Effects of the Combination of Non-Phytate Phosphorus, Phytase and 25-Hydroxycholecalciferol on the Performance and Meat Quality of Broiler Chickens

This experiment was conducted to evaluate the combination effect of low dietary non-phytate phosphorus (NPP) concentrations, phytase (PHY) levels, and 25-hydroxycholecalciferol (25-OH-D3) levels on the growth performance and meat quality of broilers. Two levels of NPP, two levels of PHY, and two levels of 25-OH-D3 resulted in a 2×2×2 factorial arrangements, with eight treatments (TRT). The birds on TRT 1-4 were fed diet 1 (NRC NPP was reduced by 0.1) and the birds on TRT 5-8 were fed with diet 2 (NRC NPP was reduced by 0.2). Each diet was mixed with different levels PHY and 25-OH-D3. Performance and meat quality parameters were measured. Results showed that during entire experiment the most advantageous effects were obtained with TRT 3 (NRC NPP reduced by 0.1 + 600 U/kg phytase + 34.5μg/kg 25-OH-D3) and TRT 4 (NRC NPP reduced by 0.1 + 600 U/kg phytase + 69μg/kg 25OH-D3). The lowest body weight gain (BWG) and feed intake(FI) were observed with TRT 5 (NRC NPP reduced by 0.2 + 300 U/kg phytase + 34.5μg/kg 25-OH-D3). Lowering NRC NPP by 0.1 to 0.2 significantly reduced weight gain (WG) (p< 0.05) and FI (p< 0.05) during the starter phase (ST), while during grower phase (GF) lowering NRC NPP by 0.1 to 0.2 did not affect WG (p>0.05) and produced small decrease in FI. BWG, FI and feed conversion ratio were not influenced (p>0.05) by different PHY or 25-OH-D3 levels. In addition, the meat color, pH, and shear force were not affected by the different NPP, PHY or 25-OH-D3 levels.


INTRODUCTION
Phosphorus (P) pollution from animal waste has become a major environmental concern in recent years.Reducing dietary phosphorus is an effective method to minimize phosphorus pollution (Angel et al., 2006;Powell et al., 2008;Rama Rao et al., 2009;Pillai et al., 2009).Recent studies showed that the National Research Council (NRC,1994) recommendations of non-phytate phosphorus (NPP) exceed the requirements of modern broiler chickens (Angel et al., 2000a,b;Yan et al., 2001 andAngel et al., 2005).It should be an important economic and environmental concern to formulate diets to precisely meet the phosphorus requirements of poultry (Summers,1997).

Effects of the Combination of Non-Phytate Phosphorus, Phytase and 25-Hydroxycholecalciferol on the Performance and Meat Quality of Broiler Chickens
utilization (Biehl et al., 1995;Janocha 2et al., 2009 andTatara et al., 2011).The 25-OH-D 3 has been successfully developed into a new feed additives to the industry today.Previous reports (Angel et al., 2005;Driver et al., 2005;Coto et al., 2008;Liem et al., 2009) suggested that cholecalciferol analog might work either alone or in combination with phytase to markedly improve utilization of dietary P.However, the combination effect of lower dietary NPP levels, PHY levels and 25-OH-D 3 levels has not been evaluated.
Supplementation of vitamin D 3 affects meat quality (Montgomery et al., 2004;Moron et al., 2008).Han et al. (2009) reported that 1α-OH-D 3 increase lightness and yellowness of the breast and thigh meat whereas it decrease the shear force and water-holding capacity of the thigh meat.
Our objective was to evaluate the combination effect of lower dietary NPP levels, PHY levels and 25-OH-D 3 levels on the growth performance and meat quality of the broilers.

