Efficiency of microbial phytases in diets formulated with different calcium:phosphorus ratios supplied to broilers from 35 to 42 days of age

ABSTRACT

The aim of this study was to investigate the efficiency of six microbial phytases (named of A to F) supplemented in diets (1500 FTU/kg) formulated with three different calcium:available phosphorus ratios (3.5:1.0, 5.0:1.0 and 6.5:1.0). Moreover, one positive control diet without phytase that was formulated with a calcium:available phosphorus (Ca:AP) ratio of 6.5:3.0 was also considered. The utilization of dietary nutrients, as well as the bone, plasma and performance parameters of broilers from 35 to 42 days of age was evaluated. Phytase D increased the P and magnesium plasma concentrations. Broilers fed diets containing phytase B showed a lower feed intake and weight gain, while the birds fed diets containing phytase E consumed more feed, gained more weight and excreted less total P. The highest Ca retention and also the best nitrogen utilization were determined when the diet contained phytase D or E. For all phytases evaluated, an increased in the dietary Ca:AP ratio increased the tibia ash content and the Ca balance. Phytase utilization reduced the phytate P excretion, improving its utilization by the broilers. For the rearing period evaluated, it is possible to reduce the AP of the diet to 1.0 g/kg when Ca is maintained at 6.5 g/kg, and the diet is supplemented with 1500 FTU of phytase A, B, C, D or E/kg. This diet allows the maintenance of performance, optimizes the bone mineralization, and it improves the Ca, total P, phytate P and nitrogen utilization in addition to reducing the P excretion.

KEYWORDS:

• Bone ash
• broiler
• calcium:phosphorus ratio
• metabolism
• performance
• phytase

1. Introduction

Phytate represents the main form of stored phosphorus (P) in the seeds and grains commonly present in broiler diets. However, only a small fraction of the phytate P (PP) of the diet is naturally utilized by these animals due to the low activity of endogenous phytase. Furthermore, phytic acid can bind to the dietary nutrients during their passage through the digestive tract, decreasing the bioavailability of these nutrients, which can compromise the broiler performance (Selle et al. 2009). In addition, there are other factors that encourage the use of exogenous phytase in poultry nutrition: (i) inorganic P is a non-renewable natural resource and represents one of the most expensive ingredients in feed; (ii) there is currently a strong call from an environmental perspective for the reduction of P deposition into the environment and (iii) the price of commercial phytases has been decreasing due to the advancement of biotechnology and fermentation techniques (Bertechini 2012).

In view of this situation, the diets that are currently in use have been formulated with low levels of available phosphorus (AP) and supplemented with phytase (Gomide et al. 2012; Jalali & Babaei 2012; Rutherfurd et al. 2012; Olukosi et al. 2013; Naves et al. 2014a). However, although the inorganic source of calcium (Ca) is relatively inexpensive, it is important to assess the optimal level of this nutrient when the feed is formulated with a specific P concentration and supplemented with a certain activity of phytase. Excessive Ca in the feed may reduce the efficiency of its utilization as well as that the other nutrients, such as P (Schoulten et al. 2002), and can also reduce the catalytic activity of the phytase utilized (Santos et al. 2008). Conversely, nutritional deficiency of Ca can compromise bone integrity and impair broiler performance (Selle et al. 2009).

Although there are many studies on the use of enzymes in poultry nutrition, the ideal Ca:AP ratio of feed supplemented with phytase has not been established (Selle et al. 2009; Bertechini 2012), mainly for feed without supplementation of inorganic phosphate. Further studies on mineral metabolism in broilers are therefore needed to generate scientific knowledge that contributes to the formulation of feed without significant excess P and with an adequate Ca:AP ratio (Slominski 2011).

There are several studies about the utilization of phytases in feeds for broilers during initial rearing periods (Santos et al. 2008; Gomide et al. 2012; Rutherfurd et al. 2012; Olukosi et al. 2013), however it is important to consider that during the last week of rearing there is a high feed intake (Rostagno et al. 2011). Therefore, an important nutritional strategy to reduce the cost of the feed and the P excretion in the environment is to evaluate the possibility of feeding broilers from 35 to 42 days of age with feed formulated without inorganic P, supplemented with phytase.

Moreover, distinct microbial phytases may exhibit different structures and different physicochemical and catalytic properties (Mullaney & Ullah 2003), which in turn can lead to different results when added to broiler feeds (Tamim & Angel 2003). Therefore, this study was conducted with broilers from 35 to 42 days of age to indicate which microbial phytases and which Ca level should be used in the feed when it is formulated with 1.0 g of AP/kg, considering bone, plasma and performance parameters, as well the utilization of dietary nutrients.

2. Materials and methods

2.1. Experimental procedures

One experiment was conducted with broiler chickens in the Poultry Sector of the Animal Science Department at the Federal University of Lavras. All procedures employed while conducting the experiment were approved by the Ethics Committee on Animal Use of this University (protocol number 004/11). The experimental design was completely randomized in a factorial arrangement (6 × 3) + 1 corresponding to the six microbial phytases that were added to the feeds formulated at three different Ca:AP ratios. There was also a positive control diet (PCD) without phytase, which was formulated according to the nutritional recommendations of Rostagno et al. (2011). There were four replicates with three broilers for each evaluated treatment.

