Standardized ileal amino acid digestibility of plant-based feedstuffs and phytase supplementation in broiler chicken diets

The objective was to determine the standardized ileal amino acid digestibility (SIAAD) in soybean meal (SBM), corn + SBM, wheat + SBM and a protein free diet (PFD) associated with phytase. A total of 672 Cobb 500 (493±10g) male chicks were distributed in a completely randomized design with twelve treatments (PFD, PFD + SBM, PFD + corn + SBM and PFD + wheat + SBM, supplemented with 0, 500 and 1000 FTU), eight replicates and seven birds per cage from 14 to 23 days. Phytase supplementation at 500 FTU increased the SIAAD of methionine in SBM ( P <0.05) and threonine in corn + SBM ( P <0.05). There was no significant effect ( P >0.05) for the SIAAD of methionine, arginine and histidine in wheat + SBM as the phytase supplementation. However, the SIAAD of lysine, threonine, isoleucine, phenylalanine, valine, cystine, alanine, aspartic acid, glutamic acid, glycine, serine and tyrosine differed ( P <0.05). In general, the SIAAD for SBM, corn + SBM and wheat + SBM are 90.32, 88.65 and 83.97% (0 FTU); 91.31, 88.81 and 88.36% (500 FTU); and 91.36, 87.09 and 87.87% (1000 FTU). In conclusion, the efficacy of phytase for improve the SIAAD vary according to the feedstuff and level of supplementation.

Phytase supplementation in cereal-based diets can be used as a strategy to overcome endogenous enzyme deficiencies and improve the utilization of dietary nutrients, especially in early life stages of poultry.This practice is adopted in broiler chicken farming to increase the digestibility of raw materials, improve production performance, and increase nutrient utilization efficiency, ultimately reducing production costs (Meng et al., 2005;Dessimoni et al., 2019).Phytase inclusion improves the digestibility of amino acids in ingredients individually and in compound diets (Rutherfurd et al., 2002;Cowieson et al., 2017;Walters et al., 2019).Rutherfurd et al. (2012) found that the amino acid digestibility of wheat, corn and SBM can increase by 11.7%, 4.0% and 6.5%, on average, when phytase is added to the diet.The use of the exogenous phytase enzyme in diets can not only allow for a reduction in inorganic phosphorus supplementation, but also promote the release of amino acids and other nutrients.This process is known as the extraphosphoric effect of phytase.
The objective of the present study was to determine the standardized ileal amino acid digestibility in soybean meal, corn + soybean meal, wheat + soybean meal and a protein free diet associated with different levels of the phytase supplementation in diets for broiler chickens.

MATERIAL AND METHODS
The experiment was conducted at the Unit for Teaching, Research and Extension in Poultry Production and Nutrition at the Department of Animal Science at the Federal University of Viçosa, located in Viçosa -MG, Brazil.All experimental procedures complied with the norms established by the Ethics Committee in the Use of Production Animals -CEUAP-UFV (approval no.056/2013).
A total of 672 male broiler chicks of the Cobb 500 line with an average weight of 493 ± 10 g were used in the study.The birds were evaluated in a completely randomized experimental design with twelve treatments (PFD, PFD + SBM, PFD + corn + SBM and PFD + wheat + SBM, supplemented with 0, 500 and 1000 FTU of phytase), eight replicates and seven birds per cage during the experimental period of 14 to 23 days of age, to determine the standardized ileal amino acid digestibility (SIAAD) in SBM, corn + SBM and wheat + SBM, by the ileal content collection method.From the 1st to the 13th day of age, the birds were reared as recommended in the manual for the management of the line, with water and feed available ad libitum.At 14 days of age, the birds were weighed and transferred to cages in metal structures.The cages were equipped with nipple drinkers and trough feeders.Water and feed were available ad libitum throughout the experimental period.
A PFD with supplementation of 0, 500 and 1000 FTU of phytase are described in Table 2.The ingredients in the proper proportions (SBM, corn and wheat) were added to replace starch in protein-free diets with supplementation of 0, 500 and 1000 FTU of phytase.Celite was used as acidinsoluble ash (AIA) as a marker to determine the digestibility of amino acids and protein as well as quantify endogenous amino acid excretion.The premix of minerals and vitamins that were used in the diets did not contain rice hulls as an excipient and diluent.Thus, there was no interference from silica content in the AIA analysis.Provides per kg of diet: vitamin A -7500 IU, vitamin D3 -1900 IU, vitamin E -28 IU, K3 -1.5mg, vitamin B1 -2.0mg, vitamin B2 -5.0mg, vitamin B6 -2.8mg, nicotinic acid -30mg, pantothenic acid -10.0mg, vitamin B12 -0.012mg, biotin -0.07mg; folic acid -0.07mg.
3 Butylated hydroxytoluene 99%. 4 Phytase -AB Vista Quantum.*Soybean meal replaced 30% of the starch in the diet, whereas wheat and corn replaced 50% of it in the diets.*The diets were enriched with 0.13% available P and 0.143% Ca, respectively.
On the 23rd day of age, all birds were sacrificed by cervical dislocation.The ileal digesta was harvested, stored in plastic bottles, and subsequently lyophilized.
Endogenous correction to estimate the standardized ileal digestibility of crude protein and amino acids was calculated using the PFD without phytase addition.Endogenous excretion for each enzymatic treatment (i.e., PFD with 0, 500 and 1000 FTU) was also analyzed, with the SIAAD adjusted for the enzyme-free diets and the diets with 500 and 1000 FTU, respectively.
The data were subjected to analysis of variance and means were compared by the Student-Newman-Keuls test at the 5% probability level (SAS, 2014).

