INTRODUCTION
Poultry meat accounts for 30% of global meat consumption (FAO, 2010). This high consumption is associated with the affordable price and high nutritional value of chicken meat. However, with the genetic improvement of birds, bone development has not been proportional to weight gain in these animals, which has resulted in a higher incidence of locomotion problems, this has become a major concern due to reduced performance and increased carcass condemnations in slaughterhouses. Nutritional factors are known to improve locomotion problems. Among these is vitamin D, due to its important role in calcium and phosphorus metabolism.
To be used by the body, vitamin D3 must be metabolised following ingestion into 25-hydroxycholecalciferol (25(OH)D3) in the liver and subsequently into its active metabolite 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in the kidneys. These metabolites are currently commercially available, as 1-α-hydroxycholecalciferol (1α(OH)D3), a synthetic analogue of the active metabolite 1,25(OH)2D3, which is converted into its active form in the liver.
Vitamin D supplementation is closely related to a decreased incidence of bone disorders because vitamin D is involved in various physiological processes, including the absorption of calcium and phosphorus, bone mineralisation and mobilisation (Rennie and Whitehead, 1996; Driver et al., 2005; Korver, 2005; Kasim et al., 2006). In the body, vitamin D is required for the absorption of calcium and phosphorus in the intestines, increasing its utilisation efficiency and consequently increasing the bone ash density. Furthermore, vitamin D regulates the secretion of parathyroid hormone (PTH) and stimulates several tissues with vitamin D receptors (Norman, 1985). Therefore, vitamin D deficiency can further aggravate these factors, leading to decreased productivity and emergence of metabolic disorders.
Studies suggest that vitamin D may also affect growth performance (Yarger et al., 1995; Brito et al., 2010) and meat quality (Enright et al., 1999; Han et al., 2012), altering water retention (Montgomery et al., 2000), colour (Wilborn et al., 2004) and shear force (Rider et al., 2004). Vitamin D supplementation increases the intestinal absorption of calcium and phosphorus, stimulating the production of calcium-binding proteins in the mucosa, which activates the calcium activated tenderisation (CAT) complex through the increase in plasma calcium. This complex regulates the enzymatic activity of calpain and other proteases involved in the process of meat tenderisation (Santos, 2006).
The purpose of using these substances is to provide the animals with metabolised forms of vitamin D, increasing its efficiency in the body and decreasing energy expenditure. The absorption rate of 25(OH)D3 is approximately 20% higher than that of vitamin D3 (Applegate and Angel, 2005), and 1,25(OH)2D3 and 1α(OH)D3 do not require renal metabolism.
Therefore, the objective of this study was to assess the use of different vitamin D metabolites in the feed of broiler chickens and their effect on performance, bone parameters and meat quality.
MATERIALS AND METHODS
Animals and experimental design
The experiment was conducted in the poultry section of the Iguatemi Experimental Farm, State University of Maringá, under the approval of the Animal Experimentation Ethics Committee (Comitê de É tica de Animais em Experimentação/Universidade Estadual de Maringá-CEEA/UEM-Registration No. 034/2011). The study included 952 Cobb male broiler chicks, distributed into a completely randomised experimental design of four treatments (diets containing different vitamin D3 metabolites), seven replicates and 34 birds per experimental unit.
Diets
The treatments consisted of four different sources of vitamin D: cholecalciferol (D3), 25-hydroxycholecalciferol (25(OH)D3), 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 1α-hydroxycholecalciferol (1α(OH)D3). The different sources of vitamin D were included into the diet in place of the vitamin D, providing 2,000 IU of vitamin D3 in the pre-starter and starter phases and 1,600 IU of vitamin D in the growth phase, according to the recommendations described by Rostagno et al. (2005). The metabolites of vitamins D were added to the diet in replacement for the inert component. The vitamin supplement contained no vitamin D.
Diets were formulated using corn and soybean meal, according values based on the food chemical composition and nutritional requirements for male broilers in each phase (Rostagno et al., 2005). Water and feed were provided ad libitum in a feeding program divided into three phases: pre-starter, from the first to the seventh day of life; starter, from the eighth to the 21st d; and growth, from the 22nd to the 42nd d. Percentage and calculated compositions of the experimental diets are shown in Table 1.
