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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 skin (in most species), the intestinal wall and other tissues. Vitamin D precursors have no antirachitic activity.

 

Illustration 3-1: Vitamin D Deficiency, Soft Beaks

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 D3 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 D3 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 D3 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 D3is 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 photochemically produced by 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 D 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 D 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 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). Heuser and Norris (1929) showed that 11 to 45 minutes of sunshine daily were sufficient to prevent rickets in growing chicks and that no further improvements in growth were obtained under these conditions by adding cod liver oil (a rich source of vitamin D).

Previously it was believed that part 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. Recently, however, it was shown in cattle that vitamin D3 synthesis takes place in all areas of the skin and is not exclusively associated with skin areas where hair coverage is scant or lacking (e.g. udder and muzzle) (Hymoller and Jensen, 2010). Vitamin D undergoes a multiple series of transformations and multi-site interactions in the living system (Deluca, 1979). 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. 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)D]. 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)D 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)2D] (DeLuca, 2008). Although the kidney is the main site of l-hydroxylation, other organs can also form 1,25(OH)2D3 including the placenta (Johnson, 2006; 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 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

3Figure_3-1_The_Functional_Metabolism_of_Vitamin_D3

One question that is still unanswered is whether the hormone form 1,25(OH)2 D3 acts alone or is there some response from a second vitamin D metabolite or hormone [e.g 24, 25 (OH)2 D3] (Feldman et al., 2005). However, this question was probably answered in a study where the 24-position of 25-(OH)2D3 was blocked with fluoro groups to prevent 24-hydroxylation (DeLuca, 2008). For two generations all systems were normal, indicating a need for only 1,25(OH)2 D3. Therefore research indicates that 1,25-(OH)2 D3appears to be the only functional form of vitamin D in biology (DeLuca, 2008).Production of 1,25-(OH)2D 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)D to 1,25-(OH)2D, and inactivates renal and extrarenal 24- and 23-hydroxylases, which convert the 25-OHD [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)2D 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)2D3can be modulated according to the calcium needs of the organism (Collins and Norman, 1991).For most mammals, vitamin D, 25-(OH)D, and possibly 24,25-(OH)2D3 and 1,25-(OH)2D 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 in feces with the aid of bile salts.

For mammals, 1,25-(OH)2D 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. It is now realized that vitamin D is not only important for mineralization and skeletal growth but has many other roles in regulation of the parathyroid gland, in the immune system, in skin, cancer prevention, in metabolism of foreign chemicals and in cellular development and differentiation. There is a regulatory role of vitamin D (1,25-(OH)2D) in immune cell functions (Reinhardt and Hustmeyer, 1987), the release of insulin in relation to glucose challenge (DeLuca, 2008), 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)2D to control blood calcium and phosphorus levels (Engstrom and Littledike, 1986; McDowell, 2000). Production rate of 1,25-(OH)2D 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 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)2D regulates gene expression through its binding to tissue-specific receptors and subsequent interaction between the bound receptor and the DNA (Collins and Norman, 1991; Norman and Henry, 2007). 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)2D-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)2D. 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)2D 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)2D 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)2D is transferred to the nucleus of the intestinal cell, where it interacts with the chromatin material. In response to the 1,25-(OH)2D, 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)2D 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). 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)2D, 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 is 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)2D 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. More recent research indicates that vitamin D has important functions in addition to mineralization and skeletal growth. The first evidence of non-calcium and phosphorus related activities of the vitamin D hormone was the demonstration of its receptor in tissue not related to bone metabolism. Many roles have been identified for vitamin D; in regulation of the parathyroid gland, in the immune system, in skin, in cancer prevention and in cellular development and differentiation.

A receptor for the active metabolite 1,25-(OH)2D 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)2D has additional functions in a wide variety of cells, glands and organs (Machlin and Sauberlich, 1994; DeLuca, 2008). To date, more than 50 genes have been reported to be transcriptionally regulated by 1,25-(OH)2D (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)2D 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)2D 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)2D 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)2D as an immunoregulatory hormone. Aslam et al. (1998) reported that vitamin D deficiency depresses the cellular immune responses in young broiler chicks. Turkey osteomyelitis, a disease that affects commercially processed turkeys, incidence in E. coli-challenged birds was decreased with vitamin D metabolites (Huff et al., 2002). Elevated 1,25-(OH)2D was associated with a significant 70% enhancement of lymphocyte proliferation in cells treated with pokeweed mitogen (Hustmeyer et al., 1994). 1,25-(OH)2D also inhibits growth of certain malignant cell types and promotes their differentiation (Colston et al., 1981; DeLuca, 1992). 1,25-(OH)2D has been reported to inhibit proliferation of leukemic cells (Pakkla et al., 1995), breast cancer cells (Vink van Wijngaarden et al., 1995) , colorectal cells (Cross et al., 1995) and to produce cells that suppress inflammation (Cantorna, 2006). A deficiency of vitamin D may promote prostate cancer (Skowronski et al., 1995). Also, 1,25-(OH)2D 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

