Efficacy of different limestone particle size and 25-hydroxycholecalciferol in broiler diets

In a 6-week experiment on 560 Cobb broilers, the response of chickens to supplementation of diets with particulate grit and 25-hydroxycholecalciferol (25-OH-D3) was evaluated. Maizeand soyabean meal-based diets contained pulverized limestone or a mixture of 70% pulverized and 30% particulate (1.2-4 mm in diameter) limestone as a source of calcium. Both diets were supplemented with vitamin D3 (cholecalciferol) or 25-OH-D3 was substituted in amounts equivalent to 20, 40, 50, 60, 80, or 100% of cholecalciferol. Incorporation of particulate forms of limestone into the diet positively affected performance and the ash and Ca contents in the tibia. Replacement of cholecalciferol by 25-OH-D3 at a level of 50 and 60% had a beneficial effect on body weight gain and feed conversion, 100% substitution, on the Ca content of the tibia, whereas 50, 60, 80 and 100% substitution, on Ca and P balance.


INTRODUCTION
Adequate dietary levels of calcium, phosphorus and vitamin D are required for chicken growth and normal bone development.Utilization of dietary calcium carbonate by chickens may depend more on particle size than origin.Guinotte et al. (1991) found that in comparison with ground calcium carbonate (<0.15 mm), coarse particles (>1.18 mm) incorporated into the diet diminished weight gain and feed conversion, tibia characteristics and ash content, and decreased calcium retention.In contrast, in a study by Zohravi (2002) the incorporation of coarse particle limestone (1.18-4.75mm) into the diet significantly improved the feed conversion ratio, tibia ossification and Ca and P retention.
Cholecalciferol is supplemented to practical diets for broiler chickens in large amounts and is transformed in tissues to active metabolites (Collins and Norman, 1991).The occurrence of signs of rickets and dyschondroplasia in chickens fed practical diets rich in vitamin D 3 may suggest that transformation of cholecalciferol in the liver and kidney to 25-OH-D 3 and 1,25-(OH) 2 -D 3 is not sufficient.Both active metabolites added to the diet were found to be effective sources of vitamin D 3 (Edwards, 1990;Rennie and Whitehead, 1996).Yarger et al. (1995) reported better performance of birds fed 25-OH-D 3 in comparison with those fed vitamin D 3 at a dietary level of 2760 IU · kg -1 .
The present study was undertaken to evaluate the responses of broilers to diets containing pulverized limestone or mixed particulate grit and a pulverized form of limestone, and cholecalciferol alone or gradually substituted by 25-OH-D 3 .

MATERIAL AND METHODS
A 6-week experiment was carried out on 560 Cobb broiler chickens.Sexed day-old chickens were allotted to 14 groups in 5 replicates, each containing 4 males and 4 females, and kept in cages with wire mesh floors.The diets provided in the first (starter, 1-21 days) and second (grower-finisher, 22-42 days) feeding periods contained pulverized limestone or a mixture of 70% pulverized and 30% particulate (1.2-4 mm mesh) limestone as the calcium source.The starter and grower-finisher diets (Table 1) were supplemented with 2000 and 1500 IU of cholecalciferol • kg -1 , respectively (as Lutavit 500, BASF).The diets for groups I and VIII were supplemented only with vitamin D 3 ; in the diets for the other groups, the 25-OH-derivative replaced 20, 40, 50, 60, 80 or 100% of the cholecalciferol (Table 2).The source of 25-OH-D 3 (Hy•D Beadlet, DSM) contained 12.5 mg 25-OH-D 3 per g, which was equivalent to 500 000 IU of vitamin D 3 .
Body weight and feed intake were measured, mortality was registered and body weight gain (BWG) and feed conversion ratio (FCR) were calculated for the first and second periods of feeding and for the whole experiment.On the basis of body weight gain, feed conversion and mortality, the performance efficiency index (EPE-index) was calculated.Between days 15 to 19 of life, feed intake was measured and total collection of excreta from each pen was carried out in groups I-VII fed pulverized limestone.Excreta were stored at -20 o C.After thawing, the excreta were dried in an oven at 50 o C, left on air for two days, weighed and homogenized.In the samples of diets and excreta, the Ca content was determined by flame atomic absorption spectrophotometry using a Philips PU 9100 apparatus and the P content was determined colorimetrically by the molybdo-vanadate method (AOAC, 1990).Lysine and methionine were analysed in the basal diet in acid hydrolysates in a colour reaction with the ninhydrin reagent, using a Beckman-System Gold 126 AA automatic analyser.Methionine was estimated after preoxidation to methionine sulphone.
On day 43 of age, 8 chickens (4 males and 4 females) from each group were slaughtered by decapitation, plucked, eviscerated and the carcasses were cooled.On the next day the carcass and gizzard were weighed, breast muscles (M.pectoralis maior and minor) from each carcass were excised and weighed.Eight tibia bones per replicate were collected, dried at 100 o C for 24 h, weighed, and dryashed at 600 o C for determination of crude ash and Ca.The data were subjected to statistical analysis using two-way analysis of variance, except Ca and P balance data, which were calculated using one-way analysis of variance.The significance of differences between means was determined by Duncan's multiple range test using the Statistica 5.0 PL software package.

