Introduction
The intake of vitamin D is low in most countries because few foods are relatively rich in this nutrient. Vitamin D deficiency is a frequent problem during the winter in Europe as the continent is located at high latitude, leading to limited ultraviolet light exposure. Consequently, all age groups of the human population are at risk of vitamin D deficiency in the winter months (Lamberg-Allardt et al., Reference LAMBERG-ALLARDT, OUTILA, KÄRKKAINEN, RITA and VALSTA2001). Besides being important for humans and other mammals, vitamin D is a key dietary factor for poultry. In fowls, vitamin D is responsible for normal growth, egg production, shell quality, and reproduction. Vitamin D3 or its active metabolite (1,25-dihydroxycholecalciferol) is involved in Ca and P absorption in the gut, bone mineralisation (bone formation) and demineralisation (bone mobilisation) and Ca and phosphate resorption by the kidneys (Combs, 1998). The existence of intestinal binding proteins for 25-dihydroxycholecalciferol (calcidiol or 25-(OH)D3) is not contradictory to the occurrence 25-(OH)D3 in a number of foodstuffs (Ovesen et al., Reference OVESEN, BROT and JAKOBSEN2003). Eggs from laying hens not fed 25-(OH)D3 have been shown to contain up to 1.0ug of this metabolite per 100g of egg yolk, while hens fed 25-(OH)D3 at recommended levels had 2.5 fold higher amounts in their eggs. In broiler meat, the deposition of 25-(OH)D3 is highly correlated with the intake of this metabolite (Yarger et al., Reference YARGER, SAUNDERS, MCNAUGHTON, QUARLES, HOLLIS and GRAY1995).
Poultry diets are normally supplemented with minerals and vitamins within the recommended ranges to avoid deficiencies or toxicities because low intakes and occasionally excesses of certain essential nutrients play major roles in the development of several metabolic and developmental disorders in poultry. The requirements for cholecalciferol in poultry have been established through experimentation and are reviewed frequently (NRC, 1994). However, these recommended values are the minimum levels required under controlled trial situations. In commercial diets, most vitamins are usually added in excess to meet the birds’ requirements under stressful conditions encountered in practical rearing conditions, and to make up for deficiencies occurring during diet preparation and storage (McDonald et al., Reference McDONALD, EDWARDS, GREENHALG and MORGAN1995). Most feed ingredients in poultry diets contain little or no vitamin D and, as a result, this vitamin is normally added to the diet in the synthetic form. As vitamin D2 (ergocalciferol) has only about 0.1 of the potency of vitamin D3 (cholecalciferol) for poultry, cholecalciferol is preferentially used in the diet as 1 g of pure vitamin D3 is 40x106 IU or one IU is 0.025μg (McDonald et al., Reference McDONALD, EDWARDS, GREENHALG and MORGAN1995). Structurally, it is related to steroids such as testosterone, cholesterol and cortisol. Vitamin D3 itself is a secosteroid. According to an industry survey of commercial broiler producers (Anonymous, Reference ANONYMOUS1997), the standard level of vitamin D supplementation in broiler starter diets in the United States commercial poultry industry was 70.48 µg/kg with the 25% of producers adding 49.70 µg/kg and 25% of producers adding 100.75 µg/kg.
The nanoparticle behaviour of vitamin D in the biology of meat-type birds is always related to serious issues in chickens. This versatile nutrient has played a beneficial role in preventing leg disorders in broilers (Dinev, Reference DINEV2012), even though leg abnormalities and bird mobility are affected by a number of different bone, tissue and several disorders as well as by other factors. Deficiencies and occasionally excesses of certain essential nutrients play major roles in the development of several leg abnormalities in poultry. A national survey conducted in Turkey reported that broiler flocks experience 15% additional mortality due to leg abnormalities (Misirlioglu et al., 2001). Mortality and number of culls and condemnations due to leg disorders have been estimated to cause 0.10-0.30 of the total losses (Julian, Reference JULIAN1995). Vitamin D related lameness in chickens occurs due to various associated problems including tibial dyschondroplasia (TD) (Dinev, Reference DINEV2012). TD ensues from inadequate vascularisation and ossification of the growth plate, resulting in an abnormal mass of cartilage under the growth plate, causing unnatural biomechanical forces, and therefore gait alteration, additional bone abnormalities, and even fractures (Julian, Reference JULIAN1998). Angular deformities of the long bones, such as varus-valgus deformities and ‘twisted legs’ caused by a lateral or medial deviation of the distal tibia or proximal metatarsus, frequently accompanied by slippage of the gastrocnemius tendon are other common leg disorders (Riddell, Reference RIDDELL1992). Rickets is characterised by an enlargement of the epiphyseal area and occasionally deformity of the soft, rubbery bone (Julian, Reference JULIAN1998) and occasionally occurs due to nutrient deficiencies and/or imbalances.