The phytases evaluated in the present study were added to the feed at a concentration of 1500 units of phytase activity (FTU)/kg because in a previous metabolism assay, Naves et al. (2014b) concluded that when the broiler feed was supplemented with increasing levels of 6-phytase (0, 750, 1500 and 2250 FTU/kg), the greatest PP retention coefficient (88.45%) was determined when the feed contained 1500 FTU/kg. Therefore, the following microbial phytases were added at a concentration of 1500 FTU/kg of feed: phytase A (6-phytase^® expressed by genetically modified Aspergillus oryzae with two synthetic genes derived from the gene encoding phytase ATCC 51113 in Citrobacter braakii), phytase B (6-phytase^® produced by A. oryzae that was genetically modified with the phytase gene from Peniophora lycii), phytase C (6-phytase^® produced by Schizosaccharomyces pombe ATCC 5233 that was genetically modified with the phytase gene from Escherichia coli), phytase D (a second-generation genetic improvement of the 6-phytase^® produced by E.coli), phytase E (the first generation of the genetic improvement of the 6-phytase^® produced by E.coli) and phytase F (‘wild-type’ 6-phytase^® produced by E.coli). The rations of Ca:AP in the feeds containing phytase were set at 3.5:1.0, 5.0:1.0 and 6.5:1.0, in g/kg of feed:g/kg of feed. The PCD without phytase was formulated with a Ca:AP ratio of 6.5:3.0.

In total, 228 male Cobb-500^® broiler chickens were used in this experiment. The chicks were acquired at one day of age and reared in a conventional broiler shed until they were 34 days of age. During this period, the chicks were fed a corn and soybean meal-based diet that was formulated to meet their nutritional requirements (Rostagno et al. 2011). At 35 days of age, the birds were individually weighed, separated by weight ranges and transferred to cages (with dimensions of 50  cm  × 50  cm  × 50 cm; which is defined as an experimental unit) in a metabolism room. Each experimental unit had a similar average initial broiler weight (2.204 kg ± 0.080). The room had constant lighting and a controlled temperature, with each metabolic cage containing a low-pressure poultry waterer, an individual trough-type feeder with a border to avoid waste and, moreover, an excreta collection tray.

The experimental diets (Table 1) and water were offered for ad libitum consumption during seven days, including four days of adaptation to the diets and facilities followed by three days of total excreta collection (Rodrigues et al. 2005) per experimental unit.

Table 1. Ingredients and nutrient composition (as fed basis) of the experimental diets.

Ingredients (g/kg)	PCD	Phytases A, B and C			Phytases D, E and F
	Calcium:available phosphorus ratio			Calcium:available phosphorus ratio
	3.5:1.0	5.0:1.0	6.5:1.0	3.5:1.0	5.0:1.0	6.5:1.0
Maize	669.17	669.17	669.17	669.17	669.17	669.17	669.17
Soybean meal, crude protein 460 g/kg	263.73	263.73	263.73	263.73	263.73	263.73	263.73
Soybean oil	30.73	30.73	30.73	30.73	30.73	30.73	30.73
Dicalcium phosphate	9.78	0.00	0.00	0.00	0.00	0.00	0.00
Limestone	6.60	5.69	9.41	13.12	5.69	9.41	13.12
Salt	4.19	4.19	4.19	4.19	4.19	4.19	4.19
DL-Methionine, 990 g/kg	2.47	2.47	2.47	2.47	2.47	2.47	2.47
L-Lysine, 785 g/kg	2.47	2.47	2.47	2.47	2.47	2.47	2.47
L-Threonine, 985 g/kg	0.56	0.56	0.56	0.56	0.56	0.56	0.56
Mineral premix^a	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Vitamin premix^b	0.30	0.30	0.30	0.30	0.30	0.30	0.30
Choline chloride	0.20	0.20	0.20	0.20	0.20	0.20	0.20
Phytase	0.00	0.15	0.15	0.15	0.30	0.30	0.30
Inert (kaolin)	9.30	19.85	16.13	12.41	19.70	15.98	12.26
Analysed and calculated composition (g/kg)
Calculated metabolizable energy (MJ/kg)	13.19	13.19	13.19	13.19	13.19	13.19	13.19
Calculated crude protein	180.00	180.00	180.00	180.00	180.00	180.00	180.00
Analysed crude protein	179.80	181.20	179.95	181.50	180.75	181.06	181.49
Calculated calcium	6.50	3.50	5.00	6.50	3.50	5.00	6.50
Analysed calcium	6.65	3.69	5.06	6.75	3.74	5.13	6.48
Calculated available phosphorus	3.00	1.00	1.00	1.00	1.00	1.00	1.00
Calculated total phosphorus	5.00	3.00	3.00	3.00	3.00	3.00	3.00
Analysed total phosphorus	5.25	3.15	3.25	3.17	3.07	3.14	3.21
Calculated sodium	1.85	1.85	1.85	1.85	1.85	1.85	1.85
Calculated chlorine	3.03	3.03	3.03	3.03	3.03	3.03	3.03
Calculated potassium	6.77	6.77	6.77	6.77	6.77	6.77	6.77
Calculated lysine	10.10	10.10	10.10	10.10	10.10	10.10	10.10
Calculated methionine + cysteine	7.37	7.37	7.37	7.37	7.37	7.37	7.37
Calculated threonine	6.56	6.56	6.56	6.56	6.56	6.56	6.56
Electrolyte balance (mEq/kg)^c	168	168	168	168	168	168	168
Calculated phytase activity (FTU/kg)^d	0.0	1.500	1.500	1.500	1.500	1.500	1.500
Analysed phytase activity (FTU/kg)^e	22	1.518	1.492	1.509	1.502	1.487	1.510