The vitamin D3 requirements listed in NRC (1994) are 200 IU per kg (90.9 IU per lb) of diet for broilers, geese and leghorn classes 0 to 18 weeks. Note: ICU (International Chick Units) and IU (International Units) are considered equal for Vitamin D3, but not equal for vitamin D2. Higher levels of D3 (IU per kg; IU per lb) are required for leghorns that are in prelay (18 weeks to first egg), laying (assuming 100 g feed intake per day) and breeding (300; 136.4) and for turkeys (1,100; 500), ducks (400; 181.8) and Japanese quail (750; 340.9). Edwards (1999) suggested different quantitative requirements for vitamin D3 in broilers to maximize responses to different criteria. The vitamin D3requirement was 275 ICU per kg (125 ICU per lb) for growth, 503 ICU per kg (228.6 ICU per lb) for bone ash, 552 ICU per kg (250.9 ICU per lb) for blood plasma calcium and 904 ICU (410.9 ICU per lb) for rickets prevention. Increasing dietary levels of calcium up to 4% with a level of 4,000 IU per kg (1818 IU per lb) vitamin D3improves egg shell quality of hens without adverse effects on laying performance (Ed-Maksoud, 2010) Based on a vitamin D supplementation trial, Atencio et al. (2004) suggested that 2,800 to 3,000 IU per kg (1,273 to 1,364 IU per lb) of vitamin D3 is required for broiler breeders. However, for almost total prevention of tibial dyschondroplasia, Whitehead et al. (2004) suggested 10,000 IU per kg (4,545 IU per lb) vitamin D3in the diet. It has generally been assumed that 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 (vitamin D2) of the vitamin should not be relied on to provide sufficient vitamin D for these species. Poultry breed and age influence the requirements of vitamin D3. Different broiler strains (Elliot and Edwards, 1994) and turkey breeds (Dudley-Cash, 1999) have responded differently to leg problems with vitamin D3 supplementation.

In poultry there is a genetic foundation for incidence of leg abnormalities (Talaty et al., 2009). A comparison of four different crosses of commercial broilers demonstrated different vulnerabilities in the incidence of tibial dyschondroplasia and walking ability, implicating genotype as a contributing factor. Talaty et al. (2009) showed that purebred lines of meat-type chickens expressed large differences in bone traits, suggesting the potential to genetically select birds for increased bone mineral content.

Soares et al. (1976) concluded that, with age, there appears to be a progressive deterioration in the ability of the hen’s liver to hydroxylate vitamin D3 to 25-hydroxyvitamin D3. Stevens and Blair (1987) stated that reduced hydroxylation of vitamin D3 in the liver or kidney could result in inadequate production of 1,25-(OH)2D3 for maximum absorption of calcium and phosphorus for bone formation. The laying hen is able to obtain 1,25-(OH)2D3 through metabolism of dietary vitamin D3 sources when fed sufficient amounts to maintain normal production and eggshell quality. There is evidence, however, that the hormone is not produced from vitamin D3 at levels high enough to support or maintain tibia weight and perhaps tibia strength in the aged hen.

 

In addition to sunlight, other factors influencing dietary vitamin D requirements include (1) amount and ratio of dietary calcium and phosphorus, (2) availability of phosphorus and calcium, (3) species breed and age, and (4) physiological 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 D in skin since glass does not allow penetration of ultraviolet (UV) rays which need to reach the body. Poultry housed indoors for much or all of the year must depend on their feed for the vitamin D they need.

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. Species differences can be illustrated by the fact that adequate intakes of calcium and phosphorus in a diet that contains enough vitamin D to produce normal bone in the rat or pig will quickly cause the development of rickets in chicks. 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 dietary vitamin D or irradiation can overcome some of this interference. Phosphorus absorption is less dependent on vitamin D intake, with the inefficient absorption in rickets being secondary to failure of calcium absorption. The improvement, of rickets upon vitamin administration is a result of improved calcium absorption.

The need for vitamin D depends to a large extent on the ratio of calcium to phosphorus. The vitamin D needs of poultry are increased several fold by inadequate levels of calcium and (or) phosphorus or by improper ratios of these minerals in the diet. 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. For poultry, the optimum dietary ratio of calcium:inorganic phosphorus is approximately 2:1. The consensus opinion of NRC (1994) was that 5 µg per kg (2.3 µg per lb) was a suitable D3 requirement for chicks between hatching and 21 days of age when diets contained adequate levels of calcium and available phosphorus. However, Baker et al. (1998) reported that chicks fed diets that are severely deficient in available phosphorus continue to respond to D3 in excess of 37.5 µg per kg (17.0 µg per lb).

Phosphorus of inorganic orthophosphate tends to be well absorbed, other factors being favorable, while that of phytic acid, which is the predominant phosphorus compound of unprocessed cereal grains and oilseeds, is poorly available to poultry. Supplemental dietary microbial phytase has been shown to increase the availability of phytate phosphorus for poultry and pigs fed a commercial corn-soybean meal diet. Poultry diets supplemented with microbial phytase had increased digestibility and availability not only of phytate bound phosphorus, but also of calcium magnesium, zinc, manganese and copper (Singh, 2008). It has also been shown to increase ileal digestibility of crude protein and amino acids.

