World monkey) do not discriminate between vitamin D2 and D3 has been proven incorrect. Studies with ruminants (Sommerfeldt et al., 1981) suggests that these species discriminate between the two sources of vitamin D in metabolism and D3 is the preferred substrate for production of 1,25-(OH)2D3. Sommerfeldt et al. (1983) reported that the concentration of 1,25-(OH)2D3 in the plasma of ergocalciferol (D2)-treated dairy calves was one-half to one-fourth the level in cholecaliferol (D3)-treated calves. Discrimination against vitamin D2 in ruminants may be, in part, a result of either its preferential degradation by rumen microbes or less efficient intestinal absorption.  For most species, it has been found that cholecalciferol exceeded ergocalciferol in biological activity. Recent evidence indicates that in man, vitamin D2 has only 25 to 30% of the biological activity of vitamin D3 (Armas et al., 2004).

Hymoller and Jensen (2011) report that vitamin D2 impairs utilization of vitamin D3 in high-yielding dairy cows. Vitamin D3 given after D2 was less efficient at increasing the plasma status of 25-(OH)D3 than D3 given without previous D2administration.

Vitamin D3 is the principal source of supplemental vitamin D for livestock and poultry diets. Vitamin D3 is commercially available in resin or in crystalline forms, usually containing 24 to 40 million IU per gram. This form is used largely in human foods and vitamin supplements. New commercial products such as 25-(OH)D have been developed, as they are a more potent form of vitamin D. Vitamin D3 product forms for the animal feed industry include oil dilutions, oil absorbates, emulsions, dispersible liquid concentrates, spray- and drum-dried powders and, most recently, stabilized beadlets. Test results have shown that the spray-dried gelatin beadlet offers the greatest vitamin D3 stability. Vitamin A, D3 combination products are also produced in beadlet form. Beadlets containing both vitamin A and D are usually cross-linked by and an additional process in order to increase protection from oxidation. Antioxidants are often added to commercial vitamin beadlet products.

Some irradiated sterol resins used as a source of vitamin D in feed contain significant proportions of biologically inactive impurities, such as tachysterol and isotachysterol. The vitamin D activity in products of this type has been overestimated by the USP Chemical Assay, which does not distinguish cholecalciferol (D3) from these biologically inactive compounds (Baker, 1978). Crystalline vitamin D3 and vitamin D3 resins are very susceptible to degradation upon exposure to heat or contact with mineral elements. For this reason, resins are normally stored under refrigeration with nitrogen gas. Dry, stabilized vitamin D supplements retain potency much longer and can be used in combination with minerals in complete premixes. Vitamin D3 is more stable than D2 in feeds containing minerals, probably due to the more reactive side chain of vitamin D2.

Stability of vitamin D in storage is enhanced by (1) excluding air; (2) storing feed under cool, dry, dark conditions; (3) minimizing direct contact of vitamin D with reactive trace minerals such as copper, iron and manganese; (4) including antioxidants in the primary product form; and (5) using stabilized beadlet product forms.

Hirsch (1982) reported the results of a study in which a conventional, nonstabilized vitamin D product was mixed into a trace mineral premix or into a complete feed and stored at an ambient temperature of 68° to 77°F (20° to 25°C) for up to 12 weeks. Mash feed lost 31% of its vitamin D activity after 12 weeks. The trace mineral premix lost 66% of its vitamin D activity after only six weeks in storage.

Naturally occurring sources of vitamin D in feeds must likewise be protected. Poor handling of hay, which can otherwise be an important source of vitamin D for cattle, sheep and goats, can lead to extensive shatter and leaf loss. Leaves are richer in vitamin D than the stem. Animals fed forages harvested or stored under poor conditions are susceptible to vitamin D deficiency if there is no vitamin D supplementation in the diet (Abrams, 1978).

 