^aProvided per kg of diet: Zn 55 mg, Se 0.18 mg, I 0.70 mg, Cu 10 mg, Mn 78 mg, Fe 48 mg.

^bProvided per kg of diet: folic acid 0.48 mg, pantothenic acid 8.70 mg, biotin 0.018 mg, butylhydroxytoluene (BHT) 1.5 mg, niacin 11.1 mg, vitamin A 6000 UI, vitamin B₁ 0.8 mg, vitamin E 12.15 UI, vitamin B₁₂ 8.10 µg, vitamin B₂ 3.6 mg, vitamin B₆ 1.80 mg, vitamin D₃ 1500 UI, vitamin K₃ 1.44 mg.

^cCalculated according to Mongin (1981).

^dEnzyme supplementation expressed in FTU/kg of feed, with 1 FTU corresponding to one unit of phytase activity. Phytases A, B and C: Enzymes with declared activity of 10,000 FTU/g of product. Phytases D, E and F: Enzymes with declared activity of 5000 FTU/g of product.

^eEnzyme activity determined according to the protocol proposed by Engelen et al. (1994).

2.2. Bone and plasma parameters

At the 42nd days of age, after two hours of fasting, two broilers per replicate were slaughtered by severing the jugular vein for the removal of the left tibia. The tibias were subsequently stripped, dried at 105°C, defatted in ethyl ether and incinerated at 600°C to determine the contents of ash, P and Ca (AOAC 2005; methods 942.05, 965.17 and 927.02, respectively). The bone results were expressed in g/100 g of defatted dry matter (DDM).

Moreover, the blood of the birds was collected in tubes containing the anticoagulant heparin and centrifuged (2000 × g/15 minutes), and the resulting plasma was collected for the quantification of Ca, P and magnesium (Mg) levels using commercial colorimetric kits (Labtest^® Company, Belo Horizonte, Brazil. Methods 90, 42 and 50, respectively). The plasma fraction of the blood was used instead of the serum fraction because the quantity of the plasma sample that can be obtained is considerably larger and because the colorimetric kits allow adequate determination of plasma Ca, P and Mg concentrations.

2.3. Measurement of performance traits

The feed and leftovers were weighed on the 35th and 42nd days of age of the broilers for the calculation of feed intake in this period. For weight gain calculations, the broilers were weighed at the age of 35 and 42 days. Feed conversion ratio was calculated by dividing feed intake by body weight gain.

2.4. Balance and retention of nutrients

The feed and leftovers were weighed on the 39th and 42nd days of age of the broilers for the calculation of the feed intake during this period of excreta collection. The total excreta collection was performed daily in the morning. The excreta from each experimental unit was collected in a labelled plastic bag and stored in a freezer until the last day of the collection. At this time, the contents were weighed, homogenized and pre-dried in an oven at 55°C during 72 h. After pre-drying, the excreta were ground and stored at room temperature until that the analyses of dry matter (DM), total P (TP), Ca and nitrogen (N) were performed (AOAC 2005; methods 934.01, 965.17, 927.02 and 984.13, respectively). Gross energy was determined in a bomb calorimeter (model 1261, Parr Instrument Company, Moline, IL). The concentration of PP was determined by the colorimetric method with a 1:20 extraction ratio as described by Naves et al. (2014b). In parallel, three homogeneous aliquots of each experimental diet were collected, ground and stored until the same chemical analyses that were conducted for the excreta were performed.

The balance and retention of PP, TP, Ca and N were calculated on the DM. The nutrient intake (g/bird) was calculated by multiplying feed intake (g/bird) by the nutrient content (%) that was determined for the feed. To calculate the absolute nutrient excretion (g/bird), the amount of excreta (g/bird) was multiplied by the nutrient content (%) determined for the excreta. The nutrient retention coefficient was calculated by the following equation: coefficient of retention (%) = [(nutrient intake–absolute nutrient excretion)/nutrient intake] × 100. The apparent metabolizable energy was corrected for nitrogen balance (AMEn) according to Hill and Anderson (1958). To calculate the dry matter digestibility coefficient (DMDC) of the feed, the following equation was used: DMDC = (g of ingested dry matter–g of excreted dry matter)/g of ingested dry matter.