Birds, Diets, Feeding, and Management
The procedures of this experiment were approved by the Animal Care and Use Committee of the South China Agriculture University.
The experiment was conducted in a broiler house for 42 d during the fall.Two thousand and four hundred one-day-old yellow-feathered male broilers were randomly assigned to eight treatments according to the randomized complete block design, with five replicates per treatment.Birds were weighed and allocated to treatment groups in order to obtain similar initial average body weights among the treatment groups.The pens were located in a well-ventilated open sided house with pine-shavings.Birds were submitted to 24 h of light and same management practices.Each pen (260×320cm) was considered a replicate.The temperature inside the pen was maintained at 33 ºC on day 1 and reduced 3 ºC each week until 24 ºC was reached.
Birds were fed the experimental diets (in mash form) formulated to supply the NRC (1994) recommendations for all nutrients except for NPP.A two-phase feeding program was applied: starter diets from 1 to 21d and grower diets from 22 to 42d.The compositions of the starter and grower experimental diets are shown in Table 1.Basal diets, based on corn and soybean meal, were formulated and the treatment diets were obtained by supplementing the basal diets with monocalcium phosphate, limestone, PHY (Natuphos, BASF), or 25-OH-D 3 (DSM Nutritional Products).One unit (U) of phytase activity is defined as the amount of enzyme that releases 1 mol of inorganic P from 1.5 mM Na phytate at pH 5.5 and 37 °C.The birds in treatments 1-4 were fed with phosphorus deficient diet 1 (NPP level below NRC by 0.1 in each period), while the birds in treatments 5-8 were fed with phosphorus deficient diet 2 (NPP level below NRC by 0.2 in each period).The starter diets was formulated to contain 0.35% or 0.25% NPP and the grower diets were formulated to contain 0.3% or 0.25% NPP.Each phosphorusdeficient diet contained no Vitamin D 3 and was mixed with 300U/kg or 600U/kg phytase and 34.5µg /kg or 69µg /kg 25-OH-D 3 .Two levels of NPP concentrations (NRC NPP reduced by 0.1 or 0.2), two levels of PHY (300U/kg or 600U/kg), and two levels of 25-OH-D 3 (34.5µg/kg diet or 69µg /kg diet) resulted in a 2×2×2 Based on the tables of feed composition of NRC (1994).

Effects of the Combination of Non-Phytate Phosphorus, Phytase and 25-Hydroxycholecalciferol on the Performance and Meat Quality of Broiler Chickens
factorial design (NPP ×PHY × 25-OH-D 3 ), with a total of 8 experimental treatments (TRT), with 5 replicates (pens) per treatment (60 birds /pen).Normal level and lower level of phytase and 25-OH-D 3 was included in the diets in attempt to allow for any potential additive or synergistic effects.Samples of all feeds were assayed for crude protein, calcium, and total phosphorus.Prior to mixing the diets, corn-25-OH-D 3 premixes consisting of corn and an appropriate level of 25-OH-D 3 -premix were prepared and used in the basal diets.
The broiler house had a curtain with an ultraviolet inhibition during the study to prevent birds from being exposure to ultraviolet radiation.Feed and water were provided ad libitum during the experiment.
Mortality and leg abnormality of the birds were checked daily.Leg abnormality, including the deformed legs, valgus/varus, and tibial dyschondroplasia, was assessed by observing the walking ability (walking gait and speed) of the birds when moving spontaneously in the rearing environment.On d 21 and 42, two birds per pen were killed by cervical dislocation and their tibiae were removed to determine the presence or absence of physical leg abnormalities.Feed and broilers were weighed on d 21 and 42 for the determination of body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR).

Meat Quality Measurement
At d 42, two birds per pen were killed by cervical dislocation and their right breast and thigh muscle were removed for pH, color, and shear force determination.According to the method of Lu et al. (2006), at 45min postmortem, the pH of the breast and thigh muscles were tested with a pH meter (PHS-3C, Shanghai Precision and Scientific Instrument Co. Ltd., Shanghai, China).At 24 h postmortem, lightness (L*), redness (a*), and yellowness (b*) values of the muscles were measured using a WSC-S Chroma Meter (Shanghai Precision and Scientific Instrument Co. Ltd.).According to the procedure described by Honikel (1998), shear force of the breast and thigh raw meat was measured by using a C-LM3 Digital Meat Tenderness Meter (Northeast Agricultural University, Harbin, China).