Performance
Birds and feed were weighed weekly throughout the experimental period to assess performance (weight gain, mean weight, feed intake and feed:gain). Broiler mortality and leftover feed were recorded daily to correct the feed:gain per pen.
Bone sampling and analyses
To assess bone parameters, the left legs of two birds per experimental unit were collected at 7, 21 and 42 d of age and remained frozen (−20°C) until processing.
Once the legs were thawed, the muscle tissue was removed using scissors and tweezers, separating the femur and tibia. The bones were subsequently weighed on an analytical balance (g±0.0001), and the mid-leg length and diameter were measured using digital calipers (mm). The Seedor index (Seedor et al., 1991) was calculated using the formula Seedor index = bone weight (mg)/bone length (mm).
Bone strength analysis was performed using fresh thawed bones. The mechanism consisted of a base that supports the epiphyses of the bone and a force was applied to the central region of the bone. The values were expressed in kilograms-force (kgf).
Following the testing for bone strength, the bones were prepared for the determination of mineral content. The bones were defatted in petroleum ether, dried in a forced air oven, ground and weighed on an analytical balance (0.001 g). They were then dried at 105°C for 12 h, weighed after cooling and calcined in a muffle furnace at 600°C to obtain the ashes, following the methodology described by Oliveira (2006). The ash content of calcium and phosphorus in bone, were performed according to the methodology described by Silva and Queiróz (2004).
Muscle sampling and analyses
Meat quality variables were assessed using samples collected from the breast and thigh of two birds per experimental unit at 42 d of age, with the exception of water-holding capacity, for which samples were collected from only one bird per experimental unit.
The pH was determined directly from the left breast fillet of the birds using a TESTO® pH metre 15 min post mortem. Incisions were made and the electrode was inserted into three different regions: the upper, middle and lower breast muscle (pectoralis major). The estimate was obtained from the average of the three measured points.
The colour of the breast and thigh meats was assessed using a KONICA MINOLTA colourimeter model CR300. The characteristics under assessment were L* (lightness-from dark to light), a* (red/green intensity) and b* (yellow/blue intensity), with three replicates per point in three different regions: the upper, middle and lower breast muscle (pectoralis major) and the inner thigh, at 15 min post mortem, following the methodology proposed by Honikel (1998). The hue angle (true colour) and chroma (colour saturation) of the sample were determined at 15 min post mortem, according to the methodology described by Harder (2005).
The left breast muscle of the bird was used to determine the weight loss during cooking (WLC). The breast muscle was previously cooled and weighed to obtain the weight before cooking. The muscle was wrapped in aluminium foil and maintained on a commercial hot plate for approximately 10 min at 180°C, until the inside of the breast muscle reached 80°C. After the breast muscle reached room temperature, the sample was weighed again to obtain the weight after cooking.
The analysis of shear force was performed on the same fillets used to determine the weight loss during cooking. The samples were trimmed and cut into three rectangles (1.0×1.0×1.3 cm). The analysis described above was performed using a TAXT2i texturometre coupled to a Warner-Bratzler Shear Force mechanical probe with a 20 kg capacity and crosshead speed of 20 cm/min, providing a measure of shear force (SF) of the sample in kilograms-force (kgf/cm2).
The analysis of water-holding capacity (WHC) was performed according to the methodology proposed by Nakamura and Katok (1985). The left breast of one bird per experimental unit was collected shortly after slaughter, totalling seven samples per treatment. The method consisted of an initial weighing of 1 g of raw muscle, which was then wrapped in filter paper and centrifuged at 1,500 rpm for four min. The samples were weighed after centrifugation, dried in an oven at 70°C for 12 h and then weighed again. The percentage WHC was determined by calculating the difference between the weight of the meat sample after centrifugation and the weight of the sample after drying, then dividing this difference by the initial weight of the raw sample and multiplying by 100.
Statistical analysis
The statistical analysis of the data was performed using the Statistics and Genetics Analysis System (SAEG, 2005). Data were subjected to analysis of variance. The means of the studied variables were compared among the different treatments using the Tukey’s test for mean comparison, considering p<0.05.