2.5. Statistical analysis

The data were subjected to an analysis of variance using the general linear model of the SAS software (SAS Institute Inc 2004), and when significant, regression models were used to compare treatments with different Ca:AP ratios, while the different phytases were compared using the Student–Newman–Keuls test. Furthermore, the PCD was compared with the other experimental diets using the Dunnett test. For all statistical procedures, significant differences were considered at P < .05.

3. Results

3.1. Bone and plasma parameters

There was no interaction (P > .05) between the type of microbial phytase and the Ca:AP ratio in the feed nor was there any isolated effect (P > .05) of these factors on the P and Ca contents in the tibias of the broilers, which showed average concentrations of 7.76 ± 0.16 g of P/100 g of defatted dry matter (DDM) (coefficient of variation – CV = 3.25%) and 17.56 ± 0.93 g of Ca/100 g of DDM (CV = 4.67%). In addition, there was no interaction (P > .05) between the phytase and the Ca:AP ratio in the feed or isolated effect (P > .05) of the type of enzyme on the total mineral deposition in the tibia (Table 2). However, increasing the Ca:AP ratio from 3.5:1.0 to 6.5:1.0 linearly increased (P < .05) the tibia ash content of the broilers, resulting in an improvement of up to 2.65%.

Table 2. Ash content in the tibia and phosphorus and magnesium concentrations in the plasma of broiler chickens at 42 days of age fed diets containing different microbial phytases and different calcium:available phosphorus ratios (Ca:AP).

Phytase^1	Tibia ash (g/100 g of DDM)				Phosphorus (mg/dL of plasma)				Magnesium (mg/dL of plasma)
	Ca:AP ratio				Ca:AP ratio				Ca:AP ratio
	3.50:1.0	5.0:1.0	6.5:1.0	Average	3.50:1.0	5.0:1.0	6.5:1.0	Average	3.50:1.0	5.0:1.0	6.5:1.0	Average
Phytase A	41.62	42.74	43.99	42.79	7.23	7.30	7.22	7.25^ab	2.11	2.04	2.15	2.10^b
Phytase B	41.12	42.39	43.04	42.18	6.79	6.79	6.78	6.78^b	2.08	2.03	2.08	2.06^b
Phytase C	41.13	42.14	42.93	42.07	7.10	6.98	7.05	7.04^ab	2.12	2.11	2.12	2.11^b
Phytase D	42.46	42.48	42.31	42.42	7.55	7.46	7.40	7.47^a	2.25	2.25	2.18	2.23^a
Phytase E	42.37	42.53	42.34	42.41	6.82	7.02	6.98	6.94^b	2.04	2.04	2.04	2.04^b
Phytase F	42.17	42.40	42.90	42.49	7.16	7.09	7.36	7.20^ab	2.11	2.12	2.10	2.11^b
Average^2	41.81	42.44	42.92		7.11	7.10	7.13		2.12	2.10	2.11
PCD^3				43.41				6.68				2.04
CV (%)	3.15				6.05				6.24
P-value
Phytase × Ca:AP ratio	P > .05				P > .05				P > .05
Phytase	P > .05				P < .01				P < .05
Ca:AP ratio	P < .05				P > .05				P > .05

Notes: CV: coefficient of variation; DDM: defatted dry matter. Different letters in the column indicate significant differences (P < .05) by the Student–Newman–Keuls test.

¹1500 units of phytase activity (FTU)/kg of feed.

²Effect of the Ca:AP ratio in the feed on the tibia ash content: y = 0.36805x + 40.55209 (R² = 0.9933).

³ PCD, without supplemental phytase, formulated with 6.5 g Ca/kg of feed and 3.0 g AP/kg of feed.

All experimental diets included in the factorial design (totalizing 18 diets) resulted in levels of P, Ca and ash in the tibia similar (P > .05) to those determined in the birds fed PCD without phytase (7.99, 18.35 and 43.41 g/100 g of DDM, respectively) that was formulated to meet the nutritional requirements of the broilers in the evaluated period.

There was no interaction (P > .05) between the phytase and Ca:AP ratio in the feed, nor any isolated effect (P > .05) of the Ca:AP ratio on the plasma concentrations of P and Mg (Table 2). However, supplementation of the diet with phytase D increased the plasma concentrations of P (P < .01) and Mg (P < .05), whereas the addition of phytases B and E resulted in a decrease (P < .01) in the P content in the blood. Moreover, there was no interaction between phytase type and Ca:AP ratio nor isolated effects of these factors (P > .05) for the concentration of Ca in the plasma (10.46 ± 0.25 mg/dL; CV = 4.53%).

Compared with the results observed for broilers that received the PCD (9.98 mg of Ca/dL, 6.68 mg of P/dL and 2.04 mg/dL), none of the diets containing phytase altered (P > .05) the plasma concentrations of Ca, P and Mg under the three Ca:AP ratios evaluated.

3.2. Broiler performance

There was no interaction or isolated effect (P > .05) of the Ca:AP ratio in the feed on the feed intake and weight gain of the broilers (Table 3). On the other hand, broilers fed diet containing phytase B exhibited the lowest (P < .05) feed intake and lowest (P < .01) weight gain. However, when phytase E was used, the birds consumed more feed and gained more weight. There was no interaction or isolated effect (P > .05) of the phytase and Ca:AP ratio on feed conversion (Table 3). Moreover, all experimental diets included in the factorial design resulted in broiler performance similar (P > .05) to that of the birds fed with the PCD.