Statistical Analysis
Replicate means served as the experimental unit for statistical analysis.Analysis of variance was performed on all data for the experiment using the GLM procedure of SAS (SAS Institute, 2001) appropriate for a randomized block design.Treatment means were compared using Duncan's multiple range test (Duncan, 1955).P-values lower than 0.05 or 0.01 indicate statistical significance.

Body weight, survival rate, and leg abnormality rate
The survival rate of broilers in all treatments was high, of more than 99%, during the experimental periods, although NRC NPP was reduced by 0.2.No leg abnormality was observed during starter phase and grower phase of the experiment (Table 2).The 21 d BW of chicks on TRT 1-4 (fed with 0.35% NPP diet) was higher than that of chicks on TRT 5-8 (fed a-c Means with no common superscripts in the same column significantly differ (p ≤ 0.05).
4. Means represent five pens of sixty birds each, n=5, Average initial weight of the chicks was 35.2g.

Effects of the Combination of Non-Phytate Phosphorus, Phytase and 25-Hydroxycholecalciferol on the Performance and Meat Quality of Broiler Chickens
with 0.25% NPP diet).The broilers on TRT 4 (fed with 0.35% NPP diet + 600U/kg phytase + 69µg/kg 25-OH-D 3 ) grew better than those on any other TRT during starter phase.The BW of chicks on TRT 4 was 11.2% higher (p< 0.05) than that of chicks on TRT 8 at d 21.
The broilers on TRT 3 (fed with 0.30% NPP diet + 600 U/kg phytase + 34.5µg/kg 25-OH-D 3 ) grew better (p< 0.05) than those on any other TRT at d 42.The BW of chicks on TRT 3 was 5.84% higher (p< 0.05) than that of chicks on TRT 5 at d 42.

Grower Phase (22 to 42 d of age) Performance
In the grower phase, the BWG and FCR of chicks were not affected when NRC NPP was reduced by 0.1   4).The BWG, feed intake, and FCR of chicks were not influenced by different phytase or 25-OH-D 3 levels.
The BWG and feed intake of chicks on TRT 1-4 (NRC NPP reduced by 0.1 plus phytase and 25-OH-D 3 ) was significantly greater (p< 0.05) by 3.68% and 3.71%, respectively, compared with TRT 5-8 (NRC NPP reduced by 0.2 plus phytase and 25-OH-D 3 ) from 1 to 42 d.No significant differences (p>0.05) were found in FCR of chicks from 1 to 42 d in any of TRT.

Growth Performance
The current NPP recommendation for broilers is 0.45% from 1 to 21 d of age and 0.35% from 22 to 42 d of age.In this study, the level of dietary NPP was 0.1 and 0.2 below the NRC (1994) when phytase and 25-OH-D 3 were added in each period to spare NPP.Under this situation, no leg abnormality and deleterious effects on survival rate were observed.This is consistent with the findings of Coto et al. (2008), who reported that there were no negative effects on performance of feeding a phosphorus-deficient diet with the addition of phytase and 25-OH-D 3 .Similarly, Biehl et al. (1995) reported that the correct combination of phytase and 1α-OH-D 3 could substantially reduce the required level of inorganic P supplementation, and consequently reduce the P level in excreta.Yan et al. (2003) suggested that the NPP requirement for optimal tibia ash content was 0.15 ± 0.049% at 49 d, when diets were supplemented with phytase.
During the entire experiment, lowering NRC NPP by 0.1 to 0.2 with the addition of phytase and 25-  1. NPP =non-phytate P. From 1 to 21 d, NPP levels for broilers on treatment 1-4 and treatment 5-8 were 0.35% and 0.25% respectively.From 22 to 42 d, NPP levels for broilers on treatment 1-4 and treatment 5-8 were 0.3% and 0.2% respectively.

