The effects of dietary phytase and l , 25 ( OH ) 2 D 3 on growth and strength of bones in broiler chickens at day 21 of life

The investigation was carried out on 90 broiler chickens receiving experimental low-phosphorus starter diets until day 21 of life: C, control; D, with the addition of 750 phytase units/kg; and R, without vitamin D 3 supplemented with 3 jig l,25(OH) 2D 3 and 750 PTU/kg. On day 21,15 chickens from each treatment were killed. Humeri and femora were isolated, weighed and physical and geometrical parameters of bones were determined. Bone mass and length in both experimental groups were similar, but showed greater values in relation to controls. The femora and humeri of control and experimental chickens showed significant differences in physical parameters. The values of geometrical parameters in the experimental group were higher than in the control, but not significantly in either of the experimental groups. Bone breaking strength was significantly affected by the combined addition of l,25(OH) 2D 3 and phytase to the diet without vitamin D 3 as compared with the diet with phytase and adequate level of vitamin D 3 .


INTRODUCTION
About two-thirds of phosphorus in plant feed is in the form of phytates.Poultry and pigs do not have enzymes capable of hydrolyzing phytates.The use of microbial phytase can effectively increase digestibility of phosphorus from phytate (Perney et al., 1993;Qian et al., 1996).Phosphorus and calcium are needed for optimal growth and development of chondral and osseous tissues.The control of absorption, transportation and deposition of calcium and phosphorus, important for normal bone mineralization, is the main function of vitamin D 3 and its metabolites.Vitamin D 3 must be provided in the diet for broiler chickens to prevent rickets and other skeletal problems and to optimize growth.Vitamin D 3 must undergo two-step hydroxylation to produce 1,25-dihydroxycholecalciferol, the biologically active hormonal form, in order to carry out its physiological functions.Some conditions may impair absorption or hydroxylation of vitamin D 3 .Furthermore, some authors have indicated that 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol are two to four times as active as vitamin D 3 (Aburto et al., 1998).This provides the rationale for the use of l,25(OH) 2 D 3 in broiler diets.
The aim of the present study was to evaluate the effects of microbial phytase and 1,25(OH) 2 D 3 supplementation of the diet on growth and development of bones in broiler chickens.

MATERIAL AND METHODS
The investigation was carried out on 90 broiler chickens.From the first day of life until day 21, the chickens received an experimental low-phosphorus starter diet (calculated non-phytate P, 0.343%; Ca, 0.718%).The chickens were divided into 3 groups: C, the control group fed on the vitamin D 3 -adequate diet; group D, fed on the diet with the addition of vitamin D 3 on an adequate level plus 750 phytase units (PTU)/kg (Natuphos 5000, BASF, Germany), and group R, fed on the diet without vitamin D 3 but supplemented with 3 jLtg l,25(OH) 2 D 3 and 750 PTU/kg.The chickens were kept in standardized zoohygienic conditions.Feed and water were available ad libitum throughout the experimental periods.
On day 21, 15 chickens from each treatment were randomly selected and killed by cervical dislocation.Humeri and femora were isolated and frozen for further mechanical and geometrical analysis.The bones were later stripped of all soft tissues.Length and weight of bones were measured.Physical bone characteristics were determined by the three-point bending test using an Instron Testing Machine (Model 4302, Instron Corp., USA) (lkN 10 mm/min).The following parameters were measured: ultimate stress, elastic force, and stiffness.After the mechanical test, structural parameters were measured: second moment of inertia, cross section area, mean relative wall thickness.
The results were analyzed statistically (Anova), using Statistica Software, version 5.

RESULTS
The weight and length of the analyzed bones are presented in Table 1.On day 21 of life, birds fed the diet with l,25(OH) 2 D 3 and phytase had significantly higher femur and humerus ultimate stress and elastic force than the chickens fed on vita-    1 to min D 3 only or those fed on vitamin D 3 and phytase (Figure 1).This was not accompanied by significantly different values of geometrical parameters for these bones among experimental chickens (Table 2).The geometrical parameters of bones in experimental chickens were greater than in the control group (Table 2).The strength of bones in chickens fed on the diet with phytase and an adequate level of vitamin D 3 were also higher than in the control group.The stiffness of the femora and humeri in both experimental groups did not show significant differences, but was greater than in the control group (Table 2).
ultimate stress elastic force ultimate stress elastic force

DISCUSSION
Bone weight and length were not significantly different in experimental groups, but greater than in the control group.In contrast, Frost et al. (1990) reported that l,25(OH) 2 D 3 may have an effect on increasing bone mass in laying hens.Values of physical parameters of bones in chickens fed on the diet with the addition of phytase were higher than in the control, which is in agreement with reports of other authors (Perney et al., 1993;Qian et al., 1996;Puzio et al., 2000).In the present study, l,25(OH) 2 D 3 was more effective on the mechanical properties of bones than vitamin D 3 .These results suggest that short term l,25(OH) 2 D 3 supplementation of a diet without vitamin D 3 will improve the strength of bones.Lack of significant differences among geometrical parameters suggests that these differences in ulti-mate stress and elasticity among groups may be a result of changes in the mineralization of the bone matrix and probably are not due to differing amounts of bone material.This is not consistent with the reports of other authors (Ferretti et al., 1993;Turner and Burr, 1993;Puzio end Studzihski, 2000), who claim that the strength of bones is due to both their mineralization and structural parameters.

CONCLUSION
On day 21 of life, bone breaking strength was significantly affected by combined addition of l,25(OH) 2 D 3 and phytase to the diet without vitamin D 3 as compared with the diet with phytase and an adequate level of vitamin D 3 .

TABLE 1 g
Mean values of weight and length of bones (mean ±SE)

TABLE 2
Mean values of stiffness and geometrical parameters of bones (mean ±SE)Parameters abas in Table1C, D, R -as in Table