RESULTS AND DISCUSSION
The absolute values of endogenous amino acid losses from the digesta of broilers fed PFD diets vary according phytase supplementation levels (Table 3).However, the differences observed in the present study were not statistically significant (P>0.05).According to Moughan et al. (2014) endogenous nitrogen losses are influenced by the type of feed, body weight, content and quality of diet ingredients, amount of fiber, dry matter intake and presence of antinutritional factors (trypsin inhibitor, tannins, and lectins).In diet with SBM the SIAAD of methionine increased (P<0.05) when 500 and 1000 FTU of phytase were supplemented (Table 4).In general, the average SIAAD of SBM was 90.32, 91.31 and 91.36%.However, the SIAAD of the other amino acids of SBM were not influenced by phytase supplementation (P>0.05).Other authors also observed a significant effect of phytase supplementation on the amino acids' digestibility from plant-based feedstuffs (Ravindran et al., 2001;Rutherfurd et al., 2012;Kiarie et al., 2015).
In the diet with corn + SBM, phytase supplementation at the dose of 500 FTU improved the SIAAD of threonine (P<0.05)(Table 5).In general, the average SIAAD of corn + SBM was 88.65, 88.81 and 87.09%.However, the SIAAD of aspartic acid, glutamic acid, glycine, isoleucine, leucine, phenylalanine, serine, tyrosine, valine, and protein decreased when the phytase supplementation level was raised to 1000 FTU.The increase in P release by 1000 FTU supplementation may have caused an imbalance in the Ca:P ratio.In addition, the Ca:P ratio of the PFD with 1000 FTU supplementation was higher compared to the other diets (0 and 500 FTU).Thus, the higher Ca content may have increased the complexation with phytic acid residues forming calcium phytate, reducing the digestibility of some amino acids.Another important fact is that high-dose phytase supplementation can alter electrolyte balance, as phytase influences sodium secretion in the intestinal lumen, altering amino acid digestibility (Ravindran 2013).The SIAAD of methionine, arginine, and histidine in the diets with wheat + SBM did not differ significantly (P>0.05) as the phytase levels were increased (Table 6).However, a difference was detected (P<0.05) for the SIAAD of essential amino acids (lysine, threonine, isoleucine, phenylalanine, and valine).The SIAAD of nonessential amino acids increased (P<0.05)(cystine, alanine, aspartic acid, glutamic acid, glycine, serine, and tyrosine) between the treatments without and with phytase supplementation.The inclusion of phytase increase the mean SIAAD in the diets with wheat + SBM (P<0.05).In general, the average SIAAD of wheat + SBM was 83.97, 88.36 and 87.87%.Nevertheless, the treatments supplemented with 500 FTU and 1000 FTU of phytase were similar in this aspect.Gallardo et al. (2018) observed an increase in ileal amino acids digestibility in broilers fed diets containing 30% of wheat bran in the diets supplemented with 500 FTU of phytase, except for the amino acids isoleucine, methionine, tryptophan, alanine, glycine and tyrosine.Table 7 shows, only descriptively, the percentage contribution of phytase doses (500 and 1000 FTU) on the SIAAD of SBM.In general, for the amino acids lysine, threonine, glutamic acid, glycine, histidine, serine and tyrosine it was possible to observe a numerical reduction in the contribution to SIAAD with 1000 FTU supplementation.
In the present study, after the dose of 500 FTU, release efficiency decreases due to the reduced amount of substrate for enzymatic action.