Deficiency

Rickets, the primary vitamin D deficiency disease, is a skeletal disorder of young, growing animals generally characterized by decreased concentration of calcium and phosphorus in the organic matrices of cartilage and bone. Vitamin D deficiency results in clinical signs similar to those produced by deficiency of calcium and (or) phosphorus, because all three nutrients are required for proper bone formation. In the adult animal, osteomalacia is the counterpart of rickets. Cartilage growth in the adult has ceased, and thus this condition is characterized by a decreased concentration of calcium and phosphorus in the bone matrix (demineralization). Symptoms of rickets include the following skeletal changes, varying somewhat with species depending on anatomy and severity: (1) weakened long bones, resulting in curvature and deformation; (2) enlarged, painful hock and knee joints; (3) general stiffness of gait, arched back and a tendency to drag hind legs; and (4) beaded ribs and deformed thorax. With osteomalacia, in adults, the progressive demineralization of bones eventually results in fractures and breaks. The disturbance of calcium and phosphorus metabolism produces other symptoms such as reduced performance, hypocalcemia and reproductive failure. Although there appear to be differences among species in the susceptibility of different bones to degenerative changes, as well as differences that probably reflect body conformation (e.g., pigs compared with sheep), there is nevertheless an apparent common pattern in vitamin D deficiency (Maynard et al., n1979). The spongy and cartilaginous parts of individual bones (mainly the bone ends or epiphysis) and bones relatively rich in this tissue, such as the ribcage, are generally the first and most severely affected. As in simple calcium deficiency, the vertebrae and the skull have the greatest degree of demineralization followed by, the scapula, sternum and ribs. The most resistant bones are the metatarsals and shafts of long bones, which would have obvious survival value to the animal. Other clinical signs of vitamin D deficiency in ruminants are decreased appetite and growth rate, digestive disturbances, stiffness of gait, labored breathing, irritability, weakness and occasionally tetany and convulsions. These symptoms may precede the skeletal symptoms (i.e., enlargement of joints, slight arching of the back, bowing of legs, with erosion of joint surface causing difficulty in locomotion) (NRC, 2000). There can be an increase in the birth of dead, weak or deformed calves and lambs.

Grazing animals that have normal exposure to direct sunlight are normally protected from vitamin D deficiency. However, under most conditions of commercial livestock production, where animals have limited exposure to direct sunlight and where rates of growth or milk production are high, dietary supplementation with vitamin D is prudent and recommended.

 

A. Vitamin D Deficiency in Cattle

Vitamin D should be supplied to growing and lactating cattle housed in confinement or with limited sun exposure. In more northern latitudes during winter months, photochemical conversion of provitamin D to its active compound (cholecalciferol) in the skin can be limited because of insufficient ultraviolet radiation. Hidiroglou et al. (1979) reported that 25-(OH)D3 was higher in plasma of cattle during summer than in winter. Richter et al. (1990) found higher concentrations of 25-(OH)D3 in blood plasma when bulls were kept outdoors as compared to indoors: 21.1 vs. 14.3 ng per ml, respectively. The normal range of plasma 25-(OH)D3 in cattle is 20 to 50 ng/ml, and therefore even the bulls exposed to direct sunlight in this experiment appeared to have marginal vitamin D status. Similar relationships have been observed in human populations (Romagnoli et al., 1999), where plasma 25-(OH)D3 was significantly higher in summer than in winter. During winter 15% of young adults, 32% of postmenopausal women and 71% to 82% of hospital inpatients exhibited vitamin D deficiency based on plasma levels of 25-(OH)D3. Clinical signs of vitamin D deficiency in calves involving the skeleton begin with thickening and swelling of the metacarpal or metatarsal bones (Illus. 3-2 and 3-3). As the disease progresses, the forelegs bend forward or sideways. In severe or prolonged vitamin D deficiency, the force exerted by normal muscle tension results in bending and twisting of long bones and the characteristic bone deformity. There is enlargement of bone ends (epiphyses) from deposition of excess cartilage, giving the characteristic “beading” effect along the sternum at the point of attachment of the ribs (NRC, 1996; 2001).

 

Illustration 3-2: Vitamin D Deficiency in Cattle, Rickets

3Illustration_3-2_Vitamin_D_Deficiency_in_Cattle_Rickets

 

Illustration 3-3: Vitamin D Deficiency in Cattle, Rickets

3Illustration_3-3_Vitamin_D_Deficiency_in_Cattle_Rickets

Michigan Agriculture Experiment Station

 