Table 3. Performance of broiler chickens from 35 to 42 days of age fed diets containing different microbial phytases and different calcium:available phosphorus ratios (Ca:AP).

Phytase^1	Feed intake (kg/bird)				Weight gain (kg/bird)				Feed conversion
	Ca:AP ratio				Ca:AP ratio				Ca:AP ratio
	3.50:1.0	5.0:1.0	6.5:1.0	Average	3.50:1.0	5.0:1.0	6.5:1.0	Average	3.50:1.0	5.0:1.0	6.5:1.0	Average
Phytase A	1.048	1.099	1.097	1.081^ab	0.372	0.401	0.394	0.389^bc	2.85	2.75	2.79	2.80
Phytase B	1.088	1.052	1.038	1.059^b	0.369	0.347	0.352	0.356^c	2.96	3.11	2.95	3.01
Phytase C	1.036	1.099	1.132	1.089^ab	0.379	0.371	0.384	0.378^bc	2.75	2.97	2.97	2.90
Phytase D	1.083	1.069	1.132	1.095^ab	0.399	0.397	0.380	0.392^bc	2.72	2.71	3.00	2.81
Phytase E	1.201	1.125	1.199	1.175^a	0.428	0.453	0.454	0.445^a	2.81	2.51	2.64	2.66
Phytase F	1.158	1.115	1.095	1.123^ab	0.430	0.414	0.409	0.418^ab	2.74	2.72	2.69	2.72
Average	1.102	1.093	1.116		0.396	0.397	0.395		2.81	2.80	2.84
PCD^2				1.098				0.413				2.66
CV (%)	5.59				7.37				3.02
P-value
Phytase × Ca:AP ratio	P > .05				P > .05				P > .05
Phytase	P < .05				P < .01				P > .05
Ca:AP ratio	P > .05				P > .05				P > .05

Notes: CV: coefficient of variation. Different letters in the column indicate significant differences (P < .05) by the Student–Newman–Keuls test.

¹1500 units of phytase activity (FTU)/kg of feed.

²PCD, without supplemental phytase, formulated with 6.5 g Ca/kg of feed and 3.0 g AP/kg of feed.

3.3. Balance and retention of nutrients

There was no interaction nor isolated effect (P > .05) of the type of microbial phytase and Ca:AP ratio in the feed regarding the intake, excretion and retention of PP (Table 4). However, diets containing phytase resulted in lower excretion and higher retention coefficient of PP (P < .05), when compared to the PCD.

Table 4. Balance and retention of phytate phosphorus in broiler chickens from 39 to 42 days of age fed diets containing different microbial phytases and different calcium:available phosphorus ratios (Ca:AP).

Phytase^1	Phytate phosphorus intake (g/bird)				Phytate phosphorus excretion (g/bird)				Phytate phosphorus retention coefficient
	Ca:AP ratio			Average	Ca:AP ratio			Average	Ca:AP ratio			Average
	3.50:1.0	5.0:1.0	6.5:1.0		3.50:1.0	5.0:1.0	6.5:1.0		3.50:1.0	5.0:1.0	6.5:1.0
Phytase A	0.856	0.923	0.951	0.910	0.068*	0.058*	0.077*	0.068	0.92*	0.94*	0.92*	0.92
Phytase B	0.932	0.870	0.865	0.889	0.057*	0.075*	0.071*	0.068	0.94*	0.91*	0.92*	0.92
Phytase C	0.928	0.873	0.827	0.876	0.073*	0.087*	0.068*	0.076	0.92*	0.90*	0.93*	0.92
Phytase D	0.867	0.897	0.920	0.894	0.050*	0.078*	0.058*	0.062	0.94*	0.91*	0.94*	0.93
Phytase E	0.984	0.924	0.928	0.945	0.087*	0.066*	0.065*	0.073	0.91*	0.93*	0.93*	0.92
Phytase F	0.923	1.060	0.975	0.986	0.057*	0.056*	0.044*	0.052	0.94*	0.95*	0.95*	0.95
Average	0.915	0.924	0.911		0.065	0.070	0.064		0.93	0.92	0.93
PCD^2				0.989				0.513				0.48
CV (%)	11.68				17.07				3.85
P-value
Phytase × Ca:AP ratio	P > .05				P > .05				P > .05
Phytase	P > .05				P > .05				P > .05
Ca:AP ratio	P > .05				P > .05				P > .05

Notes: CV: coefficient of variation. Different letters in the column indicate significant differences (P < .05) by the Student–Newman–Keuls test.

*Differs from the PCD by the Dunnett's test.

¹1500 units of phytase activity (FTU)/kg of feed.

²PCD, without supplemental phytase, formulated with 6.5 g Ca/kg of feed and 3.0 g AP/kg of feed.

