MATERIALS AND METHODS

Animals and Treatments
A total of 300 one-day old Ross 308 broilers were utilized in this study. Initial weights of each chick were determined at the initiation of experiment. Then, chicks were randomly allotted into 5 treatment groups consisted of 4 subgroups. Each treatment group contained a total of 60 chicks with 15 chicks within each subgroup.

The diets (starter and finisher) used in the experiment were prepared in a private milling company and corn-soybean meal based with considerable amount of barley (14% of DM). Dietary P levels was calculated to provide 70% of required non-phytate P but total P levels were greater than the required levels (NRC, 1994). This basal diet (negative control) was supplemented with enzyme (Rovabio^®; control). Then, control diet was supplemented with 500 g ton^-1 microbial phytase (Rovaphos^®; 500 g phytase), 1000 g ton^-1 microbial phytase (1000 g phytase) and 1500 g ton^-1 microbial phytase (1500 g phytase). Chemical and botanical compositions of diets were given in Table 1. A total of 5 different diets were prepared by addition of rovabio, 500 g ton^-1 phytase+rovabio, 1000 g ton^-1 phytase+rovabio and 1500 g ton^-1 phytase+rovabio into negative control diet. Chicks were fed these diets as group of 15 chicks within each subgroup and experiment lasted 42 day. Plastic feeders and wagerers were utilized in the experiment. Chicks were maintained on a 23 h constant light schedule and in a heat-controlled room.

Determination of Animal Performance
Individual live weight gains and feed intakes were determined weekly (initiation, 7, 14, 21, 28, 35 and 42 days of experiment) using a digital scale with a sensitivity of 10 mg. Chicks had free access to fresh feed and water throughout the experiment. Chicks that died were recorded daily. Amount of feed given was recorded daily. Amount of oarts were determined at end of each week. Feed intake per week and feed efficiency were, then, calculated. During the first three days of the 5th and 6th week of trial all excreta were collected from each pen. Excreta were stored in plastic bags at -20°C. Before chemical analysis samples were defrosted, homogenized and dried at 60°C. Then they were weighed and ground to pass a 1 mm sieve. Calcium, total and phytate phosphorus contents were determined by the same chemical methods used in diet analysis.

Determination of Carcass and Internal Organ Weight
Carcass and total internal organ (liver, heart, gizzard and pancreas), intestine and abdominal fat weights were determined at the end of experiment.

Table 1:	Botanical and chemical composition of the experimental diets
*Vitamin-Mineral premix (IU or mg kg-1 diet): Vitamin A: 12000 IU; Vitamin D3: 1500 IU; Vitamin E: 30 mg; Vitamin K3: 5 mg; Vitamin B1: 3 mg; Vitamin B2: 6 mg; Vitamin B6: 5 mg; Vitamin B12: 0.03 mg; Nicotine amid: 40 mg; Calcium-D-pantothenate: 10 mg; Folic acid: 0.075 mg; Choline chloride: 375 mg; Antioxidant: 10 mg; Manganese: 80 mg; Iron: 80 mg; Zinc: 60 mg; Copper: 8 mg; Iodine: 0.5 mg; Cobalt: 0.2 mg; Selenium: 0.15 mg. Phytase: Rovaphos® contains 500 000 FTU kg-1 and isolated from Peniophora Iycii. Rovabio® contains Endo-1, 4-β-xylanase: 22,000 visco. Units per g (equivalent to 1,400 AXC units per g) Endo -1, 3 (4) β-glucanase: 2,000 AGL units per g (Trouw Nutrition Turkey)

Blood Sampling
Blood samples were randomly taken from V. subcutanea ulnaris of selected 4 chicks from each treatment groups to determine blood glucose, total protein, urea, Ca and P.

Chemical Analysis
Chemical compositions of diets were analyzed according to AOAC (1990). Blood glucose, total protein, urea, Ca and P levels were analyzed using auto-analyzer (Hitachi 912, Boehringer Mannheim). Dietary, bone, feces Ca and P levels were determined according to Combs et al. (2003).