A number of studies are available highlighting the useful effects of high levels of cholecalciferol, its metabolites and ultraviolet light irradiation on the increase of tibia ash and reduction of TD and rickets incidences (Edwards et al., 2002; Edwards, 2003; Ledwaba and Roberson, Reference LEDWABA and ROBERSON2003; Khan et al., Reference KHAN, R., MIAN, SARDAR and ANJUM2010). The application of 250 mg cholecalciferol/kg of feed had a preventive effect on TD (Whitehead et al., Reference WHITEHEAD, MCCORMACK, MCTIER and FLEMING2004) while rickets due to vitamin D3 deficiency seem to be eliminated at approximately 40-45 µg/kg of feed (Ledwaba and Roberson, Reference LEDWABA and ROBERSON2003), and doses as high as 77.5 µg cholecalciferol/kg completely eliminated rickets in broilers. The aim of this review paper is to give an overview of effective role of cholecalciferol on bird's performance, prevention of leg abnormalities and immunity.
Evaluation of cholecalciferol sources
In the commercial broiler industry, the potencies of cholecalciferol preparations have great significance as the incidence of skeletal disorders is still common (Dinev, Reference DINEV2012). The risk of toxicity is minimal because it is beyond practical means to reach dietary cholecalciferol toxicity levels of 10,000 μg/kg (Morissey et al., Reference MORISSEY, COHN, EMPSON Jr, GREENE, TAUNTON and ZIPORIN1977) and 12,500 μg/kg (Taylor et al., Reference TAYLOR, MORRIS and KIRKLEY1968). Yarger et al. (Reference YARGER, SAUNDERS, MCNAUGHTON, QUARLES, HOLLIS and GRAY1995) reported that 25-(OH)D3 did not exhibit toxicity even when a 10-fold amount of the recommended ratio of 69 μg/kg was used on a short-term basis. Results of three month toxicity studies on vitamin D3 showed that even a four-fold multiple of the usual nutritional dose was completely safe on very long term feeding basis (Papešová, Reference PAPEŠOVÁ2000). Commercially, cholecalciferol is available in several physical forms and preparations. Commercial preparations for animal feeds are usually spray-dried or drum-dried and are in beadlet or flake forms. These products are generally prepared from the same original cholecalciferol product. European Union Commission Regulation No. 1443/2006 authorises the use of 25-(OH)D3 in feeds for chickens, fattening and laying hens and turkeys (Papešová et al., Reference PAPEŠOVÁ, FUČÍKOVÁ, PÍPALOVÁ and TUPÝ2008).
From the last 40 years, there have been only two published reports on the evaluation of commercially synthesised cholecalciferol preparations which are commonly used in poultry diets. The studies carried out by Yang et al. (Reference YANG, WAIBEL and BRENES1973) and Kasim and Edwards (2000) assessed commercial cholecalciferol preparations with young male turkey poults and broiler chicks. Yang et al. (Reference YANG, WAIBEL and BRENES1973) performed two separate studies. In the first trial, out of 26 cholecalciferol supplements, 19 supplements showed potencies above 80%, six supplements had lower potencies (out of six, four showed potencies under 60%). Whereas, in the second study, all five supplements had potencies below 80%, of which three had potencies under 60%. These researchers reported that potencies of some cholecalciferol supplements had been overvalued. Similarly, Kasim and Edwards (2000) conducted three experiments to test the potencies of nine sources of cholecalciferol. The tested products were compared with a Sigma Reference Standard (SRS). Each of the products was in premix form containing cholecalciferol. They were categorised as spray-dried or drum-dried in the beadlet or flake form. For experiments one and two, three different cholecalciferol products and two levels of 200 and 400 IU/kg dietary cholecalciferol were tested. For both experiments, three additional SRS levels of 600, 800, and 1,000 IU/kg were incorporated in the studies as positive controls. In experiment three, three cholecalciferol products and four levels of dietary cholecalciferol at 150, 300, 600, and 1,200 IU/kg were used. They reported that the potencies of the products from the three experiments ranged between 86 and 118%, using slope ratio analysis. In this experiment, the requirement of chicks for cholecalciferol using the three tested products (as determined by a non-linear regression model based on bone ash) were 843, 911 and 986 IU/kg of diet as compared with 915 IU/kg when using the SRS. The results from these studies indicated that the chemical assays used to determine the cholecalciferol activity of these products were very reliable.
