Non-phytate phosphorus requirement for male broilers subjected to two calcium supply regimens from 8 to 21 days of age under thermoneutral environment conditions

The purpose of this study was to determine the non-phytate phosphorus (nPP) requirement for male broiler subjected to two calcium supply regimens from 8 to 21 d of age, based on performance and bone mineralization. Birds were distributed in a complete randomized 4 × 2 factorial design with four nPP concentrations: 0.25, 0.35, 0.45 and 0.55%, and two Ca supply regimens: Ca fixed at 9.0 g/kg of diet (CaF) or varying together with the nPP concentrations tested keeping a fixed Ca:nPP ratio (CaV), with 8 battery cages/treatment and 9 birds/battery cage. Feed intake (FI), body weight gain (BWG) and Ca:P ratio deposited in the dry defatted tibia (TibCa:TibP) were quadraticaly increased by the increasing nPP concentrations, regardless of the Ca supply regimen adopted (p<0.01). The nPP levels tested also influenced FCR and TibAsh, which presented a quadratic and linear response when CaF and CaV diets were used, respectively, and TibP and TibCa, which presented a quadratic response with both Ca supply regimen, CaF and CaV diets. Also, CaV diets provided a 2.86 and 5.02% higher FCR (p<0.05) and TibCa (p<0.01), respectively, when compared to CaF diets. Therefore, nPP nutritional requirement for male broilers reared at thermoneutral conditions from 8 to 21 d of age that provided better performance and bone mineralization were, respectively, 0.480 and 0.459% with CaF and 0.550% with CaV diets, indicating that, under thermoneutral conditions, CaV diets negatively affected growth performance of broilers, while positively affecting bone mineralization especially when low nPP levels are applied. Additional keywords: mineralization; nutritional requirement; performance; thermal comfort; tibia. Abbreviations used: aP (available phosphorus); BGTH (black globe temperature and humidity); BWG (body weight gain); CaF (fixed calcium); CaV (varied calcium according to non-phytate phosphorus level); FCR (feed conversion ratio); FI (Feed intake); nPP (non-phytate phosphorus; TibAsh (tibia ash content); TibCa (tibia calcium content); TibP (tibia phosphorus content). Authors’ contributions: Conceived and designed the experiment: EFC, JLD and RFMOD. Obtained funding and supervised the work: JLD and RFMOD. Performed the experiment: EFC, EMF, CPFA. Analyzed the data: EFC and BLS. Drafted the manuscript: EFC and BLS. All authors read and approved the final manuscript. Citation: Cardoso, E. F.; Donzele, J. L.; Donzele, R. F. M. O.; Figueiredo, E. M.; Azevedo, C. P. F.; Sufiate, B. L. (2018). Non-phytate phosphorus requirement for male broilers subjected to two calcium supply regimens from 8 to 21 days of age under thermoneutral environment conditions. Spanish Journal of Agricultural Research, Volume 16, Issue 3, e0611. https://doi.org/10.5424/sjar/201816312230 Received: 05 Sep 2017. Accepted: 05 Oct 2018. Copyright © 2018 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC-by 4.0) License. Funding: FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais). Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Evandro F. Cardoso: evandro.cardoso@ifbaiano.edu.br