In calves, the mandible becomes thick and soft, and in the worst cases, calves have difficulty eating. In calves so affected, there can be slobbering, inability to close the mouth and protrusion of the tongue (Craig and Davis, 1943). Joints (particularly the knee and hock) become swollen and stiff, the pastern straight and the back arched. In severe cases, synovial fluid accumulates in the joints (NRC, 2001). Posterior paralysis may also occur as the result of fractured vertebrae. The structural weakness of the bones appears to be related to poor mineralization. The advanced stages of the disease are marked by stiffness of gait, dragging of the hind legs, irritability, tetany, labored and rapid breathing, weakness, anorexia and cessation of growth. Calves born to vitamin D-deficient dams may be born dead, weak or deformed (Rupel et al., 1933). In older animals with vitamin D deficiency (osteomalacia), bones become weak and fracture easily, and posterior paralysis may accompany vertebral fractures. For dairy cattle, milk production may be decreased and estrus inhibited by inadequate vitamin D (NRC, 2001). Cows fed a vitamin D-deficient diet and kept out of direct sunlight showed definite signs of vitamin D deficiency within six to 10 months (Wallis, 1944). Functions that deplete vitamin D are high milk production and advancing pregnancy, especially during the last few months before calving. The visible signs of vitamin D deficiency in dairy cows are similar to those of rickets in calves. The animal begins to show stiffness in their limbs and joints, which makes it difficult to walk, lie down and get up. The knees, hocks, and other joints become swollen, tender and stiff. The knees often spring forward, the posterior joints straighten, and the animal is tilted forward on her toes. The hair coat becomes coarse and rough and there is an overall appearance of unthriftiness (Wallis, 1944). As the deficiency advances, the spine and back often become stiff, arched and humped. In deficient herds, calving rates are lower, and calves can be born dead or weak. Hypocalcemia, either milk fever (parturient hypocalcemia) or unexplained lactational hypocalcemia and paresis, may also be observed as a result of chronic vitamin D deficiency in dairy cattle. These signs are also produced by calcium, phosphorus or electrolyte deficiency or imbalances and are therefore not specific to vitamin D deficiency. Milk Fever in Dairy Cattle (Parturient Paresis)

Milk fever (parturient paresis) is a metabolic disease characterized by hypocalcemia at or near parturition in dairy cows. Goff et al. (1991b) and Horst et al. (1994) discussed milk fever and calcium metabolism of dairy cattle in detail. In essence, milk fever is a failure of calcium homeostasis in the face of increased metabolic demand for calcium. Causative and risk factors are partly, but not completely, understood (Enevoldsen, 1993; Horst et al., 1994; Liesegang et al., 1998). Milk fever is related to factors such as (a) previous calcium and phosphorus intakes; (b) previous vitamin D intake; (c) previous intakes and dietary ratios of potassium, chloride, magnesium, sulfur and sodium; and (d) age and breed of cow. Cows that develop milk fever are unable to meet the sudden demand for calcium brought about by the initiation of lactation. Milk fever usually occurs within 72 hours after parturition and is manifested by circulatory collapse, generalized paresis, depression and eventually coma and death. The most obvious and consistent clinical sign is acute hypocalcemia in which serum calcium decreases from a normal 8 to 10 mg to 3 to 7 mg (average 5 mg). Initially a cow may exhibit some unsteadiness of gait. More commonly, the cow is observed lying on her sternum with her head turned sharply toward her flank in a characteristic posture. The eyes are dull and staring, and the pupils fixed and dilated. If treatment is delayed, paresis will progress into coma, which becomes progressively deeper, leading to death. Treatment with intravenous calcium boro-gluconate is an extremely effective treatment. Some cows will relapse, sometimes with multiple episodes of paresis that indicate a severe failure of the calcium regulatory system or in some cases, severe depletion of body calcium stores. Oral calcium pastes and gels are also used both prophylactically and as an adjunct to intravenous calcium treatment. Aged cows are at the greatest risk of developing milk fever. Heifers rarely develop milk fever, which is borne out by their superior calcium status at parturition (Shappell et al., 1987). Jersey cattle are generally more susceptible than Holsteins. Older animals have a decreased response to dietary calcium stress due to both decreased production of 1,25-(OH)2D3 and a decreased responsiveness to the 1,25-(OH)2D3.In older cows, fewer osteoclasts exist to respond to hormonal stimulation, which delays the bone contribution of calcium to the plasma calcium pool (Goff et al., 1989, 1991b). The aging process is also associated with a reduced renal 1-alpha-hydroxylase response to hypocalcemia, therefore, reducing the amount of 1,25-(OH)2D3 produced from 25-(OH)D3 (Goff et al.,1991b; Horst et al., 1994). Tissue receptors for 1,25-(OH)2D3 decline at parturition (Goff et al., 1995), although there was not a significant difference between paretic and nonparetic cows. Osteoblast activity also appears to be decreased during late pregnancy and around parturition (Naito et al., 1990). This may be related to the reduced plasma calcitonin concentrations around parturition and especially in hypocalcemic, aged cows (Shappell et al., 1987). Low magnesium status is also a risk factor for parturient hypocalcemia as well as hypomagnesemia (Van de Braak et al., 1987; Van Moselet al., 1991). Infection with the common brown stomach worm (Ostertagia) has been strongly implicated as a causative agent of milk fever and displaced abomasum in dairy cows (Axelsson, 1991), apparently due to an anaphylactic reaction at parturition.