Vitamin D metabolites
Vitamin D metabolism in the bird is a multifaceted process involving several metabolites. The vitamin can either be produced in the skin by the photochemical conversion of the provitamins ergosterol and 7-dehydrocholesterol or absorbed from the diet in the intestinal tract. The absorption process of vitamin D in the intestine has two stages, with its first part consisting of a quick vitamin D supply to mucous membranes and then a slow transfer to lymphatic vessels. The first quick stage requires the presence of bile acids, while in the second slow stage it is bound to chylomicrons. The absorption takes place in the upper small intestine where the highest amount of fat is absorbed (Papešová et al., Reference PAPEŠOVÁ, FUČÍKOVÁ, PÍPALOVÁ and TUPÝ2008). The absorbed dietary vitamin D3 (calciol or inactive unhydroxylated form) are transported in the blood to the liver where they are converted into 25-hydroxycholecalciferol, the major circulating form of vitamin D3. This 25-(OH)D3 is then transported to the kidneys where it is converted into 1,25-dihydroxycholecalciferol (calcitriol or 1,25-(OH)2D3), which is the most biologically active, hormonal metabolite of the vitamin (McDonald et al., Reference McDONALD, EDWARDS, GREENHALG and MORGAN1995). This step is under tight regulation, being enhanced by parathyroid hormones, hypocalcaemia and hypophosphatemia, and suppressed by hypercalcaemia, hyperphosphatemia and impaired renal function (Davies, Reference DAVIES2000). This 1,25-(OH)2D3 supports calcium and phosphorus absorption in the intestine, affects bone calcification, co-participates in calcium and phosphorus metabolism in the birds. Receptors for 1,25-(OH)2D3 are present in many tissues, including blood elements (Fritts et al., Reference FRITTS, ERF, BERSI and WALDROUP2004). Stressful conditions, such as high bird density, heat stress, mycotoxicosis, enteritis, malabsorption syndromes, and certain immune disorders may impair absorption or liver hydroxylation of cholecalciferol, which is one rationale for the use of vitamin D metabolites in poultry feed. Moreover, as the current legal dietary limit in the European Union is 125 µg cholecalciferol/kg of feed, the feeding of vitamin D3 metabolites may be a means of providing the high intake needed by young broilers (Whitehead et al., Reference WHITEHEAD, MCCORMACK, MCTIER and FLEMING2004). The biological activity of 25-(OH)D3 is estimated to be 2–2.5-fold that of 1,25-(OH)2D3 (Edwards et al., 1994; Ledwaba and Roberson, Reference LEDWABA and ROBERSON2003). Supplementing the diet with 25-(OH)D3 caused a significant decrease in the frequency and severity of TD, although the effects are not of the degree or stability as those effects caused by 1,25-(OH)2D3 supplementation (Edwards, 1989; Rennie and Whitehead, Reference RENNIE and WHITEHEAD1996; Mitchell et al., Reference MITCHELL, EDWARDS Jr and MCDANIEL1997; Roberson, Reference ROBERSON1999). In studies showing a preventative effect of 25-(OH)D3 on TD, the supplement was added to the diet at levels between 75 and 344.5 µg/kg. Further, there appears to be an interactive effect with calcium levels in the feed. At low calcium levels, 25-(OH)D3 seems to be more effective in reducing TD incidence than at sufficient calcium levels (Ledwaba and Roberson, Reference LEDWABA and ROBERSON2003). In the same study, for chickens exposed to UV light, overall TD incidence was minimised at 10 µg 25-(OH)D3/kg feed. Concentrations of up to 690 µg 25-(OH)D3/kg have been given to chickens without adverse effects on growth rate or feed efficiency (Yarger et al., Reference YARGER, SAUNDERS, MCNAUGHTON, QUARLES, HOLLIS and GRAY1995). Also dietary supplement of the metabolite 1,25-(OH)2D3 within the range 5–10µg/kg feed has in many studies been reported to improve the incidence and severity of TD and rickets, and increase bone mineralisation, both in diets sufficient in calcium and cholecalciferol, and at suboptimal levels of calcium and phosphorus, even though results are not completely conclusive (Roberson and Edwards, 1994; Elliot and Edwards, 1997). Supplementing 1,25-(OH)2D3 with 3 to 6 µg/kg feed showed no negative effect on growth when diets contained low dietary calcium concentrations, but as dietary calcium concentrations approached 10 g/kg growth was depressed (Elliot et al., Reference ELLIOT, ROBERSON, ROWLAND and EDWARDS Jr1995; Rennie et al., Reference RENNIE, McCORMACK, FARQUHARSON, J.L., MAWER and WHITEHEAD1995; Roberson and Edwards, 1996).
