Egg quality in Haugh units (HU) was determined every 2 weeks. Albumen height was measured with a micrometer and estimated with the following equation [17] :

where H = h + (7.685 − 1.7 W^0.37);

h = observed albumen height (mm), and W = egg weight.

The specific gravity was measured with a hydrometer every 2 weeks. The values in the solutions ranged from 1.068 to 1.100 with an increase of 0.004.

The color and brightness of the egg was measured twice with a reflectance colorimeter: once halfway through the experiment and once at the end.

P content in the yolk-albumen, eggshell, and diets was determined colorimetrically; calcium content was determined by atomic absorption; and N content was determined by the Kjeldahl method [15] .

At 60 and 110 days, which were the first and second sampling days, respectively, 4 hens per treatment were selected to assess the calcium and P contents of the yolk-albumen and shell, and these samples were collected each day for 3 days. Subsequently, the shell and the yolk-albumen were separated and dried at 55˚C and the samples were incinerated for the corresponding analyses.

The ashes obtained from the samples were solubilized with 50% and 10% HC land evaporated to a volume not less than 10 ml, and the solution was transferred to 50-ml volumetric flasks for subsequent dilution. Molybdic acid and p-methylaminophenol sulfate, were used to determine the P content, and dilutions were made depending on the part of the egg to be analyzed. The samples were then read in a UV spectrophotometer at 660 nm. To determine calcium, lanthanum oxide was used, and the dilutions were made according to the part of the egg to be analyzed. Samples were then read by atomic absorption spectrophotometry at a wavelength of 425 nm.

Treatments were randomly assigned to the experimental units according to a completely randomized factorial design with a 3 × 2 × 2 arrangement: 3 levels of CA (0%, 0.6% and 1.2%), 2 levels of phytase (0 and 300 PHYU) and 2 levels of calcium (3.0% and 3.25%) with 4 replicates per treatment and 3 hens per replicate. The means were compared using Tukey’s test [18] , and the data were analyzed using the program SAS [19] .

There were no significant differences (P > 0.05) in the P and calcium contents of the yolk-albumen (Table 2). However, [9] in their study of molting laying hens, found an effect of CA and phytase on P and calcium, respectively, in yolk-albumen of egg; factors such as age and physiological state of the birds affect the response, in molting birds, calcium metabolism and its deposition in the egg increases [20] . Other important factors include the genetic line, diets, among others.

For P content in the shell, 1.2% CA × 300 PHYU × 3.25% calcium, 1.2% CA × 0 PHYU × 3.0% calcium and 0.6% CA × 300 PHYU × 3.0% calcium, increased the amount of P in the shell (P < 0.05) to 0.0067, 0.0067 and 0.0065 g respectively, in contrast with 0.0% CA × 300 PHYU × 3.0% calcium and 0.6% CA × 0 PHYU × 3.0% calcium with 0.0041 and 0.0040 g respectively (Table 3).

CA linearly increased the calcium content in the shell (P < 0.05) from 1.72 to 1.97, and 2.0 g for 0%, 0.6% and 1.2% respectively (Table 3). 1.2% CA × 300 PHYU × 3.25% calcium diets increased the eggshell calcium content (P < 0.05) to 2.25 g, in contrast with 0% CA × 0 PHYU × 3.0% and 3.25% calcium and the 0% CA × 300 PHYU × 3% calcium with 1.68, 1.69 and 1.72 g respectively (Table 3).

The pH of the diets with addition of CA decreased, from 5.4 in the control group to

*No interaction. CA = citric acid; Ca = calcium; P = phosphorus. SE = standard error.

^a,b,cAverages with distinct letters within columns are significantly different (p ≤ 0.05). CA = citric acid; Ca = calcium; P = phosphorus. SE = standard error.

4.7 and 4.3 with 0.6% and 1.2% CA, respectively, which most likely acidified the gastrointestinal tract as indicated by [21] [22] . For phytase [23] conclude that the addition of phytase increases the pH of the gastrointestinal tract, gizzard, duodenum, jejunum and ileum, which would affect the solubility of calcium, as calcium solubility depends on the chemical form of the calcium salts and the pH in the intestinal region [24] . Citric acid improved the phytase response, making calcium more available, with greater calcium deposition in the shell.