The intake of TP was not altered (P > .05) by the type of phytase and Ca:AP ratio in the feed, nor by the interaction of these factors of manner that was determined an average overall intake of 1.370 ± 0.074 g of TP/bird (Table 5). In addition, there was no interaction (P > .05) between the phytase and the Ca:AP ratio in the feed nor any isolated effect (P > .05) of the Ca:AP ratio regarding the excretion and retention coefficient of TP. However, supplementation of phytase E resulted in the lowest (P < .05) excretion and highest (P < .01) utilization of TP. Furthermore, compared to the PCD, all experimental diets included in the factorial design resulted in lower (P < .05) intake and excretion of TP, allowing for their better (P < .05) utilization by the birds.

Table 5. Balance and retention of total phosphorus in broiler chickens from 39 to 42 days of age fed diets containing different microbial phytases and different calcium:available phosphorus ratios (Ca:AP).

Phytase^1	Total phosphorus intake (g/bird)				Total phosphorus excretion (g/bird)				Total phosphorus retention coefficient
	Ca:AP ratio			Average	Ca:AP ratio			Average	Ca:AP ratio			Average
	3.50:1.0	5.0:1.0	6.5:1.0		3.50:1.0	5.0:1.0	6.5:1.0		3.50:1.0	5.0:1.0	6.5:1.0
Phytase A^4	1.282*	1.382*	1.425*	1.363	0.034*	0.089*	0.085*	0.069^bc	0.97*	0.94*	0.94*	0.95^ab
Phytase B	1.396*	1.303*	1.295*	1.331	0.120*	0.096*	0.098*	0.105^a	0.91*	0.93*	0.92*	0.92^b
Phytase C^5	1.390*	1.322*	1.239*	1.317	0.098*	0.096*	0.117*	0.104^a	0.93*	0.93*	0.91*	0.92^b
Phytase D	1.298*	1.343*	1.378*	1.340	0.129*	0.086*	0.103*	0.106^a	0.90*	0.94*	0.93*	0.92^b
Phytase E^6	1.474*	1.384*	1.390*	1.416	0.037*	0.071*	0.052*	0.053^c	0.97*	0.95*	0.96*	0.96^a
Phytase F^7	1.383*	1.550*	1.423*	1.452	0.036*	0.098*	0.089*	0.074^b	0.97*	0.94*	0.94*	0.95^ab
Average^3	1.370	1.381	1.358		0.076	0.089	0.091		0.94	0.94	0.93
PCD^2				2.518				1.240				0.51
CV (%)	12.48				19.00				4.36
P-value
Phytase × Ca:AP ratio	P > .05				P > .05				P > .05
Phytase	P > .05				P < .05				P < .01
Ca:AP ratio	P > .05				P > .05				P > .05

Notes: CV: coefficient of variation. Different letters in the column indicate significant differences (P < .05) by the Student–Newman–Keuls test.

*Differs from the PCD by the Dunnett's test.

¹1500 units of phytase activity (FTU)/kg of feed.

²PCD, without supplemental phytase, formulated with 6.5 g Ca/kg of feed and 3.0 g AP/kg of feed.

There was no phytase × Ca:AP ratio interaction (P > .05) regarding the balance and retention of Ca (Table 6). The type of phytase supplemented in the feed did not influence (P > .05) Ca intake, but the use of phytase D resulted in the lowest (P < .01) excretion of this mineral. In addition, broilers fed diets containing 1500 FTU of phytase C, D or E/kg increased (P < .01) the Ca utilization in approximately 30.0% compared to what was observed for the broilers fed PCD. Moreover, regardless of the type of enzyme, it was observed that increasing the Ca:AP ratio from 3.5:1.0 to 6.5:1.0 linearly increased (P < .01) the intake and absolute excretion of Ca. Compared to the PCD, all experimental diets included in the factorial design resulted in improvement (P < .05) in the Ca utilization.

Table 6. Balance and retention of calcium in broiler chickens from 39 to 42 days old fed diets containing different microbial phytases and different calcium:available phosphorus ratios (Ca:AP).

Phytase^1	Calcium intake (g/bird)				Calcium excretion (g/bird)				Calcium retention coefficient
	Ca:AP ratio			Average	Ca:AP ratio			Average	Ca:AP ratio			Average
	3.50:1.0	5.0:1.0	6.5:1.0		3.50:1.0	5.0:1.0	6.5:1.0		3.50:1.0	5.0:1.0	6.5:1.0
Phytase A	1.928*	2.684	3.384*	2.665	1.016*	1.432	1.778	1.409^ab	0.47*	0.47*	0.48*	0.47^b
Phytase B	2.101*	2.734	3.226	2.687	1.120*	1.472	1.724	1.439^a	0.47*	0.46*	0.47*	0.47^b
Phytase C	2.116*	2.669	3.127	2.637	0.969*	1.273*	1.536	1.259^bc	0.54*	0.52*	0.51*	0.52^a
Phytase D	1.953*	2.440	3.274	2.555	0.928*	1.165*	1.638	1.244^c	0.52*	0.53*	0.50*	0.52^a
Phytase E	2.218*	2.687	3.301	2.735	1.109*	1.342*	1.618	1.356^bc	0.50*	0.50*	0.51*	0.50^a
Phytase F	2.080*	2.749	3.292	2.707	1.120*	1.496	1.846	1.487^a	0.46*	0.45	0.44	0.45^b
Average^2,3	2.066	2.660	3.267		1.044	1.363	1.690		0.49	0.49	0.48
PCD^4				2.777				1.677				0.40
CV (%)	9.30				10.38				5.93
P-value
Phytase x Ca:AP ratio	P > .05				P > .05				P > .05
Phytase	P > .05				P < .01				P < .01
Ca:AP ratio	P < .01				P < .01				P > .05

Notes: CV: coefficient of variation. Different letters in a column indicate significant differences (P < .05) by the Student–Newman–Keuls test.