Atencio et al. (Reference ATENCIO, PESTI and EDWARDS Jr2005b) showed that 25-(OH)D3 had very high biological potency at low dietary concentrations of 1,25-(OH)2D3 and that the relative biological value of 1,25-(OH)2D3 was higher for embryos than for hens. These results suggest that further research is required to determine the optimum maternal nutritional concentrations of vitamin D and its metabolites for overall economic performance. The absorption of 25-(OH)D3 occurs irrespective of fat absorption (Sitrin and Bengoa, Reference SITRIN and BENGOA1987), thus making 25-(OH)D3 an obvious consideration in starter diets for chicks and poults at a time when pancreatic lipase might be limited for cholecalciferol absorption. Hence, as opposed to vitamin D3, 25-(OH)D3 possibly would bypass this initial insufficiency in fat digestibility at a time when skeletal changes are rapidly occurring, and bone ash percent is being maximised. In experimental studies, some other vitamin D metabolites, like 1, 24 R, 25 -trihydroxycholecalciferol, 1,25-dihydroxycholecalciferol-dihydroxy-26, 27-hexadenteniumcholecalciferol and 1,25-dihydroxycholecalciferol-dihydroxy-24R-fluorocholecalciferol have been shown to be useful in preventing the development of TD in broiler chicks (Edwards, 1990).
Vitamin D requirements for poultry
Studies indicate that birds can obtain vitamin D3 from the premix, endogenous production (UV light radiation), and animal by-products (Atencio et al., Reference ATENCIO, EDWARDS Jr, PESTI and WARE2006). In addition, as the present legal dietary limit in the European Union is 125 µg cholecalciferol/kg, the feeding of vitamin D3 metabolites may be a way of providing the high status required by young broiler chicks (Whitehead et al., Reference WHITEHEAD, MCCORMACK, MCTIER and FLEMING2004). According to the NRC (1994), the vitamin D3 requirement for egg-type white egg breeders with a feed intake of 100 g per hen per day is 7.50 µg (300 IU) of vitamin D3/kg. This interprets into a daily requirement of 0.75 µg (30 IU) of vitamin D3/kg/hen/day. Brown egg layers with a feed intake of 110 g/hen/day are estimated to require 0.83 µg (33 IU) of vitamin D3/kg/hen/day. Suggested levels for vitamin D3 for broiler breeder rations in books and commercial guidelines vary from 5.0 µg (500 IU) of vitamin D3/kg of diet (Scott et al., Reference SCOTT, NESHEIM and YOUNG1982) to 87.50 µg (3,500 IU) of vitamin D3/kg of diet (Ross Breeders, Reference ROSS BREEDERS2000). The NRC (1994) estimate for the vitamin D3 requirement of starting broilers is 5 µg/kg (200 IU/kg) but latest studies have recommended that the requirement may now be higher. Though, at commercial level, diets are normally prepared with supplemental vitamin D3 within the range 75–125 µg/kg (Whitehead et al., Reference WHITEHEAD, MCCORMACK, MCTIER and FLEMING2004). Moreover, water-soluble vitamin mixtures containing vitamin D3 may be added at early brooding period as a way of diminishing early growth problems. Whitehead et al. (Reference WHITEHEAD, MCCORMACK, MCTIER and FLEMING2004) explained that vitamin D3 requirements of broiler chicks up to 14 days of age at adequate dietary calcium and available phosphorus concentrations might be in the range 35–50 µg/kg of diet for cortical bone quality. After that, the vitamin D3 requirement for cortical bone quality reduces to less than 20 µg/kg of diet. These requirements are very high than former estimates and may be linked to high calcium requirements of modern broiler genotypes. Rickets due to vitamin D3 deficiency appear to be eradicated at approximately 40–45 µg/kg of diet (Ledwaba and Roberson, Reference LEDWABA and ROBERSON2003), although it has been reported that quantities as high as 77.5 µg cholecalciferol/kg have been needed to eliminate rickets (Elliot and Edwards, 1997). Khan et al. (Reference KHAN, R., MIAN, SARDAR and ANJUM2010) reported that feeding levels of 37.5 to 87.5 µg cholecalciferol/kg positively affected broiler performance. In mainly other studies, much lower vitamin D3 concentrations (range 25–75µg/kg) were used, which could clarify the lack of response. High levels of cholecalciferol have been shown to enhance the utilisation of phytate phosphorus and the retention of calcium and phosphorus (Mohammed et al., Reference MOHAMMED, GIBNEY and TAYLOR1991).
A difficulty in determining vitamin D3 requirement is its interaction with a number of nutrients. In modern broiler genotypes, an increase in calcium and phosphorus requirements will consequently influence vitamin D3 requirements. In broilers at three 3 weeks of age fed a diet deficient in phosphorus, very high levels of cholecalciferol (1250 µg/kg of diet) were essential to maximise growth and bone ash (Baker et al., Reference BAKER, BIEHL and EMMERT1998). In the similar study, while the diet was somewhat deficient in calcium or was sufficient in both calcium and phosphorus, adding more than 30–40 µg cholecalciferol/kg produced no response on growth, feed efficiency or bone ash values. Later information has been offered showing effects of prominent levels of vitamins A and E in depressing vitamin D status (Aburto and Britton, 1988a; 1988b). The effect of vitamin A was evident at practical dietary levels, with an increase from 37.5 to 375 µg/kg of vitamin A resulting in an approximate threefold increase in the vitamin D requirement. The effect of vitamin E on vitamin D requirement was more apparent at dietary levels of vitamin E that were significantly above dietary levels. However it must be remembered that exposure of birds to UV light can almost eliminate the requirement for dietary vitamin D.
