Optimal Available Phosphorus Levels in Diets Containing Different Dicalcium Phosphate Particle Sizes for Japanese Quails

I State University of Sao Paulo “Julio de Mesquita Filho”/ Faculty of Agrarian Sciences (FCAV), Department of Animal Science, Jaboticabal, SP, Brazil. II Federal University of Espirito Santo– Department of Animal Science, Alegre, ES, Brazil. III BM Comércio de Produtos Químicos e Biológicos Ltda. Santa Maria de Jetibá, ES, Brazil. IV Science, Technology and Innovation Agency of the state of Mato Grosso (SECITEC), Cuiabá, MT, Brazil. V COOPEAVI, Santa Maria de Jetibá, ES, Brazil. VI Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil


INTRODUCTION
Available phosphorus (avP) requirements should be taken into consideration when formulating diets for laying quails, as they have higher requirements than younger birds for bone remodeling and egg deposition (Costa et al., 2010). Phosphorus (P) is a component of the nucleic acids involved in cell growth and differentiation, and it is essential for energy use and transfer (in the form of ATP), fat transport, amino acid and protein synthesis, and also participates in the control of appetite, consequently affecting feed efficiency (Runho et al., 2001). This mineral is the third most expensive nutrient in non-ruminant diets, after energy and protein sources, in particular sulfur amino acids and lysine sources. Dietary P has been extensively researched relative to its economic and physiological importance, but also as a potential environmental polluting agent (Pinheiro et al., 2015).
However, there is limited research on the inclusion of granulated phosphate in the diet of Japanese quails. Griffith & Schexnailder (1970) observed that phosphate sources with large particle sizes increases phosphorus availability to laying hens, as it increases phosphate retention time in the gizzard, allowing its slow release in that acid environment, and consequently improving its digestibility. The solubility of P increases with the degree of grinding of phosphate sources. The eggshell is formed during the night, and part of the ingested P is directed to yolk eRBCA-2018-0878 deposition and another part is combined with calcium to be deposited in the bone.
The release of calcium from the bone is accompanied by phosphorus, significantly increasing the level of this mineral in the bloodstream. Therefore, special attention should be given to dietary phosphorus levels, because excessive P impairs the release of calcium from the bone and prevents adequate eggshell mineralization. Calcium and P absorption, metabolism, and excretion are closely linked (Salter et al., 1931). According to Furtado (1991), the best level of phosphorus (P) absorption occurs when dietary Ca levels provide for adequate Ca:avP ratio. Excessive Ca levels may reduce P absorption by producing an insoluble complex with myo-inositol and trace elements in the intestinal lumen, emphasizing the need to obtain adequate balance between Ca and avP levels in quail diets.
Therefore, the objective of this study was to evaluate the effect of dicalcium phosphate particle size and dietary available phosphorus levels on the production performance, egg quality, and bone characteristics of laying Japanese quails.

MATERIALS AND METHODS
This study was approved by the Animal Welfare Commission of the Federal University of Espirito Santo and conducted in compliance with animal welfare regulations under protocol number 024/2012.
The study was conducted at the Department of Animal Science, Federal University of Espirito Santo, Alegre, SP, from January to April 2013.
The experimental period of 84 days was subdivided into four periods of 21 days. Seven hundred and twenty 20-wk-old Japanese quails were housed in battery cages, and exposed to 16 h light/day, and had ad libitum access to feed and water. Birds were randomly distributed according to a completely randomized experimental design in a 5x2 factorial arrangement (five avP levels and two dicalcium phosphate particle sizes), with eight replicates of nine birds each.
Diets were formulated according to Rostagno et al. (2011) to contain adequate levels of all nutrients and energy, except for avP. Dicalcium phosphate (fine or coarse particles) and limestone were added to the basal diet (Table 1) in replacement of inert material (sand)in order to obtain five different avP levels (1.0, 2.0, 3.0, 4.0, 5.0 g kg -1 ) for each particle size (Table 2).  Fine (FPP) or coarse (CPP) phosphate particle sizes measured, respectively, geometric mean diameter (GMD) of 148 and 325 µm and geometric standard deviation (GSD) of 0.97 and 2.14. The determination of phosphate particle size was calculated using the equation described by Handerson & Perry (1955).
Birds were allotted to the treatments in order to obtain uniform body weight and egg production among replicates. Average egg weight, egg mass and feed conversion ratio (g of feed per g of egg mass -FCRem; and kg of feed per dozen eggs -FCRdz) were determined in the eggs collected during the last four days of each 21-d period. Feed intake was determined as the difference between feed provided during the entire experimental period and feed residues at the end of the experimental period.
