DESCRIPTION OF PROBLEM
Broiler diets are mainly composed of vegetable feedstuffs. These ingredients usually consist of high amount of P in the phytate form (60 to 80%). The phytate form that is found in plants is mostly unavailable to be used for broilers . Phytate is negatively charged under many pH conditions, and because of this property, phytate can form complexes with positively charged molecules and reduce their digestibility. Forming complexes with other nutrients can reduce the digestibility of those nutrients in the digesta, which makes them unavailable for use by animals .
Phytase (myo-inositol (1,2,3,4,5,6) hexaquisphosphate phosphohydrolases) represents a subgroup of phosphatases that are capable of initiating the phytate dephosphorylation (myoinositol (1,2,3,4,5,6) hexaquisphosphate). In theory, the enzymatic hydrolysis of phytate generates a series of lower myo-inositol phosphate esters, through a succession of dephosphorylation reactions, to produce inositol and 6 radicals of inorganic P 
The ability of phytase to degrade phytate can be affected by factors such as the amount and source of P, dietary calcium (Ca) levels, animal species and age , the presence of antinutritional factors, the type and amount of cereals used , concentrations and phytate sources of diets, and the level and type of phytase used 
Another critical factor is the location of the phytate in the seeds. In small grains, phytate is found mainly in the bran (aleurone, forehead, and pericarp layer). In maize, it is mainly in the germ, in legumes, it is accumulated in the cotyledon, and in soybean, it is distributed throughout the seed. However, in many other seeds, phytate localization is yet to be determined or has no specific location .
To maximize the feed utilization in poultry, as well as reduce the feed costs, the use of animal by-products in the diets has become a common practice in some countries because the growth in livestock and demand for animal proteins have led to large volumes of these byproducts .
According to the study by Rostagno et al. , meat and bone meal has 8.55–14.1% of total Ca and 4.59–7.54% of total P, with 4.13–6.79% of that being available P; the poultry byproducts contain 4.06–4.34% of total Ca and 2.37–2.54% of P that is available. These animal by-products have been proven to be good protein, Ca, and P sources and are relatively inexpensive that they allow nutritionists to reduce or replace the amount of inorganic P in diets
The objective of the present study was to evaluate the effects of different phytase levels in diets formulated based on vegetable feedstuffs, vegetable plus animal, and animal origin (high, medium, and low phytate, respectively) on live performance and bone characteristics of broiler chickens during the starter phase.
MATERIALS AND METHODS
The experiment was conducted at the Poultry Sector of the Experimental Station of the State University of the Western of Paraná, UNIOESTE. Experimental birds were handled with care to avoid unnecessary discomfort, and all experimental procedures were approved by the university ethical review committee.
Management of Birds
A total of 2,625 male, 1-day-old Cobb 500 broilers were housed in a controlled environment in 105-floor pens, 7 replicate pens per treatment of 25 birds per pen. Each pen was 1.96 m2 with a concrete floor covered with pine shavings as bedding and equipped with a tube feeder and nipple drinkers. Throughout the experimental period, the room temperature was maintained within the zone of thermal comfort. Feed and water were provided ad libitum during the entire experimental period.
Chicks were randomized by weight and distributed into a 3 x 5 factorial design, consisting of 15 treatments. Three diets were formulated to contain high (HP), medium (MP), and low (LP) phytate (2.45, 2.34, and 2.23 g kg−1 of phytate P, respectively) (Table 1). Fifteen experimental diets were formulated with different phytate contents combined with a positive control (PC) diet which aimed to meet the calcium (Ca) and phosphorus (P) requirements of the birds; the negative control (NC) with a reduction of 0.15% of total Ca and 0.15 of available P. The phytase product used, a fungal 3-phytase , was added to the NC at a rate of 0, 500, 1,000, or 1,500 FTU kg−1. One phytase unit is defined as the amount of enzyme that releases 1 μmol of inorganic phosphate under standard conditions (0.25 mol acetate buffer pH 5.5, 37°C and 5 mmol sodium phytate). The experimental diets were formulated according to the feed composition and nutritional requirements for a starter phase (1–21 D), proposed by Rostagno et al. .
Weight gain (WG), feed intake (FI), and feed conversion ratio (FCR) were determined at 21 D of age. Mean individual bird weight, and FI was calculated and corrected using the weight of dead birds, which was recorded daily, according to Sakomura and Rostagno .