The phosphorus equivalency of microbial phytase for 1 g of nonphytate phosphorus is reported to be 650 to 750 units of phytase in broilers (Schoner et al., 1991; Kornegay et al., 1996; Yi et al., 1996), and 520 to 700 U of phytase in turkey poults (Qian et al., 1996, 1997; Ravindran et al., 1995). Kornegay et al. (1996) suggested that phytase, D3, and the calcium:phosphorus ratio are important factors in degrading phytate and improving phytate phosphorus and calcium utilization in broilers. Results show that supplemental phytase improved body weight gain, feed intake, toe ash content, and calcium and phosphorus retention of broilers fed a corn-soybean based diet. These improvements were negatively influenced by wide calcium:phosphorus ratios, and positively influenced by higher levels of D3. High levels of D3 added to the diets resulted in an increase in the retention of phosphorus and calcium, which seemed independent of supplemental phytase. Maximum responses to supplemental phytase were achieved when broiler chicks were fed diets with 600 to 900 U of phytase per kg (272.75 to 409.1 U per lb) of diet with dietary calcium:phosphorus ratios of 1.1:1 to 1.4:1, and a D3 level of 660 µg per kg (300 µg per lb) of diet.

Apparently, there is a competitive interaction between vitamins A and D3 and vitamins E and D3. Poults that were fed a diet containing the NRC recommended level of vitamin D3 and a high level of vitamin A developed hypocalcemia and rickets (Muirhead, 1987). An extremely high level of vitamin A (45,000 IU per kg or 20,454 IU per lb) fed to broiler chicks receiving diets ranging from 0 to 3,200 IU per kg (0 to 1,454.5 IU per lb) of supplemental vitamin D3, decreased body weight, bone ash, and plasma calcium levels while increasing the incidence and severity of rickets (Aburto et al., 1998). Supplementation with high levels of either 25-(OH)D3 or 1,25-(OH)2D3 overcame the toxic effects of the excess vitamin A. Similarly, supplementing chicks and turkey poults with extra vitamin D3 partially overcame the effects of vitamin A toxicosis as measured by growth and skeletal abnormalities (Veltmann et al., 1987). The mechanism of vitamin A toxicosis, suggested in work with rats, is that excess vitamin A decreases bioactive serum parathyroid hormone (PTH) and 1,25-(OH)2D3 (Frankel et al., 1986). Supplementing a moderate level of vitamin E (150 IU per kg or 68.2 IU per lb) did not aggravate a mild cholecalciferol deficiency induced by feeding 75 IU of vitamin D3 per kg (34.1 IU per lb) (Bartov, 1997). However, exceptionally high levels of either vitamin A (>80,000 IU per kg or 36,364 IU per lb) or vitamin E (10,000 IU per kg or 4,545 IU per lb) will limit utilization of low supplemental levels of vitamin D3 if chicks have no exposure to UV light (Aburto and Britton, 1998a).

It is not clear that the feeding of any vitamin D3 metabolite will alter eggshell quality or egg specific gravity. While a few reports indicate a benefit of feeding 1,25-(OH)2D3 (Tsang et al., 1990; Tsang and Grunder, 1993), other workers were unable to observe any difference between vitamin D forms (Frost et al., 1990). Keshavarz (1996) observed an increase in the number of cracked eggs and (or) poor shell quality when supplemental vitamin D3 levels were below 2,000 IU per kg (909.1 IU per lb). Thus, it appeared levels of vitamin D3 equal to or above this level were needed to optimize shell quality in a large-egg type hen. On a positive note, the effect of dietary 1,25-(OH)2D3 on eggshell strength in older hens was evaluated. Within three weeks, the percentage of cracked or broken eggs was lower for 1,25-(OH)2D3 supplemented hens (Tsang, 1992).

 

Sources

Sources of vitamin D are feedstuff, irradiation, sebaceous material licked from skin or hair or directly absorbed products or irradiation formed on or in the skin. For dogs and cats (and presumably other carnivores), vitamin D must be obtained from dietary sources due to the inability of these species to synthesize and utilize vitamin D from precursors in the skin (How et al., 1995).

For grazing livestock in the presence of UV light, no dietary sources of vitamin D are required. The distribution of vitamin D is very limited in nature, although provitamins occur widely. Grains, roots and oilseeds and their numerous by-products for livestock feeds 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). When various plants, especially pasture species, begin to die and the fading leaves are exposed to UV light, some vitamin D2 is formed, producing vitamin D activity in hay. Its potency depends on local climatic conditions. If produced very quickly in the absence of direct sunlight and baled when still quite green, its potency will be low (Abrams, 1952). 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 (McDowell, 2000). 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).

Artificially dried and barn-cured hay contains less vitamin D than hay that is properly sun cured. Even hay dried in the dark immediately after cutting has some of the vitamin present. This is because the dead or injured leaves on the growing plant are responsive to UV irradiation even though the living tissues are not. This is also largely responsible for the vitamin D found in corn silage (Maynard et al., 1979). Under normal conditions even wilted legume silage furnishes ample vitamin D for livestock. With a few notable exceptions, vitamin D3 is not found in plants. These exceptions include the species Solanum malacoxylon, Cestrum diurnum and Trisetum flavescens (see section on vitamin safety) in which Vitamin D occurs as water-soluble beta-glycosides of vitamin D3, 25-OHD3 and 1,25-(OH)2D.

For concentrate feed mixtures, the vitamin D that occurs naturally in unfortified feed is generally derived from animal products. Saltwater fish, such as fish liver oils, are extremely rich sources. The probable origin of vitamin D in fish liver is a result of the plankton-based food chain. (Takeuchi et al., 1991). Milk contains a variable amount in its fat fraction (5 to 40 IU in cow’s milk per quart), but neither cow’s milk nor human milk contains enough to protect the newborn against rickets (Maynard et al., 1979). Cow’s milk is reportedly higher in vitamin D when produced during the summer compared to the winter.