Parturient paresis can be prevented effectively by feeding a low-calcium and adequate-phosphorus diet for the last several weeks prepartum, followed by a high-calcium diet after calving (Horst et al., 1994). Feeding low-calcium diets prepartum is associated with increased plasma PTH and 1,25-(OH)2D3 concentrations during the peripartum period (Kichura et al., 1982; Green et al., 1981). Green et al. (1981) suggested that the increased PTH and 1,25-(OH)2D3 concentrations resulted in “prepared” and effective intestinal absorption and bone resorption of calcium at parturition that prevents parturient paresis. Phosphorus deficiency did not affect plasma concentrations of vitamin D3 or its 25-OH D3 or 1,25-OH D3 active metabolites, but did elevate plasma calcium and appeared to increase 1,25-(OH)2D3 receptor binding in the duodenum of phosphorus-depleted lactating goats (Schroder et al., 1990).

Prepartal dietary cation-anion balance (DCAD) influences the degree and incidence of milk fever (Ender et al., 1971; Block, 1984; Gaynor et al., 1989; Oetzel et al., 1988; Enevoldsen, 1993). Dietary excess of cations, especially sodium and potassium, relative to anions, primarily chloride and sulfur, tends to induce milk fever, while anionic diets can prevent milk fever. The cation-anion balance of the diet affects the acid-base status of the animal, with cationic diets producing a more alkaline state and anionic diets a more acid state of metabolism. Mild metabolic acidosis, in turn, promotes calcium mobilization and excretion (Lomba et al., 1978; Fredeen et al., 1988; Won et al., 1996). Anionic diets increase the amount of 1,25-(OH)2D3 produced per unit increase in parathyroid hormone (Goff et al. 1991a). Debate remains as to the mechanisms of action and the relative importance of individual mineral ions (Enevoldsen, 1993; Horst et al., 1994).

Used correctly, anionic diets prepare the cow’s metabolism for a sudden demand for calcium at calving and reduce the incidence of subclinical hypocalcemia and paresis (Horst et al., 1994). Because most legumes and grasses are high in potassium, typical dry cow rations are alkaline. Addition of anions, usually as anionic salts, to the diet for two to four weeks prepartum has been used successfully to reduce the incidence of milk fever. Goff et al. (1991a) concluded that low calcium diets, anionic diets and PTH administration all increase renal 1-alpha hydroxylase activity, resulting in increased production of 1,25-(OH)2D3 and prevention of milk fever. Increased plasma 1,25-(OH)2D3 concentration in response to feeding acidified diets prepartum was reported by Phillippo et al. (1994).

 

Supplemental vitamin D had been used to prevent parturient paresis in dairy cows for a number of years (Hibbs and Conrad, 1976, 1983; Littledike and Horst, 1982). Feeding or injecting massive doses of vitamin D has been an effective preventive of milk fever, but toxicity symptoms and death have occurred as well. In some cows, milk fever has been induced by the treatment. Due to the toxicity of vitamin D3 in pregnant cows and the low margin of safety between vitamin D3 doses that prevent milk fever and those that induce milk fever, Littledike and Horst (1982) concluded that injecting vitamin D3 prepartum is not a practical solution to milk fever. However, more recent reports from the same laboratory have provided data suggesting that injection of 24-F-1,25-(OH)2D (fluoridation at the 24 position) delivered at seven-day intervals prior to parturition can effectively reduce incidence of parturient paresis (Goff et al., 1988).

Feeding high doses of vitamin D has been more successful than parental administration in preventing milk fever without inducing toxicity (Hibbs and Conrad, 1976). Feeding 20 million to 30 million IU of vitamin D2 for three to eight days prepartum prevented 80% of expected milk fever cases in aged, Jersey cows (Hibbs and Conrad, 1976). However, prolonging the treatment to 20 days prepartum has resulted in toxicity. The same authors fed cows 100,000 to 580,000 IU vitamin D2 per day on a continuous, year-round basis and reported a reduction in milk fever in cows with a history of the disease, but not in cows without a history of milk fever. The most practical approach to controlling milk fever appears to be through optimizing macro-mineral levels in the diet use of anionic diets, and providing continuous supplementation with vitamin D at normal levels.

 

B. Vitamin D Deficiency in Sheep and Goats

Clinical signs of vitamin D deficiency in sheep and goats are similar to those of cattle, including rickets in young animals and osteomalacia in adults (NRC, 1981; 1985, 2007b) (Illus. 3-4). An early report of rickets in Scotland referred to the condition as “bent leg,” which occurred in ram lambs seven to 12 months of age (Elliot and Crichton, 1926). The condition was prevented by administration of small amounts of vitamin D in the form of cod liver oil. Newborn lambs can receive enough vitamin D from their dams to prevent early rickets if the ewes have adequate vitamin D status (Church and Pond, 1974). Newborn kids develop rickets if the doe is fed a diet deficient in vitamin D during pregnancy (NRC, 1981). 