Introduction
Phosphorus (P) is an important mineral required in poultry diets for normal growth and development (Ankra-Badu et al., 2004).It constitutes the costliest mineral nutrient in animal nutrition and is the third most expensive component in non-ruminant diets, where the first two are energy and protein (Boling et al., 2000).
Countless studies have been focused on the P requirement for poultry, not only due to its economic importance, but also due to its environmental impact.In many countries the concern is growing about the excess of this mineral in the soil due to soil and groundwater contamination (Runho et al., 2001).The phosphorous, when leached to the fresh waters, stimulates the rapid growth of algae and cyanobacteria which, due to its high respiration rates, leads to hypoxia, impairing aquatic life (Silva et al., 2008).
Beyond its economic and environmental importance, P is an essential element in the animal body for energy metabolism, synthesis of nucleic acids, and structure of cells membranes (Saraiva et al., 2012).Along with calcium, it plays a major role in the development and maintenance of the skeletal system of the animal, being the second most abundant mineral in the body.Approximately 80% of all the P present in the body is found in the skeletal system and, when deficient, can cause rickets, retarded growth, and other skeletal deformities (Sethi et al., 2008).Additionally, it serves as a reserve to be mobilized to fulfill functions in almost all metabolic processes, playing a vital role in almost every series of biochemical reactions in the body (Cromwell, 1989).
Calcium (Ca) and P are important for the development and maintenance of the skeletal system in poultry and deficiencies in amounts or improper ratios of these two minerals usually lead to greater incidences of leg abnormalities (Xie et al., 2009).
Commonly the requirements of Ca or P are determined independently at a constant level of the other mineral (Ca or P).In fact, the requirements of Ca and P are interdependent.According to Rama Rao et al. (2003Rao et al. ( , 2006) ) and Xie et al. (2009) there is a significant interaction between dietary Ca and P on poultry nutrition and improper ratio of these two minerals can also depress growth performance.
Therefore, this study was designed to evaluate the impact of two Ca supply regimens on non-phytate phosphorus (nPP) requirement of male broilers from 8 to 21 d of age under thermoneutral conditions.

Material and methods
The protocol used in this study was reviewed and approved by the Animal Care and Use Committee of the Universidade Federal de Viçosa (MG, Brazil).The experiment was conducted at the Animal Bioclimatology Laboratory located at the Department of Animal Science, Agrarian Sciences Center, Universidade Federal de Viçosa, MG, Brazil to evaluate performance and bone mineralization.

Animal and housing
The 1-d-old male Cobb 500 × Cobb 500 broilers (slow feathering), used in these studies, were obtained from a commercial hatchery (Rio Brando Alimentos S/A, Pará de Minas, MG, Brazil) and raised in floor pens from hatch to 7 d and were fed a pre-starter diet that met or exceed Rostagno et al. (2005) recommendations for all nutrients.
On d 8, 576 birds with an initial body weight of 134.92 ± 0.32 g were transferred into environmental controlled chambers and allotted to one of the 64 metal battery cages (85.0 cm W × 85.0 cm D × 37.2 cm H), with mesh floor and supplied with hanging galvanized iron gutter type feeders and drinkers (83.0 cm L × 9.4 cm W × 5.5 cm D).
For the experimental period, the climatic chambers were set to remain with a constant air temperature of 29°C from 8 to 15 d of age and 27ºC from 16 to 21 d of age and relative air humidity between 55-65%, featuring a thermoneutral environment condition, according to Cobb Vantress (2012).
The environmental conditions inside the climatic chambers, represented by temperature and relative air humidity, were monitored twice a day (7 am and 6 pm) through thermometers of dry bulb and wet bulb, and black globe (Incoterm Industry of Thermometers Ltd., Porto Alegre, RS, Brazil) kept in the center of the room.Subsequently, these data were converted to the black globe temperature and humidity (BGTH) index, as proposed by Buffington et al. (1981).The lighting program adopted throughout the entire experimental period was continuous (24 h of artificial light).

Treatments and experimental design
On day 8, birds were assigned to eight treatments in a completely randomized 4 × 2 factorial arrangement with four nPP concentrations (0.25; 0.35; 0.45 and 0.55 %) and two Ca supply regimens, either at a fixed concentration (9.0 g/kg) (Table 1), regardless of nPP concentration, or at varying concentrations, to maintain a fixed Ca:nPP ratio (Table 2).A total of 8 replicate battery cages per treatment with 9 birds per battery cage were used.Weight of the birds was similar so that both pen to pen and within pen chick weight variation were minimized.The statistical model was as follows: where i = 1, 2, 3, 4; j = 1, 2, Y = mean of the experimental unit factor I and factor j; μ = constant inherent to all experimental units; αi = effect of level i of factor α; ßj = effect of level j of factor ß; γij = effect of interaction between factors α and ß; eij = error of the experimental unit factor i and j.