Effects of the Combination of Non-Phytate Phosphorus, Phytase and 25-Hydroxycholecalciferol on the Performance and Meat Quality of Broiler Chickens
OH-D 3 reduced BWG and feed intake of chicks.This result is consistent with previous reports (Angel et al., 2000 a,b;Karimi et al., 2013) indicating that lowering dietary NPP decreased the BWG and feed intake.
In this experiment, BWG and feed intake of starter chicks on TRT 1-4 (fed with 0.35% NPP diet) was higher by 7.87% and 6.36% than that of chicks on TRT 5-8 (fed with 0.25% NPP diet), while the BWG and FCR of grower chicks on TRT 1-4 were similar to that on TRT 5-8.This indicated that reducing NRC NPP by 0.1 to 0.2 has a stronger effect on broiler performance more during the starter phase than during the grower phase.This result is consistent with the findings of Yan et al. (2005), who studied the ability of broiler chickens to adapt to early moderate P deficiencies.Yan et al. (2005) found that broiler chickens fed a low NPP diet weighed less (p< 0.05) than those fed a control diet at 18 d; however, by 23 d, these broilers had caught up with the control birds, and no BW differences (p> 0.05) were observed on d 28 and 32.Hence, the author inferred that modern broilers have capacity to adapt when exposed to dietary P restrictions.
The information obtained in this study indicated that BWG, feed intake, and FCR were not influenced by the different phytase or 25-OH-D 3 levels.This corroborates the findings of Ana et al. (2013), who reported that the supplementation of the basal diet with 25-OH-D 3 had no effect on BW.Additionally, Angel & Mitchell (2006) reported that feeding diets low in phosphorus together with phytase and 25-OH-D 3 did not affect broiler performance.However, our results are contrary to those reported by Keshavarz (2003), Snow et al. (2004), and Emami et al. (2013), indicating that phytase addition increased ADG and ADFI in diets deficient in NPP.