RESULTS AND DISCUSSION
Growth performance
The means and standard errors for mean weight, weight gain, feed intake and feed:gain are shown in Table 2. The birds fed diets containing the metabolite 1α(OH)D3 presented lower weight gain and feed:gain for all of the evaluated time periods (p<0.05). In cases of prolonged toxicity, vitamin D can cause a decrease in feed intake and, consequently, decrease in performance (McCarthy et al., 1984; Zanuzzi et al., 2011).
Feed consumption in this study was affected by the different metabolites. In the time period of 1 to 21 days, the group of birds fed the metabolite 25(OH)D3 presented lower feed intake than the groups fed D3 and 1,25(OH)2D3. However, over the total rearing period (1 to 42 d), the group fed 1α(OH)D3 presented the lowest mean feed consumption when compared with the groups fed D3 and 1,25(OH)2D3. Nevertheless, only the group fed 1α(OH)D3 differed from the others (p<0.05) in terms of feed:gain for all of the evaluated time periods. Despite being an active analogue of vitamin D, 1α(OH)D3 has a lower utilisation efficiency in broilers, thus reducing animal performance (Han et al., 2009).
For the time period of 1 to 21 d, weight gain was greater (p<0.05) in animals fed 1,25(OH)2D3 than in those treated with 25(OH)D3; however, this difference did not persist across the total rearing period (1 to 42 d). As vitamin D undergoes hydroxylation, it is likely that the molecule tends to become more polar and more easily absorbed in the small intestines (Applegate and Angel, 2005). Furthermore, birds in the starter period do not have a complete enzymatic system with which to perform hydroxylation in the liver, which favours the administration of active metabolites (Swiatkiewiez et al., 2006), thus explaining the increased use of these already hydroxylated metabolites.
In high doses, 1α(OH)D3 can become toxic, reducing the absorption of calcium and phosphorus and thus impairing the animal performance (Reddy and Tserng, 1989). In this study, the metabolites were used in the diets to meet the vitamin D3 requirements for broilers, as recommended by Rostagno et al. (2005), replacing vitamin D3. This methodology was used to provide equivalent amounts of vitamin D3 given that each product provides different amounts of vitamin D3. Therefore, the manufacturers’ recommendations of 1α(OH)D3 were extrapolated by up to 10 times, which may have caused an overload within the animal bodies, resulting in decreased performance.
The supplementation of the different metabolites together with a source of vitamin D3 is a method for maximising animal performance because it provides the animals with a storage form of vitamin D. The metabolites will act together with the vitamin D to reduce energy expenditure, enhancing the results. The use of these compounds as a complete replacement of the vitamin D requirement could be an option with further investigation, especially considering the efficiency of each metabolite, given that the amount provided in this study may have caused toxicity.
Bone analyses
Mean bone parameters are presented in Table 3. Although 1,25(OH)2D3 increases calcium absorption in the intestines and reduces the formation of calcium complexes and therefore bone disorders (Grudtner et al., 1997; Applegate et al., 2003; Miller et al., 2006), no significant differences (p>0.05) in bone parameters, bone diameter, Seedor index, bone strength, or ash, calcium or phosphorus percentages were noted among the different metabolites used in this study (Table 3). Importantly, the values observed in this study for these characteristics were similar to those found in the literature (Aslam et al., 1998; Fritts and Waldroup, 2003; Driver et al., 2006; Han et al., 2009). Similar results were reported by Han et al. (2009), who observed that the metabolite 1α(OH)D3 decreased animal performance, although improvements in bone parameters were observed, indicating that the doses of vitamin D used in this study were inadequate for these bone parameters.
Among the metabolites tested in the present study, 25(OH)D3 is reported in the literature to be more active than vitamin D3 and to allow more efficient utilisation (Fritts and Waldroup, 2003). However, the different metabolites used in this study showed similar results for bone parameters. According to Aburto et al. (1998), the effects of D3, 1,25(OH)2D3 and 25(OH)D3 can vary depending on the biological response, with higher or lower efficiency depending on the characteristic assessed, such as greater bone strength or increased meat quality. These authors observed a greater reduction in the incidence of bone disorders with 1,25(OH)2D3 than with D3 and 25(OH)D3.