It has generally been assumed that for all but a few species, vitamin D2 and D3 are equally potent. 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. Recent evidence indicates that in man, vitamin D2has only 25-30 percent of the biological activity of vitamin D3 (Armas et al., 2004).

The dogma that mammals (other than the New Worldmonkeys) do not discriminate between vitamin D2 and D3 has proven incorrect. Data for the pig (Horst and Napoli, 1981) and for ruminants (Sommerfeldt et al., 1981) suggests that these species discriminate in the metabolism of vitamin D2 and D3, with vitamin D3 bring the preferred substrate. Pigs given oral doses of a mixture of vitamin D2 and D3 (1:w/w) had significantly higher concentrations of plasma vitamin D3, 25- OHD3, 24, 25-(OH)2D3 and 1,25-(OH)2D3, than corresponding vitamin D² counterparts. Sommerfeldt et al. (1983) reported that the amount of 1,25-(OH)2D in the plasma of ergocalciferol-treated calves was one-half to one-fourth the amount of the cholecalciferol-treated calves. Although the recent data suggests a preference for D3 by a number of animals, in practice D2 is still relatively comparable to D3 in antirachitic function except for poultry and certain monkeys.

Vitamin D3 is the principal source of supplemental vitamin D for livestock and poultry. Vitamin D3 product forms for feed include stabilized gelatin beadlets (with vitamin A), oil dilutions, oil absorbates, emulsions, and spray- and drum-dried powders. Test results have shown that the gelatin beadlets offer the greatest vitamin D3 stability. Incorporating vitamins D3 and A in the beadlet form provides physical protection from oxidation, and the selected antioxidants included in the beadlets afford chemical protection.

Due to lack of vitamin D in feeds and management systems without direct sunlight, modern poultry operations must provide a supplemental source of the vitamin. The dietary limitation of vitamin D is illustrated by the fact that a typical corn-soybean meal based diet would contain zero vitamin D.

 

Deficiency

Leg problems are a primary concern for poultry producers. These problems include lameness, bone developmental disorders, and bone breakage. Lameness is common in poultry selected for rapid growth, and its incidence within a flock can exceed 15% (Vaillancourt et al., 1999). Although lameness and bone fractures generally occur during the last few weeks before market, the underlying bone developmental abnormalities that lead to these events often occur during the first few weeks after hatch (Vaillancourt et al., 2000; van der Eerden et al., 2003; Huff et al., 2006; Dibner et al., 2007). The increased incidence of lameness in recent years (Julian, 2005) may be due in part to the change that has taken place in overall body structure and conformation of fast-growing, high-breast meat yield birds (Abourachid, 1993). Breast muscle yields have increased dramatically in recent years, thereby moving the center of gravity of the bird forward (Corr et al., 2003a,b; Havenstein et al., 2007). This body conformation characteristic of modern poultry affects gait patterns (Corr et al., 2003b) and imposes considerable stress and strain in femur and tibia bones, thus affecting bone development and increasing the risk of fractures. Vitamin D and its metabolites are the most extensively studied nutrients with respect to bone development in poultry. The outstanding 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 (Ramp et al., 1989) 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, 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 anatomy and severity: (1) weak bones causing curving and bending of bones, (2) enlarged hock and knee joints, (3) tendency to drag hind legs, and (4) beaded ribs and deformed thorax. Little difference exists among poultry species in relation to clinical signs of deficiency. Clinical signs in all poultry species would be rickets and lowered growth rate, egg production and hatchability. In addition to retarded growth, the first sign of vitamin D deficiency in chicks is rickets, which is characterized by a severe weakness of the legs. During vitamin D deficiency, growing birds develop hypocalcemia which, in turn, stunts skeletal development through widened cartilage at the epiphyses of long bones and weakened shafts (NRC, 1994). In young, growing chickens and turkeys there is a tendency to rest frequently in a squatting position, a disinclination to walk and a lame, stiff-legged gait. These are distinguished from the clinical signs of vitamin A deficiency in that birds with a vitamin D deficiency are alert rather than droopy and walk with a lame rather than staggering gait (ataxia). The beaks and claws become soft and pliable (Illus. 3-3) usually between two and three weeks of age. The most characteristic internal sign of vitamin D deficiency in chicks is a beading of the ribs at their juncture with the spinal column (Scott et al., 1982). In young turkey poults, Perry et al. (1990) reported a severe decrease in body weight gain and longitudinal skeletal growth. Femoral spiral fractures and tibia fractures are common pathologies in tom turkeys between 15 and 18 weeks of age (Vaillancourt et al., 2000; Julian, 2005).