Statistical Analysis
Experimental data were subjected to statistical analysis by using GLM of SAS for variance analysis. The differences among means were separated by Duncan t-test (SAS, 2005). All values were presented as means and standard error (SEM). The level of significantly difference was set up at p<0.05.

RESULTS AND DISCUSSION

Initial body weights of broilers were similar as a part of experimental design. However, body weight of broiler fed low available phosphorus diets supplemented with phytase were significantly higher (p<0.05) compared with broilers fed low available phosphorus diet without phytase throughout the experiment starting from second week of experiment (Table 2). There were no statistical differences on body weight of broilers fed either control or diets containing phytase throughout the experiment (p>0.05).

Table 2:	Live weight gain (g) of broiler fed different dietary treatment
Mean values with different superscripts within a column are significantly different, (p<0.05)

Table 3:	Daily feed consumption (g) of broiler fed different dietary treatment
Mean values with different superscripts within a column are significantly different, (p<0.05)

However, the highest live weight (1664.05 g) was obtained for the supplementation of 1500 g phytase per ton feed. Many studies (Ahmed et al., 2004; Pintar et al., 2004; Bozkurt et al., 2006) have reported the increases in live weight gain in broilers with increasing concentration of dietary phytase in soybean meal based diets, which are in agreement with the result of this study. Zyla et al. (2000) have noted that the growth rate of broilers fed low phosphorus diets containing microbial phytase were comparable with or even better than those fed standard phosphorus diet, supporting the concept that phytase improves not only availability of phosphorus (Bozkurt et al., 2006) but also the utilization of the essential amino acid in broilers fed soybean meal basal diets (Biehl and Baker, 1997). However, Pizzolante et al. (2002) have found that dietary phytase had no effect on live weight gain of broilers. These differences among studies might have resulted from the amount of available phosphorus used and amount of phytase added in diets.

Feed consumption of broilers fed control or diet supplemented with phytase were similar throughout the experiment (p>0.05), except second week of experiment. Broilers fed diet containing 500 g phytase/ton had significantly higher feed consumption compared with other diets containing phytase in the second week of experiment (p<0.05). Broilers fed negative control had significantly lower feed consumption compared with other groups throughout the experiment (p<0.05). Phytase supplementation to low available phosphorus diets significantly increased feed intake (p<0.05) compared with low available phosphorus diet without phytase (negative control diet) (Table 3). Similar to the result of this study, Ahmed et al. (2004) and Pintar et al. (2004) have reported that feed consumption significantly increased in broilers fed diets supplemented with phytase. Bozkurt et al. (2006) have noted that feed intake and feed efficiency of broilers fed diet containing phytase were similar to those fed control diet containing dicalcium phosphate, which support the result of the current study. The increases in feed consumption with phytase supplementation might have resulted from increases in digestibility of nutrients and partial cell wall degradation.

Feed efficiency of broilers fed control or diets containing phytase were similar during whole experimental period (Table 4, p>0.05). Although broilers fed negative control diet had significantly lower feed efficiency at 2, 3 and 4th weeks of experiment compared with other treatment groups (p<0.05), feed efficiencies were similar among all of the treatment groups last two weeks of experiment (p>0.05). The feed efficiency results are in agreement with the findings of Bozkurt et al. (2006), who noticed that feed efficiency of broilers fed diet containing phytase were similar to those fed control diet with dicalcium phosphate.

Table 4:	Feed efficiency of broiler fed different dietary treatment
Mean values with different superscripts within a column are significantly different, (p<0.05)

Table 5:	Carcass and internal organ weights (g) of broiler fed different dietary treatment
Mean values with different superscripts within a column are significantly different, (p<0.05)

Table 6:	Percentages of Ca, P in tibia and feces of broiler fed different dietary treatment, (%)
Mean values with different superscripts within a column are significantly different, (p<0.05)

However, the lower feed efficiency observed in this experiment with control diet contradicts the result of Bozkurt et al. (2006). Reduction in dietary NPP depressed live weight gain and feed intake and increased feed conversion ratio (Singh et al., 2003). Similar to present results, some researchers have also noted that even though addition of phytase into diets increased both live weight gain and feed intake, it did not have positive effect on feed efficiency (Huff et al., 1998; Brenes et al., 2003), which is in agreement with the results of the current study.

