Introduction

Exogenous enzyme supplementation in feed is used to counteract the effects of antinutritional factors present in raw materials, thus increasing nutrient availability. However, for appropriate enzyme supplementation, it is important to know the substrates on which enzymes act. The substrate of phytase is phytate, which contains 28.2% phosphorus (P), and it is the main form of P storage in plants. Phytate is poorly digested by monogastric animals, particularly poultry, which produce no, or very low levels, of endogenous phytase (Wu et al., 2014). Phytase is a phosphatase that cleaves phosphate radicals from inositol, releasing P for absorption (^{Borrmann, 1999}; ^{Fukayama et al., 2008}). Layer diets are composed mainly of plant feedstuffs and phytase supplementation releases P from phytate, increasing its availability.

Phytate is a polyanionic molecule and, in plants, typically binds cationic minerals (calcium, copper, iron, magnesium, manganese, zinc), as well as protein and energy. Therefore, the inclusion of phytase in the diet of monogastric animals releases other nutrients in addition to P, potentially resulting in significant savings in feed costs (Santos-^{Viana et al., 2009}). A meta-analysis on the responses of layers to dietary phytase supplementation showed that diets based on corn and soybean meal containing 0.22% available P and not supplemented with phytase increased egg production, egg mass, and feed efficiency. Supplementation of diets with 150, 300, and 400 phytase units (FTU)/kg diet allowed available dietary P to be reduced to 0.18, 0.15, and 0.14%, respectively (^{Ahmadi et al., 2012}). The addition of 350 FTU to a layer diet with low available P (1.8 g/kg) promoted better eggshell quality than a diet with high available P (2.1 g/kg; Englmaierová et al., 2012).

Several literature reports show that inclusion of phytase in the diet of both commercial broilers and layers promotes better performance. However, most of these studies focus only on Ca and P release from the raw materials, and there has been limited investigation of the possible release of energy, protein, and other nutrients. Consideration of the nutrient values released by phytase in the formulation of layer diets may permit changes to formulations which reduce feed costs, and therefore, production costs.

Therefore, the objective of this study was to evaluate the effects of reduced-nutrient diets supplemented with variable phytase levels on performance, egg quality, and economic parameters of layers.

Materials and Methods

Economic analysis

The output considered for the economic analysis was egg production. Fixed costs were identical for all treatment groups. The cost of feed was the only variable cost considered. Feed cost per hen, gross margin of egg sales, and total revenue per treatment were analyzed (^{Gameiro, 2009}).

Feed cost (FCi) per hen included feed and debeaking costs. The costs of diet per 30 dozen eggs were based on the historical average of monthly feedstuff prices during a 10-year period. Deflation of the monthly feedstuff prices between February 2007 and March 2017, according to the National Consumer Price Index (INPC, 2017), was applied. Debeaking costs included fees paid to the debeaking professional and the costs of two debeaking procedures. The price paid for each case of 30 dozen eggs (360 eggs) at the end of the study was US$ 22.65 dollars.

Total revenue per hen (TRi; equation 1) was calculated as the number of eggs produced per hen (NEi, egg production during the entire experimental period) multiplied by the price paid per case of eggs (PECi), and the result was divided by 360 to obtain the price per egg for each treatment. Gross margin per hen (GMi; equation 2) was calculated by dividing total revenue per hen (TRi) by feed cost per hen (FCi). Economic efficiency (EE, equation 3) was calculated by dividing TRi by FCi. The closer the TRi to FCi ratio to 1, the less efficient the treatment.

1

2

3

Results

Feed intake and feed conversion ratio per dozen eggs and per egg mass (Table 2) were not influenced by treatment (p>0.05).

The highest egg production (p≤0.05) was observed in layers fed RN300FTU, which was 2.68% higher compared with those fed the PC diet. The hens fed RN600FTU had statistically similar egg production to those on the PC diet, and the lowest egg production was in hens fed RN900FTU.

The lowest egg production occurred in hens fed RN900FTU, whereas there was a statistically similar egg production in hens fed RN600FTU and those on the PC diet.

The heaviest eggs (p<0.05) were laid by hens fed the PC diet (66.80 g). RN300FTU supplementation enhanced (p<0.024) egg weight in comparison with RN900FTU and RN600FTU, respectively.

Egg mass was not different (p>0.05) between hens fed the PC and RN300FTU diets. This suggests that reduced-nutrient diets may have compromised performance of the layers. Except for eggshell thickness (p<0.05) the egg quality traits (Table 3) were not affected by diet (p>0.05). Thicker eggshells occurred in hens on the PC diet (p<0.05). RN300FTU supplementation significantly improved eggshell thickness in comparison with RN600FTU and RN900FTU, respectively.