 

Illustration 3-3: Vitamin D Deficiency, Soft Beaks

3Illustration_3-3_Vitamin_D_Deficiency_Soft_Beaks

Courtesy of L.S. Jensen, Washington State University

 

In chronic vitamin D deficiency, marked skeletal distortions become apparent (Scott et al., 1982) in which the spinal column may bend downward in the sacral and coccygeal region. The sternum usually shows both a lateral bend and an acute dent near the middle of the breast. These changes reduce the size of the thorax with consequent crowding of the vital organs. A disease condition known as endochondral ossification defects (EOD) produces bone deformations, fractures and lameness in broiler chickens throughout the world within the first few weeks after hatching. Flocks with a high incidence of EOD have significantly lower bone ash and 1,25-(OH)2D3 compared with mildly affected flocks, and it seems probable that higher systemic concentrations of 1,25-(OH)2D3 between seven to 14 days of age will enhance the ability of broiler chickens to effectively mineralize the cartilaginous growth plates in the appendicular skeleton during early bone maturation (Vaiano et al.,1994). Tibial dyschondroplasia (TD) is one of the most prevalent skeletal abnormalities observed in avian species; it causes enormous economic losses. It is caused by lesions composed of uncalcified, unvascularized cartilage that can extend from the epiphyseal growth plate into the metaphysis. Though frequently found in the tibia, hence the name, TD can also occur in the femur and tarsus. Derangements in vitamin D metabolism have long been suspected to be intimately involved in TD (Xu and Henry, 1997). Indeed, recent research supports a role for vitamin D3 metabolism in clearance of the lesion. The hormonal form of vitamin D3, 1,25-(OH)2D3 (Elliot and Edwards, 1997) and the 1,24,25-(OH)3D3 metabolite (Edwards, 1990) have been shown effective in reducing the incidence and severity of the disease. It appears that vitamin D3 metabolites with a hydroxyl group at the 1-alpha position are most effective. The effects of 1,25-(OH)D3 on TD have been variable under laboratory conditions (Edwards, 1989; Rennie and Whitehead, 1996).

As in many other nutritional diseases of poultry, a vitamin D deficiency causes the feathers to become ruffled. In red or buff color breeds of chickens, a deficiency of vitamin D causes an abnormal black pigmentation of some of the feathers, especially those of the wings. If the deficiency is very marked, the blackening becomes pronounced and nearly all the feathers may be affected (NRC, 1994). When vitamin D is supplied in adequate quantity, the new feathers and newer part of older feathers are normal in color, although the discolored portion remains black.

Signs of vitamin D deficiency begin to occur in laying hens in confinement within four weeks of the onset of the deficiency (Tsang et al., 1990). When laying chickens are fed a diet deficient in vitamin D, the first sign of deficiency is a thinning of the eggshells. Commercial layers will continue to lay eggs with reduced shell quantity for weeks. If the diet is completely devoid of vitamin D3, egg production decreases rapidly and eggs with very thin shells or no shell will be produced.

Nys et al. (1992) observed that concentrations of circulating 1,25-(OH)2D3 and ionized calcium were inversely proportional during eggshell formation. As eggshell formation progresses, levels of ionized calcium decrease, resulting in a short-term hypocalcemia and thus levels of 1,25-(OH)2D3 increasing in the plasma. In laying hens, eggshell strength tends to decrease as the hen ages. The decline in shell strength may be due to a decrease in the hen’s ability to synthesize 1,25-(OH)2D3.

Vitamin D nutriture of the hen also influences its content in egg yolk and the subsequent need for this vitamin by the chick (Stevens and Blair, 1985). In the case of deficiency, hatchability is markedly reduced, with embryos frequently dying at 18 to 19 days of age. Malpositions are also increased dramatically, apparently as a result of reduced embryonic bone and muscular development (Narbaitz and Tsang, 1989). These embryos often show a short upper mandible or incomplete formation at the base of the beak. Eventually breast bones become noticeably less rigid and there may be beading at the ends of the ribs. Individual hens may show temporary loss of use of the legs, with recovery after laying an egg (usually shell-less) (Scott et al., 1982). During periods of extreme leg weakness, hens show a characteristic posture that has been described as a “penguin-like squat.”

 

Fortification Considerations

Synthesis of vitamin D3 in the skin of poultry, through exposure to UV radiation in direct sunlight, is limited because poultry in commercial production are confined to houses. In addition, feedstuffs supply negligible amounts of vitamin D. Consequently, all poultry diets should be supplemented with vitamin D3 (cholecalciferol from D-activated animal sterol). The D3 form of the vitamin has high bioavailability for poultry and all other food-producing animals, while the D2 form (ergocalciferol) has very limited bioavailability for poultry. Vitamin D3is the commercially available form for use in poultry rations. 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 D3 resins and supplements (Baker, 1978) can be overestimated by the USP Chemical Assay, which does not correct for these biologically inactive compounds. A commercially available vitamin product containing stabilized, high-purity vitamin D3 for feed or drinking water use should be used to assure the vitamin D3 levels needed to prevent deficiency and allow optimal performance. Continued irradiation eventually destroys the vitamin D that is produced, but the chief cause of loss from feeds is oxidation. Higher vitamin D levels in freeze-dried fish meals suggest less destruction during drying, possibly because of decreased atmospheric oxygen (Scott and Latshaw, 1994). Pure vitamin D crystals and vitamin D resin are 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 products retain potency much longer and can be used in supplements with high mineral content. It has been shown that vitamin D3 is more stable than D2in feeds containing minerals. As vitamin D3 is more available than the D2 form, likewise the metabolites of vitamin D, 25-(OH)D3 Id-OHO3 and 1,25-(OH)2D3 are the most potent sources of vitamin D. The most potent form of vitamin D, 1,25-(OH)2D3 is not approved to be added to feeds by the FDA. Feeding studies with 25-(OH)D3 suggest it has nearly twice the activity of D3 (Soares et al., 1995; Mattila et al., 2004; McDowell and Ward, 2008). Grunder et al. (1990) suggest that 5 µg of 1,25-(OH)2D3 per day (2.27 µg per lb) of diet can replace 27.5 µg of D3 per kg (12.5 µg per lb). Mireles et al. (1996) concluded that the use of 25-(OH)D3 under commercial-type environments utilizing commercial broiler strains significantly improved performance and body weight and reduced the incidence and severity of bone disorders. They recommended that for maximum performance 25-(OH)D3 should be fed from day 1 of age at a rate of 68.9 µg per kg (31.3 µg per lb) of diet. The addition of 1 alpha-OH D3 increased growth performance and tibia ash and strength in broilers by increasing the absorption and retention of phosphorus (Han et al., 2009). This form of vitamin has yet to be approved by the FDA. Considerable research has been conducted over the past few years evaluating the efficacy of vitamin D3 metabolites for poultry. Besides improving inorganic calcium and phosphorus utilization, much like vitamin D3 (Biehl and Baker, 1997a), these vitamin D3 metabolites, when supplemented to diets already adequate in vitamin D3, are very effective in improving leg disorders and in enhancing the utilization of phytate-phosphorus and trace minerals. For example, Tsang (1992) concluded that substituting 1,25-(OH)2D3 for D3 in the diet can significantly reduce egg breakage. Brito et al. (2010) reports that the addition of 25(OH)D3 in broiler rations containing vitamin D3 improves performance characteristics.