Statistical analyses
Data were analyzed using the GLM procedure of JMP PRO 10 (SAS Inst, 2012).The effects included in the model were: nPP level, Ca supply regimen and the interaction between those factors (nPP and Ca supply regimen).The results for all treatments were presented or not according to the interaction significance and the estimated nPP requirement for male broilers from 8 to 21 d of age were established by means of linear and quadratic regression model as the best fit obtained for each variable.Tukey's adjustment test was applied to the means of the Ca supply regimen factor in order to determine p-values.Significance was declared at p<0.05.

Results
During the experimental period, air temperature inside the facilities was kept at 29.08 ± 0.70°C and 27.28 ± 0.94°C and relative air humidity at 62.35 ± 4.07% and 61.32 ± 4.73 %, corresponding to a calculated BGTH index of 78.4 ± 0.66 and 76.3 ± 0.91, respectively, for the periods from 8 to 15 and 16 to 21 d of age.
The results of performance and bone mineralization of birds kept in thermoneutral environment conditions and fed diets with different levels of nPP, keeping or not a fixed Ca:nPP ratio, are shown in Tables 2 and 3, respectively.
nPP concentrations, in order to maintain a fixed Ca:nPP ratios (CaV), were used.The nPP content of feed ingredients was calculated as 33% of total P (Rostagno et al., 2005).
The levels of dicalcium phosphate, limestone and inert material (silica sand) were adjusted to obtain the desired nPP and Ca levels, then experimental diets were analysed in triplicate for Ca by atomic absorption spectrophotometry (method 4.8.03;AOAC, 2000), and total P by the colorimetric method (method 3.4.11;AOAC, 2000).Phytate P (PP) content of the basal diet was analyzed according to the ferric precipitation method as described by Ellis et al. (1977).All experimental diets were isoenergetic, isoproteic and isoaminoacidic.Animals were fed ad libitum and had free access to water throughout the experimental period.

Procedures
Pen weights were recorded at the beginning (8 d of age) and at the end (21 d of age) of the experimental period.Dead birds were weighed and removed from the pens twice daily and dead weights were used to correct feed conversion ratio (FCR).At the end of the experimental period (21 d), all birds and feeders were weighed to determine feed intake (FI), body weight gain (BWG), and FCR for the entire experimental period (8-21 d).Also, at 21 d of age, two broiler chickens from each battery cage, closest to the cage average were selected, fasted for 12 h, and then slaughtered by cervical dislocation to remove the right tibia.The right tibia was removed, forming a "pool" of two samples per replicate, and soft tissue and cartilaginous caps were removed.Samples were then pre-dried at 65°C in a drying oven for 72 h to reduce the moisture content below 15% enabling mechanical processing and sample conservation (Detmann et al., 2012).
Once dried, tibias were defatted with petroleum ether using Soxhlet apparatus for 4 h.Dry-defatted  3) FCR: feed conversion rate, based on live bird days within each period. (4)Q: quadratic effect; L: linear effect.Means followed by different letters for each variable between the Ca supply regimens employed differ by the F test (p < 0.05).*p < 0.05; **p < 0.01. (5)SEM: standard error of the mean; n = 8. regimen adopted, which increased quadraticaly to the estimated level of 0.453 and 0.480% nPP for FI and BWG, respectively (Table 2).
The nPP levels tested significantly affected (p<0.01)FI and BWG of broilers regardless of the Ca supply CaV 2.02 (1) TibP: amount of P in the dry defated tibia. (2)TibCa: amount of Ca in the dry defated tibia. (3)TibAsh: amount of ash in the dry defated tibia. ( 4 Ca:P ratio in the dry defated tibia. ( 5 Q: quadratic effect; L: linear effect.Means followed by different letters for each variable between the Ca supply regimens employed differ by the F test (p < 0.05).*p < 0.05; **p < 0.01. ( 6 SEM: standard error of the mean; n=8.  (1Ca: P ratio in the dry defated tibia. (2)TibP: amount of P in the dry defated tibia. (3)TibCa: amount of Ca in the dry defated tibia. (4)TibAsh: amount of ash in the dry defated tibia. (5)Requirement of dietetic nPP (%).
The nPP levels influenced FCR of birds, which improved (p<0.05) quadraticaly to the estimated level of 0.438% nPP, when CaF diets were used, and linearly (p<0.01) with CaV diets.Also, animals fed CaV diets had an increased (p<0.05)average FCR by 2.86%, when compared with the animals fed CaF diets.
The nPP levels tested influenced (p<0.05)TibP and TibCa content, which increased quadraticaly up to the estimated level of, respectively, 0.470% nPP for both Ca regimen tested and 0.452 and 0.440%, respectively, with CaF and CaV diets.As for TibAsh content, it was significant affected (p<0.05) by the nPP levels tested, which increased quadraticaly up to the estimated nPP level of 0.459% with CaF diets and linearly with the use of CaV diets.Also, the average TibP of broilers did not differ (p>0.05) between the groups of birds fed the CaF and CaV diets, whereas the same was not observed for the average TibCa, where a significant difference was observed (p<0.01)among animals fed the CaF and CaV diets, as the CaV diet provided an average TibCa 5.02% higher (p<0.01)when compared to the CaF diet.
Regardless of the Ca supply regimen adopted the nPP levels tested had a significant effect (p<0.01) on TibCa:TibP, which decreased (p<0.05) up to the estimated level of 0.519.