According to NRC (1994), the nutrient requirement for normal egg production and shell quality is 12.50 µg (500 IU) of cholecalciferol/kg feed. Eggs are among the few potent natural sources of vitamin D for humans. Fraser and Emtage (1976) reported that 90% of the vitamin D3 deposited in the egg occurs as 1,25-(OH)2D3, whereas, only 5% occurs as 25-(OH)D3. According to Mattila et al. (Reference MATTILA, LEHIKOINEN, KIISKINEN and PIIRONEN1999), egg content can be further increased by supplementing hens feed with additional vitamin D3. They found that it was possible to produce eggs containing seven-fold more cholecalciferol than so-called normal eggs by increasing the cholecalciferol content of the feed from the regular level (62.4µg/kg) to a level circa 3.5 times higher (216 µg/kg). In a continuation of the study, Mattila et al. (Reference MATTILA, TIMO, KAROLIINA, JARMO, LAILA and EEVA-LIISA2003) used two high levels (280 µg and 300 µg/kg feed) of cholecalciferol in hen diets. They found that the peak cholecalciferol contents in egg yolk (ca. 30 µg/100 g) were attained 8-13 days from starting the high-cholecalciferol diet. After 112 days feeding, the cholecalciferol content steadily declined to ca. 22 µg/100 g. A similar trend was observed in the next study in which the hens that received 150 or 375 µg cholecalceferol/kg feed resulted in egg yolk cholecalciferol ranging from 9.1 to 13.6 and from 25.3 to 33.7 mg/100µg, respectively (Mattila et al., Reference MATTILA, VALAJA, ROSSOW, VENÄLÄINEN and TUPASELA2004). They found that vitamin D3 was more appropriate than vitamin D2 for the enrichment of eggs, because dietary supplementation of vitamin D3 increased egg yolk vitamin D content more efficiently than vitamin D2. Moreover, Kawazoe et al. (Reference KAWAZOE, NOGUCHI, YUASA and SAITHO1997) investigated the effect of supplementation with vitamin D3 from different sources on the transfer of vitamin D to egg yolk and found a strong positive correlation. A European Union directive currently prohibits the addition of more than 75 µg (3000 IU)/kg cholecalciferol into hen feed (Mattila et al., Reference MATTILA, TIMO, KAROLIINA, JARMO, LAILA and EEVA-LIISA2003) and comprehensive research is needed before this directive can be changed because there are only a few published reports on the transfer of vitamin D3 from feed to egg yolk.
Effect of cholecalciferol on broiler performance
The main objective of the broiler industry is to produce birds at maximum performance by least cost means while having minimal mineral losses (Ramo Rao et al., Reference RAMO RAO, RAJU, A.K., SHYAM and SHARMA2009). The poor utilisation of phytate phosphorus (PP) by chickens is causing severe environmental concern because of the excretion of unutilised P and trace minerals from intensive poultry farming. The PP chelates essential minerals in cereals and oilseed by-products (Ravindran et al., Reference RAVINDRAN, BRYDEN and KORNEGAY1995) and limits their accessibility to the chicken, resulting in the excretion of these minerals into the environment. Recommended or excess concentrations of Ca and P or both in the diet decreases the utilisation of PP in the gut of chickens. Utilisation of Ca and P can be improved when they are fed at sub-optimal concentrations by supplementing high concentrations of cholecalciferol in the diet (Baker et al., Reference BAKER, BIEHL and EMMERT1998).