Bone ash, phosphorus, calcium, and magnesium contents were determined in the right tibiotarsus of three birds per repetition according to the methodology described by Silva (2009).
The obtained data were submitted to analyses of variance and of regression (polynomial models) using the software SAEG (System for Statistical Analysis and Genetics, 1997) of the Federal University of Viçosa. Independently of the effects of the interaction avP level x phosphate particle size, the results were analyzed in order to determine the effect of phosphorus levels within each studied particle size in order to obtain the avP recommendations as a function of dicalcium phosphate particle size.

RESULTS AND DISCUSSION
The results showed dietary avP levels has a quadratic effect laying rate (LR) and feed intake (FI), independently of phosphate particle size (Table 3). Q= Quadratic effect; NS= non-significant effect; CV= Coefficient of variation; CPP= coarse particle size; FPP= fine particle size; The level of 1.0 g kg -1 of available phosphorus refers to the basal diet with no addition of dicalcium phosphate.
The highest LR response was obtained with 0.44 and 0.26% avP in the diets with FPP and CPP, respectively grain (Table 3). The obtained LR values may be associated with phosphorus role in the absorption of other minerals, mainly calcium and magnesium. In addition, the lower avP recommendation estimated when CPP phosphate was added (Table 3) may be due to the longer retention time of coarse phosphate particles in the gizzard compared with FPP, which readily supply a higher concentration of ionized and solubilized phosphorus (P) in the intestinal lumen. The presence of high P levels in the intestine, as a result of high dietary Ca:P ratio, increases intestinal pH, which results in the formation of P complexes with calcium, zinc, and manganese, consequently impairing the absorption of these minerals (Oderkirk, 1998). However, Vieira et al. (2012) did not observe any significant effect of 0.10 to 0.31% avP levels on the egg production of Japanese quails, neither Amoah et al. (2012) with increasing dietary avP levels.
The highest FI was determined in birds fed 0.44% avP from FPP and 0.28% avP from CPP (Table 3), and may be explained by the higher P availability in FPP, releasing P ions in the intestinal lumen, causing greater reaction with other nutrients and forming inorganic phosphates, consequently reducing P absorption (Li et al., 2016;DiMeglio & Imel, 2019).Therefore, birds need to increase their feed intake to supply their  (Gonzalez, 2002), high intestinal P levels may saturate P absorption sites, reducing blood P levels because P absorption rate is slower when compared with that of other minerals. Consequently, birds have higher FI to attempt to achieve normal blood P levels. However, Ribeiro et al. (2016), evaluating 1.5 or 3.0 g kg -1 avP levels did not observe any feed intake inhibition or stimulation in Japanese quails.
Egg weight (EW), FCR (per dozen eggs), and egg mass (EM) showed a quadratic response to avP levels, independently of phosphate particle size, while FCR (per g) linearly decreased ( Table 4).
The heaviest eggs in birds fed FPP were determined at 0.196% avP level. These results disagree with those reported by Vieira et al. (2012), who evaluated different dietary P levels (0.10 to 0.31%) and did not observe any effect egg weight.
Egg mass is the ratio between laying rate and egg weight, and any changes in one of these parameters may have significant effects on egg mass. The estimated avP level when using FPP (0.392%) is very close to that obtained with CPP (0.385%). Therefore, the EM differences obtained are a result to laying rate rather than to egg weight, as laying rate tends to decrease and egg size to increase as birds age, and indicate that laying Japanese quails maintain egg mass for longer than laying rate (Flemming, 2005). However, the EM results disagree with those observed by Amoah et al. (2012) and Vieira et al. (2012), who evaluated increasing dietary avP levels but did not find any significant effects on egg mass. Feed conversion ratio per egg mass (g feed/g egg) and per dozen eggs (kg feed/dozen eggs) improved (p<0.05) as dietary avP levels increased, suggesting that P requirements were supplied earlier when quails were fed higher avP levels had more P available. When the diets included CPP, FCR per dozen eggs improved up to 0.427%avP. Garcia et al. (2000) observed a linear improvement in FCR per dozen eggs as avP levels increased. Phosphorus metabolism is directly related to the dietary Ca:avP ratio, which influences feed intake and laying rate in both laying Japanese quails and chickens (Almeida et al., 2009;Vieira et al., 2012).