More potent vitamin D3 metabolites, 1,25-(OH)2D3 (the active body metabolite of vitamin D3) and 1-alpha-OH D3 [the precursor of 1,25-(OH)2D3] also have been shown to improve tibial dyschondroplasia when added at low dietary concentrations (5 to 10 µg per kg or 2.3 to 4.5 µg per lb) to vitamin D3-adequate corn-soybean meal diets (Edwards, 1990; Rennie and Whithead, 1996; 1993; Elliot and Edwards, 1997; Mitchell et al., 1997; Ferket et al., 2009). However, Mitchell et al. (1997) suggested that 1,25-(OH)2D3 may not prevent tibial dyschondroplasia for a large portion of the population.

Optimum concentrations of vitamins in poultry diets (optimum vitamin nutrition, OVN) allow the birds to perform to their genetic potential. Vitamin requirements established decades ago do not take into account the modern genetically superior birds with increased growth, egg production and improved feed efficiency. Vitamin intake per unit of output is continually declining. The yearly decline for layers is around 1% per egg produced, while for broilers it has been 0.6-0.8% for body gain (Leeson, 2007). The vitamin D3 metabolite, 25-hydroxy vitamin D3 (25-(OH)D3), was used as part of an OVN investigation to evaluate egg production parameters and egg quality compared to a control of the average vitamin concentrations used in the Spanish egg production industry (Soto-Salanova and Hernandez, 2004). Vitamin D in the OVN diet was provided as 1,500 IU per kg (682 IU per lb) D3 and 1,500 IU per kg (682 IU per lb) of 25-(OH)D3 (commercial name Hy-D®). Using a combination of OVN + Hy-D® resulted in a dramatic increase of performance parameters for laying hens (Table 3-1). Hens receiving OVN + HyD® significantly improved production at a favorable cost (cost-to-benefit ratio of 1:9). Production parameters were as follows: 1) 55% benefit from higher laying rate, 2) 24% benefit from bigger egg size, 3) 15% benefit from lower feed intake and 4) 6% benefit from less broken eggs. There was also better egg quality with lower susceptibility to oxidation in fresh and 28-day stored eggs and lower egg weight losses after storing eggs for 21 days at room temperature.

3Table_3-1_Performance_parameters_of_laying_hens

A study with turkeys evaluated the impact of 25-(OH)D3 (Hy-D®) as a partial substitute for vitamin D3 in two levels of vitamin dosage, control (typical vitamin levels) or OVN (an enhanced dose of 13 vitamins) (Larroudé et al., 2005). During the first part of the experiment the birds receiving an enhanced vitamin dose (OVN), and particularly without HY-D®, grow better. However, after 12 weeks the dietary dosage of Hy-D® for the heavy turkeys, made it possible, whatever the vitamin dose, to achieve growth, bone development and body constitution and to improve percentage of fillets. It is a routine that commercial broiler diets are typically fortified with vitamin D3 at 10 to 15 times the NRC requirements (Lohakare et al., 2005b). Today 25-(OH)D3 is used routinely in commercial poultry programs. In a study with broiler chicks to compare the absorption of 25-(OH)D3 and vitamin D3, the former was found to be more efficient (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 an affinity for 25-(OH)D3 that is at least 1,000 times greater than for other D3 metabolites (Teegarden et al., 2000). Also, studies find that the intestinal uptake of 25-(OH)D3 occurs irrespective of bile acid secretion and micelle formation/fat absorption. The newly hatched bird struggles to coordinate an infantile digestive system with a rapidly developing skeleton. The 25-(OH)D3 could offer pronounced advantages to the bird under typical production challenges, not only early in the bird’s life, but also when some disease organisms are most prone to express themselves (Ward, 2004).