 

Illustration 3-4: Vitamin D Deficiency in Sheep, Rickets

3Illustration_3-4_Vitamin_D_Deficiency_in_Sheep_Rickets

Bilateral bent leg in yearling Rambouillet ram. Condition due to vitamin D deficiency.

Utah Agricultural Experiment Station, NRC, 1968

 

Vitamin D deficiency has been observed in young lambs or goat kids kept in complete confinement without access to sun-cured roughage. Hidiroglou et al. (1978) reported the clinical history of a flock of sheep kept in total confinement that showed a high incidence (8%) of an osteodystrophic condition which was vitamin D-responsive. A form of osteodystrophia has also been produced experimentally in goats (NRC, 1981). Parturient paresis occurs in ewes. It is a disturbance of calcium metabolism in pregnant and lactating ewes and is characterized by acute hypocalcemia and the rapid development of hyperexcitability, ataxia, paresis, coma and death. The disease occurs any time from five weeks before to 10 weeks after lambing, principally in overconditioned, older ewes at pasture. The onset is often triggered by abrupt changes in diet, sudden weather changes or periods of stress and fasting imposed by circumstances such as shearing or transportation. The extent of involvement of vitamin D in calcium metabolism in paretic ewes is unclear. Maternal calcium homeostasis is under stress in pregnant ewes (Paulson and Langman, 1990). Sheep differ from all other species studied thus far in that pregnancy does not result in an increase in circulating levels of 1,25-(OH)2D3(Paulson and Langman, 1990). The signs (e.g., hyperexcitability) and conditions under which this disorder occur (lush pasture, stressful events) suggest that hypomagnesemia is also involved.

 

Fortification Considerations

For ruminants in confinement in climates where the sunlight is not adequate for vitamin D production, the vitamin D content of the diet becomes important. In modern dairy operations, cattle are often completely or nearly completely confined without significant direct exposure to sunlight. These conditions, combined with higher levels of production and stress, warrant that supplemental vitamin D be provided. Cattle grazing during spring and summer months will obtain significant vitamin D from sun exposure. During fall and winter, however, sun exposure is reduced to levels that can result in marginal vitamin D status. This is a concern with both dairy and beef cows pastured during the last 60 to 90 days of pregnancy. In northern latitudes, it is unsafe to rely on exposure to sunlight to provide adequate vitamin D, at least during the six months of shortest day length (September 21 – March 21 in the Northern Hemisphere). Milk is not an especially rich source of vitamin D, but the calf, kid or lamb can obtain adequate amounts by skin irradiation if exposed to sunlight for 1 or 2 hours per day. Sun-cured forage is the best natural source of vitamin D, although the vitamin D activity of forage is variable. Due to these uncertainties and the value of replacement animals, it is prudent to provide supplemental vitamin D to young calves, lambs or kids in milk replacer and starter diets. Dairy cows fed diets high in cations, especially potassium and sodium, before calving are at increased risk of developing hypocalcemia and milk fever (parturient paresis) (Goff et al., 2004). Cows fed diets high in cations have a reduced ability to mobilize bone calcium and reduced ability to produce the hormonal form of vitamin D, 1,25-(OH)2D3 (Goff, 2000). To prevent milk fever in cows, researchers often recommended feeding or injecting pharmacological doses of vitamin D two weeks before calving. This practice increases intestinal absorption of calcium and helps prevent milk fever, but the recommended dose (up to 10 million units per day) of vitamin D is very close to the amount that causes irreversible metastatic calcification of soft tissue (Littledike et al., 1986). Hodnett et al. (1992) used a combination of 25-(OH)D3 plus 1α-hydroxycholecalciferol to reduce partiurient paresis in dairy cows fed high dietary Ca. The incidence of the disease was reduced from 33% to 8%. The 25-(OH)D is a more potent form of vitamin D, as serum 25-(OH)D3 was greater for cows dosed with 25-(OH)D3(119.0 ng/mL) compared with those dosed with vitamin D3 (77.5 ng/mL) or the controls (Taylor et al., 2008). The hypothesis of Taylor et al. (2008) is that dosing preparturent cows with exogenous 25-(OH)D3 days before parturition would allow calcium homeostatic mechanisms to be active at parturition. Presently the most common procedure for milk fever prevention is to provide acid ingredients (anions), particularly chlorine and sulfur, to prepartum diets. Diets higher in anions increase osteoclastic bone resorption and synthesis of 1,25-(OH)2D3 in cows, thereby increasing blood calcium and thus preventing milk fever. Due to lack of vitamin D in feeds and management systems without direct sunlight, modern ruminant livestock operations must provide a supplemental source of the vitamin. A commercially available vitamin product containing stabilized, high-purity vitamin D or the more potent 25-(OH)D3 for feed or drinking water use should be used to assure the vitamin D levels needed to prevent deficiency and allow optimum performance in ruminants.