The current NRC (1994) recommended concentration of dietary non-phytate phosphorus (NPP) for broilers is 0.45%. The effect of cholecalciferol on body weight gain (BWG) has been extensively studied and results show improvements in BWG, feed intake (FI) and feed efficiency (FE) of broilers when birds were supplemented with high concentrations of D3 (Whitehead et al., Reference WHITEHEAD, MCCORMACK, MCTIER and FLEMING2004; Rama Rao et al., Reference RAMA RAO, RAJU, PANDA, SHYAM and SHARMA2006; 2009). Nawaz et al. (Reference NAWAZ, SHAFIQ, YAQOOB, YOUSAF and AHMAD2008) found chicks fed diets of adequate NPP with D3 supplementation at 5.0, 25, 50 and 75 µg/kg significantly improved FI, FE and BWG from 1,706 g to 1,866 g over the 42 day production period. These results were similar to Baker et al. (Reference BAKER, BIEHL and EMMERT1998), who found chicks fed diets of 0.10 or 0.45% NPP with vitamin D3 supplementation ranging from 0 to 1250 µg/kg significantly increased FI by 70 g and 40 g and improved BWG from day 8 to 20. Atencio et al. (Reference ATENCIO, EDWARDS Jr and PESTI2005a) noted an improvement in FI, FE and BWG of chicks when fed an adequate dietary NPP with both maternal and direct supplementation of D3. Similarly, Edwards (2002) found a significant improvement of FE by feeding 220 µg/kg of D3 with 0.30% dietary NPP to chicks from day 1 to 16. Recently, Khan et al. (Reference KHAN, R., MIAN, SARDAR and ANJUM2010) evaluated four different cholecalciferol levels, using diets supplemented with 5.0 (control), 37.5, 62.5 or 87.5 µg/kg of cholecalciferol. The results showed that, at both 21 and 42 days, the BWG of birds fed 37.5 to 87.5 µg/kg of cholecalciferol was significantly greater than birds fed 5.0 µg/kg. Similarly, better FCR was observed in birds fed diets containing high levels of cholecalciferol. In this study 87.5 µg/kg had positive effects on meat yield. These results are in line with the findings of Cantor and Bacon (Reference CANTOR and BACON1978), who found that supplementation of high levels of cholecalciferol caused considerable increases in breast meat yield of broilers due to better growth. They suggested that increasing dietary vitamin D3 levels enhanced the amount of calcium within the body.
More recent research evaluated the role of cholecalciferol in maternal diets on progeny growth and skeletal development. Progeny BWG was greatest when broiler breeders were fed the highest maternal dietary concentrations of 50 or 100 µg cholecalciferol/kg, and the incidence of TD in progeny were improved in the offspring of young (30 or 37 weeks old) but not older (45 or 57 weeks old) broiler breeders (Atencio et al., Reference ATENCIO, PESTI and EDWARDS Jr2005b). These concentrations were similar to the recommended commercial supplementation of 70 µg/kg (Atencio et al., Reference ATENCIO, EDWARDS Jr, PESTI and WARE2006). Ding et al. (Reference DING, PIRONE, LENZI, BAGLINI and ROMBOLI2011) investigated the effect of hen dietary 25-(OH)D3 on the morphological changes of the small intestine of chicks during the pre- and post-hatch periods. After two weeks of feeding, eggs from hens fed the experimental diets were collected and incubated. Results showed that the weight, length, thickness of small intestine and the height, perimeter and surface area of villi in the small intestine increased with age, and they changed faster in the jejunum and duodenum than in the ileum. In comparison with the control diet, 25-(OH)D3 supplemented into the maternal diet caused lower weight and shorter length of the small intestine of offspring after hatch. Meanwhile, 25-(OH)D3 of maternal diet induced the increase of the villi height and the crypt depth in all three gut segments after hatch, in particular in the jejunum and duodenum. They suggested that dietary 25-(OH)D3 (75µg/kg) can be used as a feed additive in the maternal diet to stimulate morphological maturation and, consequentially, improve intestinal function in chicks during later incubation and in the first week of life.
Effect of cholecalciferol on layer performance
Vitamin D3 and its metabolites have similar effects on layers as they do on broilers. Frost and Roland (1990) reported that FI and BWG significantly increased in hens (65 to 75 weeks of age) with supplementation of 1,25-(OH)2D3 at 1.0 μg/kg into a diet containing 3.75% Ca and 0.55% P. However, these results disagree from Keshavarz (Reference KESHAVARZ2003) and Mattila et al. (Reference MATTILA, VALAJA, ROSSOW, VENÄLÄINEN and TUPASELA2004), who did not observe any effect on FI and BWG during the production period when diets were supplemented with either 1,25-(OH)2D3 or 25-(OH)D3.
Most studies conducted with D3 supplementation on laying hens have focused on egg quality. Frost and Roland (1990) determined a significant improvement (10 and 5%) in egg production by supplementing 37.50 μg/kg of 25-(OH)D3 and 1.0 μg/kg of 1,25-(OH)2D3, respectively, to a diet containing 0.55% P and 3.75% Ca fed to hens aged 65 to 75 weeks. These results are in agreement with Carlos and Edwards (1998), who saw a significant improvement (5%) in egg production when hens (59 to 68 weeks of age) were fed a diet containing 5 μg/kg of 1,25-(OH)2D3 with 3.0% Ca and 0.33% P. Marrett et al. (Reference MARRETT, FRANK and ZIMBELIMAN1975) also noted a 3 to 10% increase in egg production when hens were fed a diet supplemented with 22 μg/kg of 25-(OH)D3 and Ca levels ranging from 2.75 to 4.00%. However, some studies showed no significant improvement in egg production when vitamin D3 was applied (Keshavarz, Reference KESHAVARZ2003, Mattila et al., Reference MATTILA, VALAJA, ROSSOW, VENÄLÄINEN and TUPASELA2004 and Musapuor et al., Reference MUSAPUOR, POURREZA, SAMIE and SHAHRBABAK2005).