RESULTS
In the first period of feeding (1-21 days of age) the performance of chickens was not affected by the kind of calcium source and vitamin D 3 substitution (Table 3).
In the second period of feeding (Table 4), the replacement of pulverized limestone by particulate limestone had a positive effect on BWG (P≤0.001),feed intake (P≤0.05) and FCR (P<0.01).Chickens fed the diet supplemented with cholecalciferol had the lowest weight gain and feed intake.Substitution of 25-OH-D 3 for cholecalciferol at a level of 50, 60, 80 and 100% significantly improved BWG and FCR.
For the whole period of feeding (Table 5) the improvement of BWG and FCR was confirmed when the diet contained 30% limestone in particulate form (P≤0.001).Replacement of cholecalciferol at a level of 50 and 60% also positively affected BWG and FCR.Improvement of the performance efficiency index was observed when 30% of the particulate calcium source was incorporated into the diet and 25-OH-D 3 substituted for 50, 60 and 100% of the cholecalciferol added to the diet.Experimental factors had no effect on the results of slaughter yield and on the percent of breast muscle and gizzard in the carcass (Table 6).The crude ash content in tibia bones (Table 7) differed significantly between chickens fed different forms of limestone, but substitution of 25-OH-D 3 for vitamin D 3 made no difference.The bone ash content in chickens fed pulverized limestone was lower than in groups fed mixed forms of limestone (P≤0.05).The calcium content in tibia bones (Table 7) was significantly affected by both treatment factors.In chickens fed pulverized limestone the Ca content in bones was lower than in groups fed mixed forms of limestone (P≤0.001).Complete replacement of cholecalciferol by 25-OH-D 3 significantly increased the Ca content as compared with groups fed only vitamin D 3 (P≤0.05).An interaction was observed between limestone source and D 3 substitution by 25-OH-D 3 .
A balance trial was conducted only in groups I-VII of chickens fed pulverized limestone (Table 8) because the excreta from groups fed the particulate limestone contained grit and were not susceptible to homogenization.The daily calcium retention in chickens was lowest when 100% D 3 was added to the diet as cholecalciferol and increased (P≤0.05) when 25-OH-D 3 substituted for 60-100% of vitamin D 3 .Daily amounts of Ca excreted in excrements, and relative Ca retention (in % of Ca ingested) did not differ statistically, however.Nevertheless a numerical tendency was observed for 3-8% higher values of relative Ca retention when the 25-OH-D 3 metabolite was supplemented to the diet.Daily retention of phosphorus and relative P retention (in % of P ingested) were the lowest when the diet was supplemented with cholecalciferol (Table 8).Daily P retention significantly increased (P≤0.05) when 70-80% vitamin D 3 was replaced by 25-OH-D 3 .The relative P retention in chickens (as % of P intake) increased at vitamin D 3 substitution levels of 20, 50 and 60%.