Studies with different classes of poultry have shown benefits from considerably higher dietary concentrations of the vitamin D. Atencio et al. (2004) indicates that 2,800-3,000 IU per kg (1,273 to 1,364 IU per lb) vitamin D3 should be present in the diets of broiler breeders for maximum production and the requirement may be higher for optimum bone ash of progeny. Chicks fed 3,200 IU vitamin D3 perkg (1,455 IU per lb) feed had the highest body weight and tibia ash and the lowest tibial dyschondroplasia and Ca rickets incidences (Atencio et al., 2005). Fritts and Waldroup (2003) observed a decrease in tibial dyschondroplasia incidence and severity by supplementing vitamin D3 up to 4,000 IU per kg (1,818 IU per lb) in diets of broiler chicks. McCormack et al. (2004) reported that 10,000 IU of vitamin D3 per kg (4,545 IU per lb) of diet can prevent tibial dyschondroplasia almost completely. Since the activity of the vitamin D metabolite 25-(OH)D3 is almost twice that of vitamin D3, it would seem logical to use 25-(OH)D3 alone or in combination with vitamin D3 for improved supplementation programs. Research indicated that 25-(OH)D3 was effective at reducing tibial dyschondroplasia in broilers when Ca was less than 0.85% (Ledwaba and Roberson, 2003). Optimum vitamin nutrition programs have used 3,000 IU per kg (1,364 IU per lb) of vitamin D3 (½ D3 and ½ 25-(OH)D3) to evaluate egg production parameters. Hens that receive the OVN (including 25-OHD3) had greatly improved laying rates and egg sizes, reduced broken eggs, better feed efficiency and lowered susceptibility to oxidation of fresh and stored eggs. Turkeys receiving 25-(OH)D3 (Hy-D®) were able to achieve growth with good bone development, without lameness disorders.

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 the exogenous supplement phytase in poultry diets to enhance phytate-phosphorus utilization and reduce the flow of phosphorus into the environment is now common (Onyango et al., 2006). The efficiency of supplemental microbial phytase depends on its rate of inclusion, dietary calcium and phosphorus ratio, vitamin D3, nature of diet, age and genotype of birds (Singh et al., 2008).

Vitamin D metabolites improve phosphorus utilization from phytate. Intestinal brush border phytase could possibly contribute to phytate-phosphorus digestibility and may be subject to regulation by the vitamin D3 and phosphorus status of the chicken (Maenz and Classen, 1998; Carlos and Edwards, 1998; Ward, 2004). Hydroxylation at the 1-alpha position of vitamin D3 appears important to the vitamin’s efficacy in improving phytate utilization (Biehl and Baker, 1997b; Biehl et al., 1998; Singh, 2008). Yet, though both 1,25-(OH)2D3 and 1-alpha-(OH) D3 markedly improve the release of phytate-bound phosphorus, Biehl and Baker (1997a) report that it is apparently not caused by increased intestinal phytase activity. The response of vitamin D is much like the response that occurs when microbial phytase is added to these same diets (Biehl et al., 1997a; Denbow et al., 1995; Yi et al., 1996). However, Onyango et al. (2006) concluded that vitamin D3 administered to broiler chicks did increase the activity of intestinal mucosa phytase. The ability of 1,25-(OH)2D3 and 1-alpha-(OH)D3 to improve phytate-phosphorus utilization is potentially very important to the poultry industry. The hydroxylated form of vitamin D (25-(OH)D3) can increase phytate phophorus retention for broilers and turkeys (Angel et al., 2001; Applegate et al., 2003). A combination of 1,25 (OH)2D3 and phytase was more effective in improving the performance of birds fed a low phosphorus diet (Angel et al., 2006; Yan and Waldroup, 2006). Phosphorus is the third most expensive ingredient in poultry diets, behind energy and protein, and phosphorus excretion in waste products is becoming a major concern, especially in countries where intensive livestock production is practiced.

Stabilization of vitamin D can be achieved by: (1) rapid compression of the mixed feed, for example, into cubes, so that air is excluded; (2) storing feed under 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 micropellets.

Stability of dry vitamin D supplements is affected most by high temperature, exposure to moisture and contact with trace minerals such as ferrous sulfate, manganese oxide and others. In complete feeds and mineral-vitamin premixes, Schneider (1986) reported activity losses of 10% to 30% after either four or six months’ storage at 22°C (72°F). 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°C or 72°F) for up to 12 weeks. The mash feed had lost 31% of its vitamin D3 activity after 12 weeks and the trace mineral premix had lost 66% of its activity after only six weeks of storage.

Cost of vitamin D3 supplementation to animal and poultry feeds is very nominal (Rowland, 1982). In contrast, the cost of not adding enough vitamin D3 to prevent deficiencies is very high. Supplemental levels of vitamin D3 administered to poultry through the feed or drinking water should be adjusted to provide the margin of safety needed to offset the factors influencing the vitamin D3 needs of poultry. This is important to prevent deficiency and allow optimum performance. Factors that increase the amount of vitamin D3 needed to maximize productive and reproductive responses often are not reflected in the NRC requirements. Consequently, nutritionists often use more than the minimum levels of vitamin D3 in feeds. Successful nutrition programs contain two to five times the NRC minimum of vitamin D3. However, no amount of vitamin D3 can make up for lack of enough calcium or phosphorus in the diet.