The economic analysis was based on egg production and on the historical average monthly prices of feedstuffs during a 10-year period. The results are shown in Table 4 and presented in US dollars (USD). Feed cost per hen fed the PC diet was significantly higher (p>0.05) compared with those calculated for the reduced-nutrient diets supplemented with phytase. The feeding cost of the RN900FTU diet was approximately 9% lower compared with that of the PC diet due to reduced levels of expensive feedstuffs, such as dicalcium phosphate. Total revenue, gross margin, and total cost to gross revenue ratio (TC/TR) were not significantly different between treatments (p>0.05).

Discussion

These results indicate that layers fed diets with reduced nutrient levels and supplemented with phytase maintain the same feed intake and achieve the same FCR as those fed diets with standard nutrient levels. In a study evaluating diets with reduced available P (0.15%) supplemented with 200-600 FTU phytase, no differences in feed intake were observed (^{Viana et al., 2009}).

Phytase has shown to increase the availability of P and other nutrients, including phytate-bound cations (iron, magnesium, and zinc), energy, and amino acids (^{Fukayama et al., 2008}; ^{Ferreira et al., 2015}). Phytase inclusion in our study may have contributed to the increase in egg production in layers fed the reduced-nutrient diet with 300 FTU supplemental phytase (RN300FTU) which produced 2.68% more eggs compared with those fed the PC diet. In the study of ^{Borrmann (1999}), commercial layers fed a diet with a commercial phytase (300 FTU/kg of feed) and reduced P levels (0.18% available P) had higher egg production compared with those fed 0.30 and 0.36% available P. Overestimation of nutrient release by dietary phytase supplementation can have deleterious effects on the performance of birds and, in our study, the layers fed RN900FTU had the lowest egg production. Further research into the effects of phytase on the availability of nutrients other than phosphorus is needed. Studies evaluating amino-acid digestibility in phytase-supplemented diets show variable results, and the underlying mechanisms of nutrient release by phytase are not well understood (^{Selle et al., 2007}). However, it is important to emphasize that optimal results with the supplementation of digestive enzymes can only be achieved with adequate provision of specific substrates, correct enzyme dosage, and specific pH and temperature conditions. These results suggest that the problem is complex in laying hens due to the high Ca requirement for eggshell development and the detrimental consequences of Ca:P metabolic interactions. The increase in feed conversion ratio and decrease in hen-day egg production and egg mass production in dietary treatments with 1,500 FTU/kg at a Ca concentration of 35 g/kg rather than 42 g/kg Ca revealed the dependence of phytase superdosing on the Ca content (^{Skřivan et al. 2018}).

The egg quality results corroborate the findings of Costa et al. (2014), who reported no significant differences in egg quality traits when brown layers were fed diets with reduced available P and supplemented with several phytase levels. Eggshell thickness was, however, influenced (p<0.05) by the treatment. This result is consistent with the findings of ^{Englmaierová et al. (2015}), who fed layers with diets containing increasing levels of a phytase produced by Aspergillus niger (0, 150, 250, or 350 FTU/kg) and reduced non-phytate P (1.8 or 2.1 g/kg) and observed thinner eggshells in layers on the diet with the lowest non-phytate level and supplemented with phytase.

Based on the economic analysis results, reduced-protein diets supplemented with phytase decrease production costs. According to ^{Plumstead (2008}), the inclusion of 600 FTU phytase/kg and a 30% reduction of dicalcium phosphate in layer diets reduced feed costs by approximately US$ 0.89/t. In our study, the cost of the diet with 30% reduction of dicalcium phosphate and supplemented with 600 FTU phytase was US$ 0.103/100 g less than the PC diet.

Dietary inclusion of phytase is a common practice in the poultry industry, as it allows reduction of inorganic phosphorus sources, consequently reducing environmental contamination (^{Silva et al, 2012}). The analysis of the economic impacts (in terms of production) of different dietary formulations allows us to determine the optimum phytase supplementation to maximize economic benefits.

In conclusion, laying hens fed a reduced-nutrient diet supplemented with 300 FTU phytase improve egg production performance and reduce total cost production while egg quality traits are not affected by the diet. Nevertheless, the other diets formulated assuming higher nutrient release from the feedstuffs by phytase and supplemented with 600 and 900 FTU, do not improve layer performance compared with the conventional diet. However, neither of those diets have any negative effects on egg quality or economic results.