*Differs from the PCD by the Dunnett test.

¹1500 activity units of phytase (FTU)/kg of feed.

²Effect of the Ca:AP ratio on calcium intake: y = 0.40038x + 0.66267 (R² = 0.9999).

³Effect of the Ca:AP ratio on calcium excretion: y = 0.21547x + 0.28819 (R² = 0.9999).

⁴PCD: positive control diet, without supplemental phytase, formulated with 6.5 g calcium/kg of feed and 3.0 g available phosphorus/kg of feed.

There was no interaction (P > .05) between the microbial phytase and Ca:AP ratio in the feed, nor any isolated effect of these factors regarding the intake and excretion of N, for which the determined average values were of 41.377 ± 1.456 g/bird and 16.882 ± 0.813 g/bird, respectively (Table 7). On the other hand, regardless of the Ca:AP ratio in the feed, phytases D and E resulted in the better (P < .05) N retention, while that the phytase F was the enzyme that resulted in the worse (P < .05) utilization. However, all experimental diets included in the factorial design improved (P < .05) N retention, compared to that observed when broilers were fed PCD. There was no interaction (P > .05) or isolated effect (P > .05) of the microbial phytase and Ca:AP ratio in the feed on AMEn and DMDC, for which the determined average values were of 14.11 ± 0.21 MJ/kg of feed (CV = 3.49%) and 0.75 ± 0.01 (CV = 2.43%), respectively. Furthermore, all experimental diets included in the factorial design provided similar results (P > .05) to those observed for the broilers that received the PCD (13.83 MJ AMEn/kg of feed and 0.74 of DMDC).

Table 7. Balance and retention of nitrogen in broiler chickens from 39 to 42 days of age fed diets containing different microbial phytases and different calcium:available phosphorus ratios (Ca:AP).

Phytase^1	Nitrogen intake (g/bird)				Nitrogen excretion (g/bird)				Nitrogen retention coefficient
	Ca:AP ratio			Average	Ca:AP ratio			Average	Ca:AP ratio			Average
	3.50:1.0	5.0:1.0	6.5:1.0		3.50:1.0	5.0:1.0	6.5:1.0		3.50:1.0	5.0:1.0	6.5:1.0
Phytase A	41.517	41.549	42.816	40.961	17.324	17.002	17.170	17.165	0.58*	0.59*	0.60*	0.58^ab
Phytase B	41.962	39.648	38.918	40.176	17.233	16.291	15.883	16.469	0.59*	0.59*	0.59*	0.59^ab
Phytase C	41.774	42.599	41.302	41.892	16.707	17.136	16.793	16.879	0.60*	0.60*	0.59*	0.60^ab
Phytase D	39.005	40.366	41.422	40.265	16.095	15.767	15.963	15.942	0.59*	0.61*	0.61*	0.60^a
Phytase E	44.310	41.593	41.764	42.556	18.165	16.265	16.409	16.946	0.59*	0.61*	0.61*	0.60^a
Phytase F	43.185	41.686	39.365	41.412	18.735	17.844	17.085	17.888	0.57*	0.57*	0.57*	0.57^b
Average	41.459	41.240	40.931		17.376	16.718	16.550		0.58	0.59	0.59
PCD^2				37.763				18.888				0.50
CV (%)	9.74				10.62				4.65
P-value
Phytase × Ca:AP ratio	P > .05				P > .05				P > .05
Phytase	P > .05				P > .05				P < .05
Ca:AP ratio	P > .05				P > .05				P > .05

Notes: CV: coefficient of variation. Different letters in the column indicate significant differences (P < .05) by the Student–Newman–Keuls test.

*Differs from the PCD by the Dunnett's test.

¹1500 units of phytase activity (FTU)/kg of feed.

²Positive control diet, without supplemental phytase, formulated with 6.5 g Ca/kg of feed and 3.0 g AP/kg of feed.

4. Discussion

Even when the Ca:AP ratio in the feed was increased from 3.5:1.0 to 6.5:1.0, all evaluated microbial phytases were effective in acting on the dietary phytate. This activity released PP in a sufficient quantity so that it could be used by the bone tissue without any negative effect on the deposition of P in the tibia of the broilers, which would most likely occur in the absence of supplementation of the diet with phytase because no source of inorganic P was included in the experimental diets, which were formulated with only 1.0 g of AP/kg. Moreover, the levels of Ca and ash in the bone were not reduced by the highest Ca:AP ratio evaluated, reinforcing the hypothesis that a higher Ca content in the feed did not compromise the catalytic efficiency of the phytases. These results contradict the findings of Schoulten et al. (2002), who reported that high Ca content in feed (high Ca:P ratio) usually reduces the phytase activity due to an increase in the rate of the formation of the calcium–phytate complex in the digestive tract of birds, which is resistant to enzymatic hydrolysis.