Rowland (1982) noted that diets for young, rapidly growing chickens must contain liberal amounts of vitamin D3 to prevent field problems. He further observed that the NRC level of 200 IU per kg (90.9 IU per lb) of feed for young chickens is extremely unrealistic for broilers. Even under low-stress research conditions, three to five times the NRC level is required to support maximum weight gain of broilers, and under commercial conditions, 10 times the NRC level is prudent for broiler feed. The NRC vitamin D3 levels for laying hens and turkeys are somewhat more realistic, with a factor of five times the NRC level generally supporting optimum performance and providing some margin of safety (Rowland, 1982). The most logical approach is to adjust supplemental vitamin D3 levels to expected production conditions.

Besides inadequate quantities of dietary vitamin D3, 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 under environmental conditions causing vitamin D3 loss (Hirsch, 1982).

Supplementation considerations are dependent on other dietary ingredients. The requirements for vitamin D3 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 D3 (Cunha, 1977). For example, when corn contains the mold Fusarium roseum, a metabolite of this mold prevents vitamin D3 absorption from the intestinal tract in chicks. Other molds may also be involved, and may result in a large percentage of birds with bone disorders. A number of flocks have been successfully treated by adding water-dispersible forms of vitamin D3 to drinking water at three to five times the normally recommended vitamin D levels. This “field rickets syndrome” in poultry is apparently not cured by adjusting vitamin D3 allowances in the feed, but it is frequently cured (clinical signs) by administering high levels of vitamin D3 from a water-dispersible vitamin product in the drinking water.

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. The possibility exists that poultry with certain diseases or a heavy infestation of internal parasites might 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 (Mac-Auliffe and McGinnis, 1976). Vitamin D supplementation may be needed for an optimum immune response. Vitamin D deficiency results in a marked depression in the cellular immune responses of young broiler chicks with negligible apparent impact on humoral immunity (Aslam et al., 1998).

Eggs are among the few potent natural sources of vitamin D for humans. Recent research has indicated that the vitamin D3 content of eggs can be further increased by supplementing hen feed with vitamin D3. For groups of hens that received 6,000 or 15,000 IU per kg (2,727 or 6,818 IU per lb) feed, egg yolk vitamin D3 ranged from 9.1 to 13.6 and from 25.3 to 33.7 µg/100 µg, respectively (Mattila et al., 2004).

 

Vitamin Safety

Besides the toxicity resulting from excess vitamin A, vitamin D is the vitamin next most likely to be consumed in concentrations toxic to poultry and livestock. Although vitamin D is toxic at high concentrations, as much as 100 times the requirement level may be tolerated. Young broiler chicks have a tolerance for excess vitamin D3 as high as 50,000 IU per kg (22,727 IU per lb) with no apparent negative effects on bone mineralization and growth (Baker et al., 1998). On the basis of renal calcification and body weight, 25-(OH)D3 was found to be five to 10 times more toxic than vitamin D3 (Yarger et al.,1995).Table 3-2 provides estimations of vitamin D tolerance for poultry. Under practical conditions vitamin D toxicosis for poultry would only be expected as a result of ration-mixing errors and then over a relatively long period of time. Excessive intake of vitamin D produces a variety of effects, all associated with abnormal elevation of blood calcium. Elevated blood calcium is caused by stimulated bone resorption, as well as increased intestinal calcium absorption and renal resorption. The main pathologic effect of ingestion of massive doses of vitamin D is widespread calcification of soft tissues. For poultry, clinical signs of vitamin D toxicity include anorexia, reduced eggshell quality, reduced egg production, renal tubular calcification, muscular atrophy and emaciation (NRC, 1987). Egg weight, shell quality, feed intake and fertility were significantly decreased in hens given 200,000 ICU vitamin D3 compared to controls that received 960 ICU (Ameenuddin et al., 1986). Commonly, anorexia is noted with vitamin D3 toxicity. Kidney tubule calcification is usually fatal. Renal tubular calcification proved to be the most sensitive index of toxicity and 0.1 mg 25-(OH)D3 per kg (0.045 mg per lb) of diet (approximately 8,000 IU per kg or 3,636.4 IU per lb) was the minimal level causing initial calcification (Soares et al., 1995). In contrast, it was necessary to feed 10 mg per kg (4.55 mg per lb) vitamin D3 to get full renal calcification.

3Table_3-2_Estimation_of_Safe_Upper_Dietary_Levels_of_Vitamin_D3_for_Poultry

Over-supplementation of vitamin D is a risk factor for chronic heart failure in fast growing commercial broilers (Nain et al., 2007). A diet containing 80,000 IU per kg (36,364 IU per lb) vitamin D3resulted in birds with cardiac arrhythmia and negative QRS axis on lead-II (an indication of left heart failure) compared to controls. Leg problems may arise with growing birds because of bone calcium loss (Cruickshank and Sim, 1987), but few obvious changes occur with hens other than a general depression in performance (Ameenuddin et al., 1986). Toxic levels of vitamin D may be transferred into the egg to create similar problems for the embryo. However, hypercalcemia occurs from shell resorption, and bone mineralization is enhanced (Narbaitz and Fragiskos, 1984). Vitamin D toxicity can be reduced with vitamin A supplementation. Aburto et al. (1998) reported that 45,000 IU per kg (20,455 IU per lb) of dietary vitamin A ameliorated the potential toxic effects of feeding high levels of vitamin D3, 25-(OH)D3, and 1,25-(OH)2D3 to young broiler chickens.