Moreover, all metabolites were provided in the feed so that equivalent amounts of vitamin D were offered, which may explain the lack of significant differences observed among the various metabolites because vitamin D acts to maintain homeostasis in the body.
Vitamin D3 deficiency can impair bone formation, considering vitamin D, calcium and phosphorus are extremely important for bone mobilisation (Pereira, 2010). None of the bone parameters under assessment were affected, suggesting that the levels used in the present study were in accordance with the requirements for bone tissue maintenance.
Meat analyses
The variables WHC, SF, WLC and the pH of the meat did not differ (p<0.05, Table 4) among the different vitamin D3 metabolite treatments. Differences among the treatments were expected for the studied characteristics because vitamin D is involved in calcium metabolism and the activation of calpains, calcium-dependent proteases that act in the process of meat tenderisation (Montgomery et al., 2000).
The lightness of breast meat 15 min post mortem was greater in animals fed 1,25(OH)2D3 than in those fed vitamin D3 (p<0.05). Lightness is related to the denaturation of meat protein; therefore, the greater the denaturation, the greater the release of intracellular fluid and the less light transmitted through the fibres. These changes in biochemical reactions result in paler meat and higher values of L* (Duarte et al., 2007). However, the greater lightness in this case did not reach the values characteristic of Pale, Soft and Exudative (PSE) meat (L*<53.0), given that meat considered to be PSE is characterised not only by values of L* but also by a sharp decline in pH. A greater calpain activity and consequently higher values of L* are expected with the use of vitamin D.
The shrinkage of muscle fibres is caused by the decrease in pH, which increases the loss of liquid and the reflectance of the meat. However, despite the differences (p<0.05) in lightness (L*) observed among the treatments in this study, the water-holding capacity of breast meat was not affected (p>0.05). In general, higher values of lightness are positively correlated with lower water-holding capacity (Castellini et al., 2002). The latter is related to the formation of lactic acid and decrease in post mortem pH, which reduces the enzymatic degradation of the myofibrillar structure, thereby worsening the water-holding capacity (Roque-Specht et al., 2009).
The red/green intensity (a*) of the thigh meat 15 min post mortem was greater in birds fed 25(OH)D3 than in those fed vitamin D3 (p<0.05). The high intensity of a* is related to the formation of the carbon-myoglobin complex, attributed to the reduction of the iron ion present in myoglobin molecules combining with oxygen to form oxymyoglobin, which confers a darker colour to the meat, influencing the type of muscle fibre.
Vitamin D acts by regulating the concentrations of calcium in animal blood and muscles. Therefore, after the animal is slaughtered, there is a decrease in pH that activates calcium-dependent proteases, with increased activity of calpains and other proteolytic enzymes, causing a consequent effect on colour, texture, tenderness and water-holding capacity. However, in this study, the pH of the meat was similar among animals fed different vitamin D metabolites, indicating that these act in a similar manner on calcium concentrations. Values of pH are negatively correlated with yellow/blue intensity (b*) and lightness (L*). Therefore, a decrease in pH reduces the myoglobin, resulting in meat that is less red and more yellow (Han et al., 2012).
However, at 24 h post mortem, the highest values of a* in thigh meat were observed in the treatment containing D3, and the lowest values were noted for 1,25(OH)2D3, with no significant difference in relation to the other treatments. The yellow/blue intensity (b*) in thigh meat, 24 h post mortem, was higher in animals fed vitamin D3 and 25(OH)D3 than in those fed 1α(OH)D3 (p<0.05). The metabolite 1α(OH)D3 is metabolised in the liver to form 1,25(OH)2D3, which has a shorter half-life (6 to 8 h) than 25(OH)D3 (2 to 3 wks). Therefore, despite having a higher rate of intestinal absorption, its use can be impaired by the lack of body reserves (Silva et al., 2008; Castro, 2011).
Although the different metabolites influence these parameters, the true colour and hue angle of the meat cuts were similar among the treatments because the colour of the meat is a result of the selective absorption of light and depends on numerous factors, such as the concentration of myoglobin, fibre composition, proteins and the presence of liquid in the meat (Gaya and Ferraz, 2006).
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