On day 21, 2 birds per pen were randomly chosen and fasted for 6 h, and blood samples were collected via brachial puncture. Blood was coagulated and centrifuged  at 1,008 x g rpm for 10 min to obtain serum, which was stored at −20°C . To perform the analyzes, the serum was thawed at room temperature and centrifuged at 1,008 x g for 5 min, and then, Ca, P, and alkaline phosphatase (ALP) analyses were performed using a highperformance automatic spectrophotometer  with specific kits, calibrated with standards .
For the evaluation of bone development, 2 birds with mean group weights (±5%) were euthanized by electronarcosis followed by exsanguination, according to Normative Resolution No. 37 of February 15, 2018, of CONCEA, establishing the Euthanasia Practice Guidelines of the National Council for Animal Experimentation Control. Legs were separated and deboned to obtain tibia. After deboning, the left tibia was weighed to the nearest ±0.0001 g, and their lengths were determined using a digital caliper (accuracy of 0.01 mm). The Seedor Index  was calculated by dividing the bone weight (mg) by its length (mm). After this determination, the tibia was individually stored at −20°C for further analysis.
Determination of bone-breaking strength (BS) was performed after bone thawing at room temperature. The tibia was individually supported on the epiphysis. A force load of 200 kgf at the speed of 5 mm s−1 was applied in the central region of each bone using a probe TATPB and a texturometer .
The broken tibia was used for tibia ash determination. The tibia was weighed on an analytical balance (±0.0001 g), and DM analyzed , after which the samples were weighed, ashed overnight at 600°C, and weighed again . The percentage of bone ash (BA) was calculated as the proportion of the dry, preashed tibia multiplied by 100.
To determine the amount of Ca and P concentration in the bones, the ashes were placed in a sand bath (250°C) with HCl (6 mol) solution to solubilize the minerals. Ca was measured using an atomic absorption apparatus  and P using a spectrophotometer .
To evaluate the incidence of tibial dyschondroplasia (TD), the left leg tibia was decalcified with 50% formic acid and 20% sodium citrate . After decalcification, the bone was embedded in paraffin . The sections were made with microtomes at 5-μm thickness and stained with hematoxylin-eosin, for observation of the epiphyseal disk area and measurements of the regions to characterize the incidence of TD. For analysis of tibial epiphyseal cartilage slides, 2 distinct areas characterized by the morphological appearance were considered: growth plate (A1) and hypertrophic cartilage zone (A2). The images were measured with the aid of a computerized image analyzer .
The left tibias were used to determine radiographic bone mineral density (BMD), which was performed at the Dentistry Clinic of the University Hospital of Cascavel. The tibia was used to determine the optical densitometry in radiographic images compared with an aluminum scale with 10° for 1 mm (penetrometer). The bones were radiographed , and the digital images were analyzed using Adobe Photoshop CS6. Five areas of each penetrometer degree (1–5 mm) were analyzed, and an equation was used from the values obtained. Also, 6 regions of each bone were evaluated, and the earned value was applied in the equation to determine BMD expressed as millimeters of aluminum (mmAl). Higher values indicated greater radiopacity and greater bone density.
Statistical analysis was performed using SAS University Edition . An analysis of variance and subsequent polynomial regression between the inclusion levels of the enzyme was performed excluding the PC treatment. Also, the Dunnett’s test was performed at the 5% probability level to compare the PC treatment with the other treatments. Tukey’s test was performed to compare the means of each phytate content.
No significant interaction (P > 0.05) was found on WG and FCR (Table 2). The FI was higher, and FCR was poorer (P < 0.05) in broilers fed with diets of high phytate (HP) content compared with those fed with diets of low phytate
(LP). Broilers that received diets NC, without phytase supplementation, showed the lowest WG and reductions in FI, compared with the PC treatment, according to Dunnett’s test. Broilers receiving 1,000 and 1,500 FTU kg−1 had the best WG and FCR compared with the PC treatment. Regression equations for FI and WG had the best fit with quadratic adjustment, and the levels that provided the maximum responses were estimated at 233 and 1,180 FTU kg−1, respectively. FCR showed a linear improvement with increasing levels of phytase (P = 0.01).