Pure vitamin D3 crystals or vitamin D3 resin is very susceptible to degradation upon 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 D2 in feeds containing minerals.

Stabilization of the vitamin can be achieved by: (a) rapid compression of the mixed feed, for example, into pellets so that air is excluded; (b) storing feed under cool, dry, dark conditions; (c) preventing close contact between the vitamin and potent metallic oxidation catalysts, such as manganese; and (d) including 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 non-stabilized 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.

Naturally occurring sources of vitamin D in feeds must also be protected from loss. Poor handling of hay, which can otherwise be an important source of vitamin D and various other nutrients for cattle, sheep and goats, can lead to extensive fragmentation and loss of the leaves, which is much richer in vitamin D than the stem. Animals fed on grain, silage and hay that is poorly produced or kept are extremely liable to have dietary deficiency of the vitamin (Abrams, 1978).

The cost of vitamin D supplementation in livestock diets is nominal (Rowland, 1982). In contrast, the cost of inadequate vitamin D supplementation is very high. For optimum animal performance, vitamin D levels in feeds and mineral supplements should be adjusted to provide a margin of safety needed to offset the factors influencing the vitamin D needs of the animal and effects of storage. Factors that increase the amount of vitamin D required to optimize animal productivity are often not reflected in the NRC minimum nutrient requirements. Consequently, many nutritionists use greater than the minimum levels of vitamin D in feeds. Successful nutrition programs may greatly exceed the NRC minimum vitamin D requirement. However, no amount of vitamin D can compensate for lack of enough calcium or phosphorus in the diet.

Vitamin D deficiencies may result from: (1) errors in addition of the vitamin to diets; (2) inadequate mixing and distribution in feed; (3) separation of vitamin D particles after mixing; (4) instability of the vitamin D content of the supplement; or (5) excessive length of storage of feeds under environmental conditions causing vitamin D loss (Hirsch, 1982).

Supplementation considerations are also dependent on other dietary ingredients. The vitamin D requirements are increased several fold by inadequate levels of calcium and (or) phosphorus or by improper calcium:phosphorus ratios in the diet. Several reports have indicated that molds in feeds interfere with vitamin D metabolism (Cunha, 1987). For example, a toxin produced by the mold Fusarium roseum interferes with vitamin D3 absorption from the intestinal tract of the chick. Similar deleterious effects of mycotoxins on vitamin D metabolism may occur in other species, including ruminants.

Other factors that influence vitamin D status are diseases of the endocrine system, intestinal, liver or kidney disorders, and certain drugs. Liver or kidney disease or degeneration can limit production of the active form of vitamin D3 [1,25-(OH)2D3]. Enteric disease or heavy infestation by internal parasites can reduce absorption of vitamin D and the other fat-soluble vitamins. Unknown factors in feeds, possibly other mycotoxins, may increase vitamin D requirements. For example, there is evidence of a factor in rye grain and soybeans that interferes with vitamin D absorption from the intestine (MacAuliffe and McGinnis, 1976).

Optimum levels of vitamin D recommended for ruminants are shown in Table 3-3. Although 15,000 IU vitamin D per day appeared adequate in lactating cows (Reeve et al., 1982), improvements in milk yield (Hibbs and Conrad, 1983) and reproduction (Ward et al., 1971) have been recorded when higher levels, up to 50,000 IU per day, were fed to lactating cows (Weiss, 1998). Given these data and the uncertainties of sun exposure and rumen metabolism on vitamin D, the levels indicated are believed to represent a safe optimum.