According to Ravindran (2013), in the presence of adequate concentrations of enzyme, the reaction rate increases as the substrate concentration is elevated up to a maximum value.This is because there is more substrate than the enzyme can cleave.In this way, as the amount of substrate is reduced, no linear increase in cleavage occurs with the growing phytase levels.On this basis, higher phytase levels are not significantly effective, thus obeying the "law of diminishing returns".Some important points must be clarified to understand the effects of the action of supplemented phytase in diets for broilers with different plant-based feedstuffs.First, in the present study, FTU analysis of phytase added to diets was not performed.The phytase nutritional matrix information described in the methodology at the bottom of table 2 was used.Second, recovery analysis of phytase supplemented in different concentrations (500 and 1000 FTU) in SBM, corn + SBM and wheat + SBM.And third, the levels of phytic acid in feedstuffs have not been analyzed.Data from the Brazilian Tables for Poultry and Swine were used (Rostagno et al., 2011).Thus, these factors are important to increase the accuracy of the real impact of supplementing different doses of phytase on the SIADD in plant-based feedstuffs used as a basis for broiler diets.(250, 500, 750, 1000, 2000 and 3000 FTU/kg) in diets based on corn and SBM for broilers.These authors observed improve in digestibility of amino acids with the supplementation of increasing levels with phytase, with a more pronounced effect in increasing digestibility of tryptophan, threonine, valine, arginine, methionine, and histidine in broilers at 28 days of age.For wheat + SBM, the better SIADD were achieved when 500 FTU of phytase were added, for both essential and non-essential amino acids, except for threonine, cystine, alanine, aspartic acid and glutamic acid (Table 9).respectively ((Rostagno et al. 2017).However, it is important to point out that these values can vary from 0.17 to 0.22% for corn, 0.35 to 0.45% for SBM and 0.18 to 0.29% for wheat (Selle and Ravindran 2007).In addition, the crude protein content of SBM, corn and wheat in the present study was 44.95, 8.22 and 11.07%, respectively.This explains the different effects found for the different levels of phytase supplementation in diets.After all, the phytate content of feedstuffs and the interactions between amino groups and phytate and starch-protein in the diet can influence endogenous loss and mucus production (Cowieson et al., 2004).Qaisrani et al. (2018) report that mucus is made up of about 5% mucin that is mainly formed by threonine, serine, and proline.Therefore, the increase or depletion in the production of mucus alters the ileal digestibility of amino acids in the diet.
Therefore, the lack of substrate impedes the progressive release of amino acids; however, the extra-phosphoric effects are not limited to phosphorus release or amino acid digestibility, but also to other nutrients (Ca, Mg and Na) and enzymes (pepsin) (Liu et al., 2009;Kiarie et al., 2015).Considering that endogenous losses change as the amount of phytase addition is increased, the strategy of using different PFD should be better investigated to prevent under-or overestimates of the phytase contribution values.In addition, for new studies evaluating endogenous losses and SIADD associated with supplementation with phytase for broiler chickens, it is necessary to carry out FTU analyzes of the enzyme, enzymatic recovery in the diets and levels of phytic acid in the feedstuffs used in formulation of diets.

CONCLUSIONS
The standardized ileal amino acid digestibility of plant-based feedstuffs varies according to the phytase supplementation level.

Table 3 .
Mean values of ileal endogenous amino acids (mg/g of dry matter intake) determined using proteinfree-diet (PFD) supplemented with 0, 500 and 1000 FTU of phytase for broilers 1 Phytase -AB Vista Quantum; 2 Coefficient of variation.

Table 4 .
Standardized ileal amino acid digestibility coefficients in diets based on soybean meal supplemented with 0, 500 and 1000 FTU of phytase for broiler chickens

Table 5 .
Standardized ileal amino acid digestibility coefficients in diets based on corn + soybean meal supplemented with 0, 500 and 1000 FTU of phytase for broiler chickens A,B -Means followed by different letters in the same row are significantly different according to the SNK test (P<0.05); 1 Phytase -AB Vista Quantum; 2 Coefficient of variation.

Table 7 .
Phytase contribution (FTU/kg) to the standardized ileal amino acid digestibility in soybean meal for broiler chickens (%)

Table 8 .
Phytase contribution (FTU/kg) to the standardized ileal digestibility of amino acids in corn + soybean meal for broiler chickens (%)