Many studies have been conducted with vitamin D3 and its metabolites which have resulted in inconsistent data on egg weight. Tsang and Grunder (Reference TSANG and GRUNDER1993) concluded that supplementation of 25-(OH)D3 rather than 1,25-(OH)2D3 produced a heavier egg, but supplementation of any vitamin D3 resulted in a heavier egg when compared to non-supplemented control. Ameenuddin et al. (Reference AMEENUDDIN, SUNDE, DELUCA and COOK1986) and Mattila et al. (Reference MATTILA, VALAJA, ROSSOW, VENÄLÄINEN and TUPASELA2004) found a marginal (P<0.09) reduction in egg weight when vitamin D3 was supplemented in the diet at levels of 2500 to 5000 µg/kg and 375 µg/kg, respectively. Other studies, however, have noted no significant differences in egg weight when diets were supplemented with D3 or its metabolites (Carlos and Edwards, 1998; Keshavarz, Reference KESHAVARZ2003; Musapuor et al., Reference MUSAPUOR, POURREZA, SAMIE and SHAHRBABAK2005).
Egg shell weight has been shown to increase when either vitamin D3 or its metabolites were supplemented in the diet, but heavier shells were observed when vitamin D3 metabolites were supplemented (Frost and Roland, 1990). Some studies have found that supplementation of vitamin D3 or its metabolites resulted in a significant increase of shell thickness or specific gravity (Tsang and Grunder, Reference TSANG and GRUNDER1993; Carlos and Edwards, 1998; Keshavarz, Reference KESHAVARZ2003).
Effect of cholecalciferol on hatchability
The transfer of nutrients from a hen to the egg follows two pathways i.e. through the ovary to the yolk or via the oviduct to the albumen, eggshell and membrane. It has been recognised for some time that vitamin D is necessary for the hatchability of eggs. Lack of this vitamin causes embryos to have a shortened mandible and other skeletal deformities, with death occurring after day 18 of incubation. The maximum values for fresh weight and calcium content of embryo tibiae take place by day 19 of incubation (Kubota et al., Reference KUBOTA, ABE, SHINK and SUDA1981). The 1,25-(OH)2D3 is not moved into the egg, and does not maintain hatchability better compared to the absence of vitamin D supplementation in hen diets (Abdulrahim et al., Reference ABDULRAHIM, PATEL and MCGINNIS1979). On the other hand, 25-OH-D3 metabolite given to hens is known work best in regards to hatchability (Hart et al., Reference HART, SCHNOES and DELUCA1986) relative to any other known vitamin D metabolite. The chick embryos absorb large amounts of Ca in their bones in a short time, and as the yolk holds inadequate amounts of Ca, this must be derived from the eggshell. The 25-(OH)D3 is simply transferred into the chicken and turkey egg, and is then converted to 1,25-(OH)2D3 by the embryonic kidney after day eight to support the transfer of Ca from the eggshell, and absorption into the embryonic skeleton. A large-scale field study (involving three million breeders supplemented diets with 25-(OH)D3 and reported increased hatchability of fertile eggs from 79.5 to 82.5% (Hargis, Reference HARGIS2000). In this study, 25-(OH)D3 was given in addition to vitamin D3 in the diet, thus demonstrating that vitamin D3 supplementation under field conditions may not be enough to optimise hatchability in broiler breeders.
The mechanism after the improvement in hatchability is not explicable but could be connected to an enhanced transfer of 25-(OH)D3 into the egg. Thus, despite there being no studies that have revealed the mode of action by which 25-(OH)D3 can improve hatchability, the prevalence of data indicate that this metabolite has a place in breeder feeding programs.