3Table_3-3_DSM_Guidelines_for_Optimum_Vitamin_Nutrition

Tenderness has been identified as the single most important palatability factor affecting consumer satisfaction of beef. A number of studies have evaluated supplemental dietary vitamin D as a method to improve meat tenderness. Vitamin D plays a vital role in maintaining blood concentrations of calcium. The major effect of supplementing high levels of vitamin D is an increased calcium absorption and the release of calcium from bone stores (Littledike and Goff, 1987). Elevated calcium triggers the tenderizing process by activating postmortem muscle enzymes (proteases/calpains). Raising cattle’s blood calcium 20-30% by feeding the animals extra vitamin D, beginning 2-3 days before slaughter, resulted in an increase in muscle calcium and more tender cuts of meat (Montgomery et al., 2002). Dietary vitamin D3improves tenderness of beef from younger cattle by accelerating the postmortem aging process (Swanek et al., 1999; Montgomery et al., 2000, 2002; Karges et al., 2001). Electron microscopy visualization of bound calcium indicated that vitamin D3 mobilized calcium from the sarcoplasmic reticulum and transverse tubule system into the myofibrils (Montgomery et al., 2004a). Feeding large doses of vitamin D3 to beef steers results in substantial vitamin D3 and 25-(OH)D3 residues in muscle and plasma. A 24-fold increase in consumption of steak could meet human requirements for vitamin D, however, excess vitamin D can cause soft tissue and arterial calcification. Supplementation with 25-(OH)D3 versus vitamin D3 increased postmortem proteolysis and tenderness in muscle, without accumulation of large concentrations of residual vitamin D3 and its primary metabolite 25-(OH)D3 in beef (Foote et al., 2004).

Researchers have demonstrated that feeding a supranatural dosage (0.5 to 7.5 million IU) of vitamin D3 or 25-(OH)D3 to beef cattle for 7 to 10 days before slaughter resulted in more tender beef (Swanek et al., 1999; Montgomery et al.,2000, 2002, 2004a, b, c; Karges et al., 2001; Foote et al., 2004; Gutierrez et al., 2007). Contrary to beef (Scanga et al., 2001; Rider Sell et al., 2004; Wertz et al., 2004; Cho et al., 2006), lamb (Wiegand et al., 2001) and pork (Wilbornet al., 2004) show no benefit on tenderness with vitamin D3 (or 25-(OH)D3) at the levels and durations of their studies. Cho et al. (2006) found no effect on tenderness in beef, but suggested in their experiment that the amount of 25-(OH)D3 administered was insufficient. Feeding vitamin D3 (or 25-(OH)D3) to increase muscle calcium seems to be a cost-effective means of improving beef tenderness (Karges et al., 2001; Montgomery et al., 2002). Intramuscular collagen contributes to the variation in background toughness in meat.   Vitamin E may reduce intramuscular collagen maturity (Archile et al., 2010). Carnagey et al. (2008) fed both 25-(OH)D3 (500 mg) and 1,000 IU of vitamin E for 104 days to cattle to evaluate beef tenderness. Both nutrients were effective in improving tenderness (Warner-Bratzler shear force) when fed alone, but not in combination.

Typically, vitamin D is supplemented in the diet or injected. Variations in dry matter intake may lead to less than optimal doses of the vitamin being consumed. There are also concerns regarding injection site lesions (Roeber et al., 2002). Transmucosal delivery by the buccal route (side of the oral cavity toward the cheek) is an effective means of delivering various human medications. Rivera et al. (2005) reported that buccal administration of 100 and 1000 mg 25-(OH)D3 increased vitamin D3 metabolites in serum and tissues, and it should be an effective method of delivering the vitamin. Moreover, Okura et al. (2004) recently demonstrated effective absorption of 1,25-(OH)2D3 after vaginal administration in Holstein heifers.

 