The interaction between phytate and phytase significantly influenced the FI (Table 3). Feed consumption was increased (11%) (P = 0.003) in birds that fed on HP diets with nutritional reduction of Ca and P without phytase, when compared with birds on the LP diets. Phytase supplemented to the NC diet at 500 FTU increased (P = 0.031) (4.1%) the FI of birds fed HP, when compared with birds on the LP diets. With 1,000 FTU kg−1 of inclusion, there was no differentiation between FIs. A quadratic effect was observed only between phytase supplementation and LP diet, and the level of phytase that provided the maximum FI responses was estimated at 1,051 FTU kg−1.
Minerals on Blood and Bone
A significant interaction between phytase supplementation and phytate content was detected in serum Ca, P, and ALP (Table 4). Serum Ca and P of birds receiving LP were lower (P < 0.05) than those of birds receiving MP and HP, only for the NC treatment. Broilers receiving HP and supplementation of 1,000 FTU kg−1 of phytase had a higher concentration of serum P than those receiving the LP. Blood Ca linearly increased (P = 0.043) in broilers fed with HP, and the quadratic effect was observed (P= 0.013) when broilers were fed with LP diets; the level that was determined as providing the maximum response value was 1,029 FTU kg−1 phytase. A quadratic response of blood P was observed for broilers fed with different phytase levels for all phytate concentrations (HP, MP, and LP) in the diets, and the levels that were determined as providing the maximum response value were 1,067, 995, and 992 FTU kg−1 phytase, respectively. For ALP, only broilers fed with MP had a quadratic behavior (P = 0.018), and the levels that were determined as providing the maximum response value was 934 FTU kg−1 phytase.
A significant interaction between phytase supplementation and phytate content was detected in bone Ca and P (Table 5). Bone P content of broilers fed with LP diet and receiving NC was lower (P = 0.007) than that of birds fed with MP diet. Broilers fed with diets with HP and receiving 1,000 and 1,500 FTU kg had higher (P < 0.05) P tibia content. Tibia Ca content in broilers fed with LP diets had a linear adjustment, and upon increasing phytase levels, there was an increase in Ca content (P = 0.048).
No effects of phytate and phytase levels (Table 6) were observed (P > 0.05) for Seedor Index, growth plate (A1), and BMD. Broilers fed with NC diets without phytase supplementation exhibited the lowest BS, DM, and BA content and a higher value for hypertrophic cartilage zone (A2), by Dunnett’s test. Most measurements had the best fit with quadratic adjustments, and the equations derived showed the most significant values for supplementation at 1,140 (BS), 1,008 (DM), and 1,304 (BA) FTU kg−1. For A2, 1,308 FTU kg−1 may provide a tendency of TD.
However, the phytate and phytase interaction significantly influenced the DM and BA (Table 7). Tibia DM of broilers fed with diets of HP and receiving the NC without enzyme was 7.52% and 8.34% higher than that of broilers fed with diets of MP and LP, respectively; BA was higher in birds fed with NC plus 1,000 FTU kg−1 with LP than that of broilers fed with diets of MP. A quadratic effect was observed in DM content in broilers receiving diets with MP (P = 0.005) and LP (P = 0.001), and the higher level of phytase was 1,074 and 1,049 FTU kg−1, respectively. An increasing linear effect (P < 0.007) was observed in content BA in broilers receiving HP diets; on the other hand, BA percentage of broilers receiving LP was obtained with the addition of 1,101 FTU kg−1.
Diets with reduced Ca and P levels had negative effects on broilers performance. The reductions in WG and FI and poorer FCR were due to the reduction of 0.15% of Ca and 0.15% of P, levels much below the recommendation for broilers’ diets at 21 D. It was clear that regardless of the phytase level, broilers fed with diets of HP had a worse FCR.
Phytase supplementation was responsible for attenuating the negative effect of reducing Ca and P while keeping a similar performance to the birds fed with PC treatment and also promoting an improvement in FCR. This is due to the increased availability of P, other minerals, and nutrients, allowing a better-quality diet and due to higher nutrient digestibility as a result of phytase action [27, 28].