The addition of CA could promote calcium solubility by creating an acidic environment in the gastrointestinal tract that facilitates the absorption of calcium ions, thus decreasing calcium excretion increasing its availability. This is supported by [13] in their study of laying hens as well as by the higher calcium content in the shell observed in the present study. Calcium is completely soluble under an acidic pH and does not bind to phytic acid [25] .

On the other hand [26] increased calcium levels from 2.5% to 3.5% and 4.5% in the diets of laying eggs observed a significant improvement in shell quality; therefore, birds supplemented with higher levels of calcium retained more of this mineral for shell composition.

In terms of phytase, in a study with laying hens in the tropics, [27] indicate that phytase does not affect shell parameters, [3] [28] find that eggshell quality tends to decrease in hens fed phytase. In the current study, 0% CA, 300 PHYU and 3% calcium decreased eggshell calcium, possibly due to increased P availability, which interferes with calcium and thereby reduces the quality of the shell [29] . High blood serum P levels are associated with low-quality shells, which can be attributed to the inhibited precipitation of calcium ions in the serum due to the presence of P ions, and at high levels, the phosphate ion competes with the carbonate ion for calcium by inhibiting the formation and development of calcite crystals [30] .

There were no differences in the specific gravity of the eggs (P > 0.05) (Table 4). For phytase a similar result was found by [31] . [28] report that the specific gravity of the eggs in birds fed phosphate-deficient diets supplemented with phytase is lower than that in a control group. In contrast, [32] indicate that the specific gravity of eggs was reduced in chickens fed P-deficient diets, and the combination of 0.1% available P with addition of phytase improved the specific gravity.

1.2% CA × 300PHYU × 3.25% calcium and 1.2% CA × 0PHYU × 3.0% calcium diet yielded the highest specific gravity values, without differences (P > 0.05). [33] conclude

^a,bAverages with distinct letters between rows and columns are significantly different (p < 0.05). SE = standard error.

that organic acids and prebiotic, that lower pH in the diet and intestinal content, may have a positive effect on the quality of the shell. In contrast, [34] report that organic acids do not improve shell quality.

Haugh units were not influenced by levels of CA, phytase and/or calcium (P > 0.05) (Table 4), however [35] indicates that acidifying substances increase Haugh units and primarily act on the pH and the proportions of albumen calcium and magnesium and reports that few are the substances food-related factors.

Similar results for phytase were found by [28] , who detected no difference in albumen quality. In contrast, [36] in a study of 22- to 35-week-old hens fed low levels of available P (0.20%) supplemented with phytase, found significant differences in the height of the albumen, which decreased with high levels of available P (0.40%) and phytase.

For the yellow pigmentation of the yolk, 1.2% CA × 0 PHYU × 3% calcium, 0.6% CA × 0 PHYU × 3% calcium and 0% CA × 300 PHYU × 3% calcium improved (P < 0.05) yellow pigmentation, with 41.5, 41.8 and 41.4 respectively, in contrast with 0.6% CA × 300 PHYU × 3% calcium with 36.6 (Table 4). Phytase negatively interacts with CA at low calcium levels and interferes with the yellow pigmentation of the yolk, associated with the possibly interacting effects that both compounds have on the availability of certain nutrients.

In animals that do not make good use of fat, the digestibility of carotenoid esters is low, so the efficiency of fat absorption in birds influences the rate of carotenoid deposition [37] .

Research in this area involving CA is scarce, However [7] found that lipid metabolism is not modified by the addition of organic acids.

About, [38] report that extracting pigment from marigold flowers, Tagetes erecta, pre-treatment with a CA solution produces higher pigment yield and greater carotenoid retention. Moreover in some carotenoid-producing fungi, the addition of organic acids raises the content of β-carotene [39] , the activity of the CA cycle and a high respiratory rate is associated with an increase in the production of compounds that improve the production of carotenoids [40] .

On the other hand, there was a better response of yolk pigmentation to low levels of calcium, which is consistent with another researcher [41] , who indicates that increasing the levels of calcium in the diet negatively affects the absorption of carotenoids.