Vitamin Safety

Aside from vitamin A, vitamin D is the most likely cause of vitamin toxicity in livestock. Although vitamin D is toxic at high concentrations, short-term administration of as much as 100 times the requirement level can be tolerated. For most species, the presumed maximal safe level of vitamin D3 for long-term feeding conditions (more than 60 days) is four to 10 times the dietary requirement. For cattle and sheep, the upper safe dietary level for short-term exposure is 25,000 IU per kg (11,364 IU per lb) of diet, and for over 60 days, it is 2,200 IU per kg (1,000 IU per lb) of diet (NRC, 1987). Studies in a number of species, including ruminants, indicate that vitamin D3 is 10 to 20 times more toxic than vitamin D2when provided in excess amounts (NRC, 1987). Excessive intake of vitamin D produces a variety of effects associated with hypercalcemia. Serum calcium concentration is elevated due to increases in bone resorption and intestinal absorption of calcium. The main pathological effect of vitamin D toxicity is widespread calcification of soft tissues. Pathological changes in these tissues include inflammation, cellular degeneration and progressive calcification. Diffuse calcification affects joints, synovial membranes, kidneys, myocardium, pulmonary alveoli, parathyroid glands, pancreas, lymph nodes, arteries, conjunctivae and cornea. In advanced cases, cartilage growth is disrupted. As a result, the skeletal system undergoes demineralization that results in loss of bone mass and strength. Other common observations of vitamin D toxicity are anorexia, extensive weight loss, brachycardia, reduced rumination, depression, polyuria, muscular weakness, joint pain and stiffness, elevated blood calcium and lowered blood phosphate concentrations. Vitamin D toxicity in neonatal lambs may be characterized by clinical signs consisting solely of unthriftiness and weakness or an inability to stand (Roberson et al., 2000). Cows receiving 30 million IU of vitamin D2 orally for 11 days developed anorexia, reduced rumination, depression, premature ventricular systoles and brachycardia (NRC, 1987). Littledike and Horst (1982) reported that moderate toxicity was characterized by delayed shedding of the winter hair coat; rough, dry hair coat; poor appetite and milk production; muscle and joint stiffness; excessive thirst; and air pockets under the skin of the neck and back with crepitation (crackling) of these areas. The same authors reported that severe toxicity resulted in pasty ocular discharge, flaccid udder, labored breathing, rapid, pounding pulse, fever, ketosis, severe anorexia and death. Adipose tissue may provide some buffering effect against vitamin D toxicity. Brouwer et al. (1998) reported that in rats given large oral doses of vitamin D3, adipose tissue accumulates and slowly releases vitamin D3, thus mitigating the increase in plasma 25-(OH)D3 and calcium.Vitamin D toxicity is enhanced by increased supplies of dietary calcium and phosphorus and is reduced when the diet is low in calcium. Route of administration also influences toxicity. Parenteral administration of 15 million IU of vitamin D3 in a single dose caused toxicity and death in 71% of pregnant dairy cows (Littledike and Horst, 1982). On the other hand, oral administration of 20 to 30 million IU of vitamin D2 daily for seven days resulted in little or no toxicity in pregnant dairy cows (Hibbs and Pounden, 1955). Rumen microbes are capable of metabolizing vitamin D to the inactive 10-keto-19-nor vitamin D. This may partially explain the difference in toxicity between oral and parenteral vitamin D. The toxic dose of vitamin D is variable, with an important factor being duration of intake, since this is a cumulative toxicity. Pregnant cows are more susceptible to vitamin D toxicity than nonpregnant cows, possibly due to placental production of 1,25-(OH)2D3 (Littledike and Horst, 1982; DeLuca, 1992).Although it is usually assumed that living plants do not contain vitamin D2, certain plants contain compounds that have vitamin D activity. Grazing animals in several parts of the world develop calcinosis, a disease characterized by deposition of calcium salts in soft tissues (Carrillo, 1973; Morris, 1982). Ingestion of leaves of the shrub Solanum malacoxylon by grazing animals causes enzootic calcinosis in Argentina and Brazil, where the disease is referred to as “enteque seco” and “espichamento,” respectively. Consumption of as few as 50 fresh leaves per day (200 g of fresh leaves per week) over a period of eight to 20 weeks will produce this toxicity disease in cows (Illus. 3-5) (Okada et al., 1977). The calcinogenic factor in S. malacoxylon is a water-soluble glycoside of 1,25-(OH)2D (Wasserman, 1975). The sterol is released during digestion, which results in a massive increase in the absorption of dietary calcium and phosphorus such that normal physiological processes are unable to compensate and soft tissue calcification results. Other plants that cause calcinosis in grazing animals are also reported in the alpine regions in Europe, New Guinea, Florida, Hawaii, Australia and Jamaica (McDowell, 2000).

 

Illustration 3-5: Vitamin D Toxicity in Cattle

3Illustration_3-5_Vitamin_D_Toxicity_in_Cattle

Courtesy of Bernardo Jorge Carillo

 

During development of plant-induced calcinosis diseases, destruction of connective tissues precedes tissue mineralization in which calcium, phosphorus and magnesium are involved. Clinical signs of the disease are a stiff and painful gait and progressive loss of weight. If animals are removed in the early stages from the affected areas, they recover quickly, but mortality is high after prolonged exposure. In advanced cases, joints cannot be extended completely, and animals tend to walk with an arched back, carrying their weight on the forepart of the hooves.

It is clear that the calcinogenic plants are economically important, due to losses in meat and milk production (Morris, 1982). In some fields in Argentina, between 10% and 30% of cattle show signs of “enteque seco,” and S. malacoxylonis now regarded as one of the most important poisonous plants of that country. Other than these examples, most natural feedstuffs do not contain high enough levels of vitamin D to cause toxicity. Marine fish oils are a rich source of vitamin D, but amounts present in livestock diets are not high enough to be of concern. In practice, vitamin D toxicity is unlikely