Birds that received NC and HP had higher FI than broilers fed with LP treatment. However, broilers receiving 500 FTU kg−1 achieved a FI similar between that of HP and MP, showing the effect of phytase on making similar diets with different phytate contents. The FI response peaked at 1,050 FTU kg−1 in broilers fed with LP and MP diets and may be associated with the diet composition, which had a lower fiber content in relation to a HP diet. High dietary fiber results in higher viscosity of the digesta, reducing intake and consequently nutrient digestibility and bird performance . However, this phytase-induced improvement in FI was not reflected in the WG and FCR of the same group of birds.
The positive effect of the enzyme also happens with some bone characteristics such as BS, DM, BA, and A2. Bone mineralization increased because of the availability of minerals released from the phytate mineral complex diets , meeting the requirements of skeletal development; these data are in agreement with previous studies [28, 31–34]. The abnormal bone development is a sign of a P deficiency and no phytase supplement, and this can affect the degree of conversion of cartilage to bone in the tibia and the histological development of tibia . Broilers fed with NC diet had defective or disorganized mineralization of the extracellular matrix of cartilage compared with those fed with PC, confirmed by A2 results. However, with supplementation of phytase, this effect reverts because of the improvements of phytate P and Ca digestion, and its utilization by broilers chickens .
The higher DM concentrations were observed in broilers fed with NC and HP diets, which match with FI results. The maximum achieved DM bone concentration in broilers in MP and LP groups was very similar ˜1,062 FTU kg−1. BA of broilers fed with LP diets had a quadratic effect (P < 0.0001), and the highest BA was obtained using 1,101 FTU kg−1 and agrees with a higher BA deposition observed in broilers supplemented with 1,000 FTU kg−1 into LP treatment. On the contrary, broilers into HP treatment had an increase in BA concentration with increased phytase inclusion; this may be due to the more significant substrate content in vegetable origin diets. Morgan et al.  found that 47% of phytate in wheat bran was susceptible to the effects of phytase, that is, it could be removed if there was sufficient phytase; this suggests that our vegetable origin diets (HP) with 3% wheat bran could be improved with the use of high doses of phytase.
The concentration of serum P responded in relation to dietary Ca and nonphytic P levels; birds receiving P-deficient diets had a high concentration of Ca and a lower concentration of P in plasma. Phytase supplementation causes an increase in P levels and decrease of Ca levels in plasma, restoring the homeostatic balance between these minerals . The enzyme ALP is an indicator of increased bone formation activity; high concentrations of ALP are associated with increased formation of bone tissue. However, the reduction of this enzyme associated with diets supplemented with phytase may reflect the decrease of ALP because of the increase of P availability.
High P contents in the blood are supposed to be related to a dynamic bone growth; when bone growth decreases, P is transferred to a lesser extent into the bones, and thus the serum contents are higher. However, the P content may show broad variations, which may be due to a difference in FI and difference in digestibility of feedstuffs, and thus, despite equal P concentrations in the diet, the availability of this mineral can vary, which will be noticeable in blood concentrations .
The calculated phytate P concentrations in diets used in the present study were around 2.45 (HP), 2.34 (MP), and 2.23 (LP) g kg−1 diets; therefore, it could be expected that phytase responses would be more pronounced in HP diets because of the higher amount of available substrate. However, the phytase effect was more pronounced into LP diet, affecting the most of variables in a quadratic manner, confirming the presence of a maximum value that can be considered the recommended dose to obtain maximal technical performance. However, at some point, the enzymes become saturated, and the reaction rate levels off, not leading to additional effect.
High phytase levels appear to be more effective in diets with LP content. At LP concentrations, more phytase is required to maintain the product supply as the available substrate is depleted, while at HP concentrations, even a low level of phytase is saturated with phytate, and thus most of the degradation of the substrate originates from high molecular weight (and more antinutritional) .
CONCLUSIONS AND APPLICATIONS
1. Diets with reduced calcium (Ca) and phosphorus (P) levels without phytase supplementation had negative effects on broilers’ performance and BS, DM, and BA content and led to a higher value for hypertrophic cartilage zone.
2. Our study findings revealed that phytase supplementation improves broiler’s performance and bones quality.
3. The use of 1,101 FTU kg−1 is recommended for better bone characteristics in low-phytate (LP) diets; this recommended level should not negatively affect the other parameters evaluated.
This article was originally published in 2020 Journal of Applied Poultry Research 29:240–250. https://doi.org/10.1016/j.japr.2019.10.010. This is an Open Access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).