Discussion
According to the Roncbi (2004) and Cobb Vantress (2012), air temperatures ranging from 26 to 29°C with relative air humidity of 60% characterize an optimal environmental condition for broiler chickens from 8 to 21 d.In addition, according to Valério et al. (2003) and Lana et al. (2005), BGTH index values ranging from 74 to 83 are suitable for this category.Therefore, based on this information, we can infer that in this study all birds were kept in a thermoneutral environment condition.
Similar FI response has also been reported by Runho et al. (2001), Persia & Saylor (2006), Kill et al. (2008), Puppo et al. (2008) and Maia et al. (2009), who also found that available phosphorus (aP) levels influence voluntary FI of broilers fed diets with fixed Ca, independently of sex and strain of birds.On the contrary, when working with broilers, keeping a fixed 2:1 Ca to aP ratio, Yan et al. (2000), Bünzen et al. (2008) and Mello et al. (2012) failed to observe any aP effect on FI.
According to Rama Rao et al. (2006), the negative effect of low aP concentration on the voluntary FI of broilers is dependent on the Ca level used.Also, Qian et al. (1997) stated that the wider the Ca:aP ratio, the lowest is the FI of broilers.Several authors (Kill et al., 2008;Maia et al., 2009;Cardoso et al., 2010) identified that the highest FI for broiler chickens were obtained with diets in which the Ca:aP ratio were, respectively, 2:1, 1,96:1 and 2,12:1 while working with fixed Ca concentrations and increasing aP levels.
The estimated nPP levels that provided the best BWG results of broilers did not differ (p>0.05) between groups of birds fed CaF and CaV diets.According to Tamin et al. (2004), birds nPP requirement is influenced by the Ca level of the diet, due to its possible interaction with phytic phosphorus, negatively influencing the digestibility of both.
The nPP level that provided best BWG result in this study (0.480%) was superior to those obtained by Brugalli et al. (1999) and Maia et al. (2009), 0.45% and 0.46%, respectively, for broilers from 8 to 21 d of age fed CaF diets, and Mello et al. (2012), corresponding to 0.19%, for broilers from 11 to 21 d of age fed CaV diets.Rostagno et al. (2011) proposed the level of 0.401% nPP as the requirement for broilers from 8 to 21 d for best performance.The low Ca level (8.41 g/kg), when compared to the present study (8.99 g/kg), proposed by those authors is one of the factors that would justify the lower nPP requirement value proposed.
Similarly to our results, Brugalli et al. (1999), Runho et al. (2001), andMaia et al. (2009) also found a positi ve influence of the aP levels on FCR of broilers from 8 to 21 d of age while working with fixed Ca concentration.In contrast, Kill et al. (2008), Puppo et al. (2008) andCardoso et al. (2010), under the same circumstances, did not identify any significant FCR variation.Also, in the case of diets with fixed Ca:nPP ratio, a similar result was also obtained by Mello et al. (2012).
The inconsistency of the results showed can be attributed, among other things, to the variation in initial body weight of chicks used in the different studies.As reported by Angel (2011), there are several factors that can justify the different results in determining the nutritional nPP requirements, especially the growth rate of birds during the experimental period.
It was also evidenced in this study that in the two lowest nPP levels (0.25 and 0.35%) tested, the absolute FCR values were, respectively, 6.94 and 5.07% higher in birds fed CaV diets.This may indicate Ca deficiency when diets with CaV were used.