Effects of the Combination of Non-Phytate Phosphorus, Phytase and 25-Hydroxycholecalciferol on the Performance and Meat Quality of Broiler Chickens
Meat Quality There are no reports on the combined effects of NPP, PHY, and 25-OH-D 3 on the meat quality of broilers.More studies previously reported that the supplementation with vitamin D 3 before slaughter to cattle improved meat color (Wilborn et al., 2004;Lobo et al., 2012), decreased shear force (Tipton et al., 2007;Moron et al., 2008).Another study indicated that supplementation with vitamin D 3 improved steak tenderness by affecting muscle Ca concentrations, μ-calpain activities, and muscle proteolysis (Montgomery et al., 2004).
Some paper reported the effect of 25-OH-D 3 on meat quality.Lawrence et al. (2006) and Carnagey et al. (2008) reported that the supply of an oral bolus of 25-OH-D 3 to cows before slaughter influenced some muscle characteristics known to enhance beef tenderness, despite no effect on tenderness was not observed.Cho et al. (2006) studied the effects of 25-hydroxy-vitamin D 3 (25-OH-D 3 ) on carcass traits and beef tenderness and observed that, although Ca concentrations of the lean muscle numerically increased in response to 25-OH-D 3 supplementation, no statistical differences in Warner-Bratzler shear force or sensory traits were detected.In the present experiment, lightness (L* value), redness (a* value), yellowness (b* value), pH, and shear force of thigh meat were not affected by different NPP, phytase, or 25-OH-D 3 levels.This result is consistent with those mentioned above.
The different effects of 25-OH-D 3 and vitamin D 3 on meat quality are probably due differences in supplementation route, supplemental dose, animal type, etc.In most studies, vitamin D 3 was orally supplemented before slaughter and the experimental animals were cattle, whereas in the present study,   2. In the starter phase, reducing NRC NPP by 0.1 to 0.2 with the addition of phytase and 25-OH-D 3 reduced broiler BWG and feed intake.In the grower phase, reducing NRC NPP by 0.1 to 0.2 and adding phytase and 25-OH-D 3 to the diet did not affect BWG or FCR, produced a small decrease in feed intake of broilers.
3. Different levels of NPP, phytase, or 25-OH-D 3 did not have significant effects on meat color, pH, or shear force.
a-c Means with no common superscripts in the same column significantly differ (p ≤ 0.05).1. NPP = non-phytate P 2. Natuphos, BASF 3. 25-OH-D 3 =25-hydroxycholecalciferol, Hy-D, DSM Vitamins, shanghai, Ltd, 69µg/kg diet=2,760 IU/kg diet.4. Means represent five pens of sixty birds each, n=5, Average initial weight of the chicks was 35.2g ; BWG = body weight gain; FCR =feed conversion ratio.5. Weighted average of the SEM Zhang Z, Ye H, Xia W, Feng D Effects of the Combination of Non-Phytate Phosphorus, Phytase and 25-Hydroxycholecalciferol on the Performance and Meat Quality of Broiler Chickens to 0.2, but feed intake decreased by 2.67%(from 1461 g/bird/d to 1422 g/bird/d) when NRC NPP was lowered by 0.1 to 0.2 (Table 2. Natuphos, BASF 3. 25-OH-D 3 =25-hydroxycholecalciferol, Hy-D, DSM Vitamins,Shanghai,Ltd, 69μg /kg diet=2,760 IU/kg diet.4. Means represent five pens of sixty birds each, n=5 ; BWG = body weight gain; FCR =feed conversion ratio.5. Weighted average of the SEM Effects of the Combination of Non-Phytate Phosphorus, Phytase and 25-Hydroxycholecalciferol on the Performance and Meat Quality of Broiler Chickens force (p=0.142) of breast meat among TRT (Table Means with no common superscripts in the same column significantly differ (p ≤ 0.05).
-OH-D 3 =25-hydroxycholecalciferol, Hy-D, DSM Vitamins,shanghai,Ltd, 69 μg /kg diet=2,760 IU/kg diet.4. Means represent five pens of sixty birds each, n=5, Average initial weight of the chicks was 35.2g ; BWG = body weight gain; FCR =feed conversion ratio.5. Weighted average of the SEM Zhang Z, Ye H, Xia W, Feng D Means with no common superscripts in the same column significantly differ (p ≤ 0.05).1. NPP =non-phytate P 2. Natuphos, BASF 3. 25-OH-D 3 =25-hydroxycholecalciferol, Hy-D, DSM Vitamins, shanghai, Ltd, 69µg/kg diet=2,760 IU/kg diet.4. Means represent five pens of sixty birds each,n =5. 5. Weighted average of the SEM Zhang Z, Ye H, Xia W, Feng D Effects of the Combination of Non-Phytate Phosphorus, Phytase and 25-Hydroxycholecalciferol on the Performance and Meat Quality of Broiler Chickens 25-OH-D 3 was supplemented in the diet from the beginning to the end of the experiment and the experimental animals were broilers.Hence, further investigations on these factors are needed.

CONCLUSION 1 .
During the entire experimental period, lowering NRC NPP by 0.2 and adding phytase and 25-OH-D 3 did not affect the survival rate of broilers or caused leg abnormalities.

Table 1 -
Composition and nutritional levels of phosphorusdeficient basal diets 3

Table 2 -
Effect of the combination of NPP, phytase and 25-OH-D3 on BW, mortality rate and leg abnormality rate of broilers

Table 3 -
Effect of the combination of NPP, phytase and 25-OH-D 3 on the performance of starter broilers (1 to 21 days).

Table 4 -
Effect of the combination of NPP, phytase and 25-OH-D 3 on the performance of grower broilers (22 to 42 days).
a-c Means with no common superscripts in the same column significantly differ (p ≤ 0.05).1.NPP =non-phytate P

Table 5 -
Effect of the combination of NPP, phytase and 25-OH-D 3 on the performance of 1-to 42-d-old broilers

Table 6 -
Effect of the combination of NPP, phytase and 25-OH-D 3 on breast meat quality of 42-d-old broilers 5. Weighted average of the SEM Zhang Z, Ye H, Xia W, Feng D

Table 7 -
Effect of the combination of NPP, phytase and 25-OH-D3 on thigh meat quality of 42-d-old broilers