The effects of dietary avP levels on albumen (ALW and ALRW), yolk (YW and YRW) and eggshell (ESW and ESRW) absolute and relative weights are presented in Table 5. Albumen (FPP) and eggshell (CPP) absolute weights and eggshell relative weight (CPP) present a quadratic response to dietary avP levels, whereas linear responses were obtained for albumen (FPP), yolk (FPP) and eggshell (FPP) relative weights. No influence of avP levels were detected on eggshell (FPP) and albumen (CPP)absolute weights. However, Ribeiro et al. (2016) did not find any effect of dietary avP levels on eggshell weight. There was no effect (p>0.05) of dietary avP levels on the absolute weights of the albumen and yolk, independently of dicalcium phosphate particle size, or of the eggshell of birds fed FPP. Albumen and yolk relative weights of birds fed CPP, and eggshell relative weight of birds fed CPP also did not respond to avP levels (p>0.05).
Dietary avP levels had a quadratic effect on ESRW when the diets contained CPP, but no changes were observed when FPP was added to the diets. These results are consistent with those of Costa et al. (2010), who observed quadratic eggshell at 0.43, 0.41, 0.42, and 0.27% avP in the diet.
The obtained quadratic response of albumen absolute weight (ALW) to increasing avP level in the diets containing CPP disagrees with Costa et al. (2011), who did not find any influence of avP levels on albumen absolute or relative weights.
Dietary avP levels had a linear influence (p<0.05) on albumen relative weight (ALRW) and on yolk relative weight (YRW) of quails fed FPP. However, Ceylan et al. (2003) and Costa et al. (2010) did not find any significant changes in YRW in response to dietary P levels.
The regression equations are shown in Table 6. The level of 1.0 g kg -1 of available phosphorus refers to the basal diet with no addition of dicalcium phosphate. ESW= eggshell weight; ARW= albumen relative weight; YRW= yolk relative weight; ESRW= eggshell relative weight; CPP= coarse particle size dicalcium phosphate; FPP= fine particle size dicalcium phosphate The analysis of tibiotarsus parameters (Table 9) shows no influence of dietary avP levels on bone Ca percentage of birds fed CPP, or on P and Mg percentage, independently of dicalcium phosphate particle size. Vieira et al. (2012) evaluated four avP levels (0.10 to 0.31%) combined with three Ca levels (2.0, 2.5, or 3%) in quail diets and did not find any influence on Ca bone content (p>0.05). However, those authors reported a linear effect (p>0.05) of avP levels in the diets containing 3.0% calcium on tibial calcium content, whereas Costa et al. (2007), evaluating five avP levels (0.15 to 0.55%) in a diet with 3.2% Ca, obtained a quadratic response of P bone percentage.
Bone ash content presented a quadratic response to increasing dietary avP levels (Table 7), with an estimated maximum point at 0.,377% avP. This result is not consistent with the findings of Araujo et al. (2010), who fed commercial laying hens with diets three avP levels (0.28, 0.38, or 0.48%) from two fine and coarse particle size phosphate and did not detect any significant changes in bone ash content. ------> 0.5 CPP= coarse particle size dicalcium phosphate; FPP= fine particle size dicalcium phosphate.
The level of 1.0 g kg-1 of available phosphorus refers to the basal diet with no addition of dicalcium phosphate.
On the other hand, Ca bone content linearly decreased as dietary avP levels increased (Table 9) when birds were fed FPP, but not CPP. This may be explained by the fact that, when diets with increasing inclusion levels of fine dicalcium phosphate are fed, P absorption sites are saturated due to the high availability of ionic P (Angel, 2010;Pereira, et al., 2009), compromising Ca to P ratio and leading to the formation of insoluble phosphates, causing the animal to mobilize bone hydroxyapatite to meet its daily requirement (Pereira, et al., 2009).However, Garcia et al. (2000) evaluated similar avP levels (0.27 to 0.42%) at four Ca levels (2.5 to 4%) in quail diets, but did not detect any influence of avP levels on Ca bone percentage (p>0.05), possibly because Ca:P ratio was maintained in the evaluated diets.
The results of the present study indicate that the recommended dietary available phosphorus levels for 20-to 32-wk-old laying Japanese quails depends on dicalcium phosphate particle size. When using fine particle size dicalcium phosphate (148 µm), the recommended dietary avP level is 0.44%, which corresponds to an intake of 113 mg avP/bird/day, whereas for coarse particle size dicalcium phosphate (0.325 µm), the avP recommendation is 0.385%, corresponding an intake of 95.3 mg avP/bird/day.