Also, in the lowest levels of nPP (0.25%) tested, the absolute values of TibP were 13.24% higher in birds fed diets with CaV what indicates the importance of a proper Ca:nPP ratio for an ideal bone mineralization.The obtained data on TibCa and TibP in the lowest nPP level tested (0.25%) showed that birds fed diets with CaV prioritized the deposition of both minerals in the tibia at the expense of growth.
As for TibCa, when observing the lowest (0.25%) and highest (0.55%) nPP levels tested, it was found that Ca deposition was compromised when using diets with CaF.Thus, it can be deduced that the deposition of calcium in the bone with deficiently nPP levels or above the requirement for optimal growth (0.45%) is dependent on a proper Ca:nPP ratio, which in this study was 1.6:1.
As for TibAsh content, a significant effect of the increasing nPP levels tested was also observed by Gomes et al. (1993), Lima (1995) and Runho et al. (2001).The results obtained for TibAsh when the lowest nPP concentration was tested (0.25%), suggests that once the Ca:nPP ratio is maintained at 1.6:1, birds tend to improve bone mineralization even if those minerals are deficient.
Although bone mineralization occurs most efficiently when Ca:nPP ratio is maintained, Cardoso et al. (2010) found that broiler chickens fed diets deficient in Ca and aP had a lower TibAsh percentage due to insufficient quantity of those minerals studied for a proper mineralization.
The average values of TibCa:TibP (2.00:1 × 2.02:1) did not differ (p > 0.05) when using diets with CaF or CaV.Since Ca:nPP ratio in the diet which has been used CaF ranged from 1.63 to 3.60, it can be inferred that tibia P deposition occurs simultaneously with Ca in a proportion close to 2:1, characterizing interdependence between these minerals.Studying the nPP levels in broiler chicks from 8 to 21 d old on thermoneutral environment conditions, Maia et al. (2009) also found proportionality in TibCa:TibP ratio (2:1), regardless of their ratio in the experimental diets.
Based on the results, it is clear that the levels of dietary Ca and nPP are not responsible for this regulation in the TibCa:TibP ratio deposited.In this sense, Crenshaw et al. (2011) identified the bone as the main site of synthesis of Fibroblast Growth Factor 23 (FGF 23), responsible for both renal P transport and by regulating vitamin D 3 activation, characterizing the bone tissue as an endogenous gland.
The best performance and bone mineralization of male broilers reared at thermoneutral conditions from 8 to 21 d of age were at nPP concentration of, respectively, 0.480 and 0.459% with the diet in which the level of Ca was kept fixed, corresponding to the estimated nPP intake of 4.46 and 4.26 g, and 0.550% independently of the Ca supply regimen adopted with the diet in which Ca varied proportionally to the nPP level, keeping a fixed Ca:nPP ratio of 1.6:1, corresponding to the estimated nPP intake of 5.14 g.In conclusion, the results from the present study indicate that, under thermoneutral conditions, CaV diets negatively affected growth performance of broilers, while positively affecting bone mineralization especially when low nPP levels are applied.

Table 1 .
recommendations, except for nPP and in certain cases Ca, when diets varying Ca and Ingredient and chemical composition of experimental diets fed to broilers from 8 to 21 d of age.

Table 2 .
Growth performance of male broilers fed diets containing different non-phytate phosphorus (nPP) concentrations, keeping or not the Ca:nPP ratio of diets, under thermoneutral conditions.

Table 3 .
Bone mineralization of male broilers fed diets containing different non-phytate phosphorus (nPP) concentrations, keeping or not the Ca:nPP ratio of diets, under thermoneutral conditions.

Table 4 .
Non-phytate phosphorus (nPP) requirement of male broilers from 8 to 21 d of age, keeping or not the Ca:nPP ratio, under thermoneutral conditions.