DISCUSSION
The kind of calcium source and range of 25-OH-D 3 substitution for cholecalciferol affected chicken performance only in the second period of feeding and for the whole experiment.Incorporation of 30% of limestone as particulate grit had a positive effect on weight gain, feed intake and the feed conversion ratio.Grit can probably stimulate the digestive tract and nutrient utilization.Compared with diets containing pulverized limestone, a mixture of pulverized and particulate limestone forms increased the percentage ash and calcium contents in the tibia.These findings agree with the results of Zohravi (2002), who used 0.6 to 1.18 mm and 1.18 to 4.75 mm particle sized limestone versus less than 0.15 mm.Guinotte et al. (1991) used coarse particles of limestone (greater than 1.18 mm) as the sole source of Ca added to the diet and found a negative effect on performance and bone quality of chickens.These results and the results of the present study suggest that for good performance, chickens require the presence of limestone in powdered form in the diet and that particulate grit should make up only a part of the limestone.
Introduction of the 25-OH-derivative as a partial or complete (50 to 100%) substitute for cholecalciferol increased body weight gain and improved the feed conversion ratio as compared with chickens fed only vitamin D 3 .No positive effect was observed at lower levels of substitution.A significant increase of the Ca content in the tibia was noted when 25-OH-D 3 completely replaced D 3 in the diet.This may suggest that synthesis of the active form of cholecalciferol in the liver is not sufficient for chicken performance and bone calcification.Improvement of body weight in chickens fed 25-OH-D 3 as a complete substitute for D 3 was reported by Fritts and Waldroup (2003) for diets with added 125-1000 IU D 3 and by Yarger et al. (1995) for diets with 2760 IU D 3 · kg -1 .A greater percentage of bone ash and a lower incidence and severity of tibial dyschondroplasia in chickens fed 25-OH-D 3 were reported by Fritts and Waldroup (2003).
The data from the present experiment may suggest that a mixture of pulverized and particulate limestone is a more effective source of calcium than pulverized limestone alone and that addition of the 25-OH vitamin D 3 derivative to the diet better covers chickens' requirements for calcium-vitamin D 3 metabolism than cholecalciferol.Improved performance was noted when chickens were fed a mixture of particulate and pulverized limestone and when 50 and more percent of the added vitamin D 3 was replaced by 25-OH-D 3 .
The results of the balance trial were consistent with performance and bone ash and Ca content.Retention of Ca and P in chickens increased when cholecalciferol added to the diet was substituted by 25-OH-D 3 at a level of 60-100 and at 70-80%, respectively.Relative P retention grew significantly at 50-60% of substitution, but daily P excretion in excrements was not changed.Substitution of 25-OH-D 3 for cholecalciferol did not decrease the emission of P into the environment.Fritts and Waldroup (2005) reported that phosphorus utilization was not improved as a result of 25-OH-D 3 incorporation into a diet with a reduced nonphytate P content and relatively high vitamin D 3 level, supplemented with microbial phytase.This could suggest that the efficacy of 25-OH-D 3 is greater at lower levels of dietary vitamin D 3 supplementation.At levels typically used by the poultry industry, e.g., 10 to 20 times above NRC (1994) recommendation, the effect of 25-OH-D 3 substitution for cholecalciferol on P utilization by chickens is negligible (Fritts and Waldroup, 2005).

CONCLUSIONS
Replacement of 30% of pulverized limestone in the diet by particulate grit had a beneficial effect on performance, ash and Ca content in the tibia.Partial or complete substitution of 25-OH-D 3 for the cholecalciferol added to the diet positively affected performance, Ca content in the tibia, and results of Ca and P balance.

TABLE 3
Performance in the first period of feeding (1-21 days of age)

TABLE 8
Retention and excretion of Ca and P, mg per chicken per day