Carcass weights of broilers fed control or diets containing phytase were statistically similar (p>0.05), but broilers fed negative control diet had significantly less carcass weights compared with other groups (p<0.05). The highest carcass weight was obtained in broiler fed diet containing 1000 g ton^-1 pytase (Table 5). A positive correlation between dressed weight and live weight and age has been earlier reported by Howlider and Rose (1989) and Ahmed et al. (2004). Dressed weight was a function of live weight, thus, as live weight increased carcass weight also increased accordingly. Preston et al. (2000) reported an increased carcass yield with addition of phytase into diet, which coincided with the result of the current study. Weights of intestine and abdominal fat were similar among treatment groups, but weight of internal organs was less in broilers fed negative control compared with other groups. The differences in internal organ weights could be due to the differences in size of broiler in different treatment groups.

The percentages of phosphorus and calcium in tibia increased with the addition of phytase into diet (Table 6, p<0.05). Calcium contents of broiler fed negative control were less compared with those fed either control or diets supplemented with phytase (p<0.05). Similar to this study, Midilli et al. (2003) have reported an increased Ca and P content of tibia with addition of phytase into diet.

Table 7:	Serum glucose, total protein (g dL-1), P, Ca and urea levels (mg dL-1) of broiler fed different dietary treatment
Mean values with different superscripts within a column are significantly different, (p<0.05)

An increase in ash and phosphorus content of tibia has been described as a good indication of increased availability of phosphorus (Bozkurt et al., 2006). The increases in tibia phosphorus and calcium content of broilers fed diets containing phytase clearly indicated that availability of phosphorus increased by the action of phytase in this study. The improvement in phosphorus availability by phytase also significantly reduced amount of phosphorus excreted in manure, which was supported by the results of Pintar et al. (2005) (p<0.05). However, phytase did not affect amount of calcium excreted in manure (p>0.05). Simons et al. (1990) have shown that the viability of phosphorus increased over 60% and the amount of phosphorus in manure decreased by 50% with microbial phytase supplementation of low phosphorus diet. Plumstead et al. (2007) also showed that phytase inclusion in a broiler breeder laying diet at the expense of all added P from dicalcium phosphate reduced the manure total P and water soluble P concentrations by 42%, which support the result of this study.

Total serum glucose, protein and phosphorus levels were statistically similar among groups (Table 7) (p>0.05). However, both total serum protein and phosphorus levels linearly increased with increasing level of phytase in diets. Brenes et al. (2003) have reported that serum Ca (4%), P (12%) and total protein (7%) increased with phytase supplementation into broiler diet. Even though increases in serum total P and protein were not significant, they support the results of Brenes et al. (2003). While serum calcium levels decreased serum urea level increased with addition of phytase into diet (p<0.05).

The results of the current study showed that microbial phytase in broiler diets enhanced the availability of phosphorus. Therefore, the growth performance and carcass yield of broilers increased and phosphorus content of manure decreased with addition of phytase into broiler diet, which supplementation of phytase did not only improve animal performance but also reduced potential environmental pollution. Thus, it can be concluded that dietary available phosphorus can be reduced to 30% in broiler diet with 1000 g phytase/ton supplementation without affecting animal performance.

ACKNOWLEDGMENTS

This study was supported by a grant from the Scientific Research Projects Fund of Yuzuncu Yil University, Van, Turkey with a grant No. 2006-VF-YTR27. We thank to Trouw Nutrition, Turkey for their great effort to supply us necessary requirements in our Project. They should note that we appreciate their aim in contributing to the scientific activities of Turkey.