The tibia ash contents determined in this study could not be correlated with the bone deposition of Ca and P because the feed containing the Ca:AP ratio of 6.5:1.0 did not result in corresponding increases in the deposition of these minerals. Therefore, it is important to conduct further experiments to evaluate other minerals that play a role in bone formation, such as Mg, potassium, fluorine, zinc, sodium and even copper (Rath et al. 2000; Santos et al. 2008).

The average concentration of plasma P determined in broilers fed diet containing phytase C was lower (7.04 mg/dL) than was reported by Liu et al. (2010) for the same phytase (7.41 mg/dL). However, the diet evaluated in this study was formulated with a lower level of P (1.0 g versus 2.8 g of AP/kg of feed), which could explain the lower circulating concentration of P. Moreover, according to Ansar et al. (2004), the P concentration in the plasma of broilers decreases as the Ca:AP ratio in the diet increases, but this pattern was not observed in this study.

None of the diets containing different Ca:AP ratios supplemented with the different evaluated phytases altered the plasma Ca concentration compared to the level determined in the birds fed PCD. This finding can be explained by the efficient control mechanism for Ca homeostasis in the organism, which works to maintain a narrow range of calcemia (McDowell 1992). Thus, in cases of Ca deficiency, the animal induces an increase in the intestinal absorption of this mineral, along with a reduction in its excretion by the kidneys and, in addition, mobilizes a portion of the Ca stored in bone for use in other tissues through the bloodstream if necessary. Conversely, under situations of excess Ca, the intestinal absorption of this mineral is reduced through the saturation of membrane proteins involved in the Ca transport associated with increases in its excretion (Maiorka & Macari 2002).

Regardless of the Ca:AP ratio in the feed, only phytase D was able to simultaneously increase the blood levels of P and Mg, corroborating with Han et al. (2009), who reported that distinct microbial phytases may exhibit different catalytic efficiencies under the same conditions. This improved Mg absorption, based on the increase in its plasma levels, can be explained by the action of phytase on the phytate, reducing its ability to bind cationic minerals, such as Mg (Selle et al. 2009).

The broilers feed conversion was not altered by the type of phytase supplemented in the feed because the increased weight gain observed was result of a higher feed intake.

Compared to findings obtained for the broilers fed PCD it was observed, for all phytases and Ca:AP ratios evaluated, that it is possible to reduce the AP content in the diet to 1.0 g/kg without any negative effect on the broiler performance when the diet is supplemented with 1500 FTU/kg. This result is important because it demonstrates the possibility of using a feed based on corn and soybean meal without adding any source of inorganic P, during the rearing period from 35 to 42 days of age.

The maintenance of the broiler performance observed in this study can be explained by the improvement in the PP utilization promoted by the use of any of the evaluated phytases. Considering that the PP average content in the feed was 2.02 g/kg and that its average retention coefficient was 0.93, it can be concluded that the phytase released 1.88 g of P/kg of feed, which added to the P provided by the corn and soybean meal (1.0 g of AP/kg of feed) represents a total of 2.88 g of P/kg of feed, demonstrating that this level was sufficient to meet the P requirements of the birds. According to Rostagno et al. (2011), the P requirement for male broilers for average performance at 34–42 days of age is 2.98 g of P/kg of feed. However, according to Slominski (2011), it is likely that the currently established P requirement for broilers is overestimated.

The retention coefficients of TP, Ca and N were influenced by the type of phytase utilized, reinforcing the previously noted idea that different microbial phytases may exhibit different structures and varying physicochemical and catalytic properties (Mullaney & Ullah 2003), which can lead to differing results when these enzymes are added to the broiler feeds. Nevertheless, supplementation of the feed with phytase A, B, C, D or E under the three Ca:AP ratios evaluated and also supplementation of the feed formulated with Ca:AP ratio of 3.5:1.0 with phytase F improved TP, Ca and N utilization in comparison to the PCD.

Finally, it was found that supplementing the diet with phytase did not change the AMEn or the DMDC of the diets, corroborating with the results of Gomide et al. (2012) obtained for broilers reared in the period from 22 to 35 days of age.

5. Conclusions

In the rearing period from 35 to 42 days, it is possible to reduce the level of available phosphorus in broiler feed to 1.0 g/kg when this feed is supplemented with 1500 activity units of phytase A, B, C, D or E/kg. Furthermore, the calcium level should be fixed at 6.5 g/kg to maintain performance and to optimize the bone mineralization of the birds, as well as to improve the retention coefficients of calcium, phytate phosphorus, total phosphorus and nitrogen and also decrease the phosphorus excretion into the environment.

Acknowledgements

The authors extend their appreciation to the companies DSM, Nutron and ABVista for providing the phytases.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

The authors thank the Brazilian Institutions CNPq, FAPEMIG and INCT-CA for the financial support.