Effect of cholecalciferol on immunity
The presence of vitamin D receptors on lymphocytes cells is the first evidence of an interrelationship between it and the immune system. Immature and potentially active helper T cells, B cells, regulatory cells and dendritic cells are directly affected by the active vitamin D form (Takeda et al., Reference TAKEDA, TOMOYA, NAOTO, KENJI, TOMOYUKI, MASAKAZU, TATSURO and KEN-ICHI2010). The active form procures immunological effects by binding to nuclear vitamin D receptors (VDR), which are in the majority of immune cell types including both innate and adaptive immune cells. VDR is found in monocytes and activated macrophages, dendritic cells, NK cells, T and B cells. The activation of VDR has strong anti-proliferative, pro-differentiative, and immunomodulatory functions including both stimulating and immunosuppressive effects (Nagpal and Rathnachalam, 2005). VDR ligands have been exposed to enhance the action of natural killer cells, and increase the phagocytic activity of macrophages. The active vitamin D hormone improves the production of cathelicidin (antimicrobial peptide) that is formed in macrophages triggered by bacteria, viruses, and fungi (Janet, Reference JANET2006). Vitamin D deficiency is likely to raise the risk of infections, for example influenza (Cannell et al., Reference CANNELL, VIETH, UMHAU, HOLICK, GRANT, GARLAND, MADRONICH and GIOVANNUCCI2006). The effects of VDR-ligands (vitamin D hormone) on T-cells include suppression of T cell activation and stimulation of regulatory T cells and changes in cytokine secretion patterns (Yee et al., Reference YEE, CHINTALACHARUVU, LU and NAGPAL2005). The VDR-ligands have been shown to influence maturation, differentiation, and migration of dendritic cells, and restrain DC-dependent T cell activation, commonly seen in immunosuppression (Van Etten and Mathieu, Reference VAN ETTEN and MATHIEU2005). Such immunoregulatory properties show that ligands with the potential to make active the VDR, accompanied by supplementation with 1,25-(OH)2D3, may have therapeutic clinical applications in the treatment of inflammatory diseases, dermatological conditions, osteoporosis, cancers, autoimmune diseases and in preventing organ transplant rejection (Nagpal and Rathnachalam, 2005).
Munger et al. (Reference MUNGER, LEVIN, HOLLIS, HOWARD and ASCHERIO2006) explained the facts of a relationship between Vitamin D deficiency and the start of multiple sclerosis, as the researchers thought that this may be caused by the immune response suppression properties of vitamin D. Advanced research shows that vitamin D is essential to stimulate a histocompatibility gene (HLA-DRB1*1501) essential for distinguishing between self and foreign proteins in a subgroup of individuals genetically inclined to multiple sclerosis (Tina, Reference TINA2009). Recently, Khan et al. (Reference KHAN, R., MIAN, SARDAR and ANJUM2010) reported that cholecalciferol supplementation improved disease resistance and enhanced the relative weights of lymphoid organs in broiler chicks. Komori et al. (Reference KOMORI, NAKANO, OHSUGI and SUGAWARA1985) and Huff et al. (Reference HUFF, HUFF, BALOG and RATH2000) concluded that 1-α hydroxyvitamin D3 enhanced immune responses by the activation of helper cells and this effect was reliant on the extent of the immune response. Aslam et al. (Reference ASLAM, GARLICH and QURESHI1998) reported that low levels of vitamin D in the diets of female broiler chickens caused a decrease in thymus weights, cutaneous basophil hypersensitivity response and phagocytosis by sephadex-elicited abdominal macrophages. Similarly, Garlich et al. (Reference GARLICH, GARLICH, FERKET and ASLAM1992) added 20 µg/kg supplemental cholecalciferol to turkey diets and reported an enhanced antibody response to sheep erythrocytes, raised phagocytosis by sephadex-elicited abdominal macrophages and a boost to the percentage of tumour cells killed by lipopolysaccharide-stimulated abdominal macrophages. Deluca and Cantorna (Reference DELUCA and CANTORNA2001) discovered that the major role of vitamin D compounds as selective immunosuppressant is showed by their ability to either prevent or stifle animal models of autoimmune diseases like encephalomyelitis and rheumatoid arthritis. These workers proposed feasible mechanisms of suppression of these autoimmune disorders, proposing that vitamin D hormone stimulates the conversion of growth factor TGFβ-1 and interleukin 4 production, which consecutively may suppress inflammatory T cell activity. The ability of supplemental vitamin D3 to influence the physiology and disease resistance of birds supports the present conceptual observation that vitamin D3 is a prohormone concerned in the homeostasis of various biological systems (Jones et al., Reference JONES, STRUGNELL and DELUCA1998).
Conclusions
The main functions of cholecalciferol are to maintain the blood levels of calcium and phosphorus. It is necessary for the absorption of these substances from the small intestine and for the mineralisation of bones. It regulates the parathyroid growth and parathyroid hormone production. It has a considerable effect on the immune system and can assist in the suppression of certain autoimmune diseases.
For poultry reared under commercial conditions, cholecalciferol may provide assurance for improving the economics of production in terms of body weight gain, feed efficiency and hatchability. As a way of decreasing disease problems in broilers, a modified feeding programme containing higher levels of vitamin D3 supplementation than NRC (1994) recommendations may be appropriate to allow for sufficient skeletal growth in the starter and finisher diets of broilers.
