Introduction

Phytic acid represents 60 to 80 % of phosphorus (P) stored in vegetables,¹ and is essential for the germination process. Phytic acid plays a critical anti-oxidant role in dormant seeds,² but in diets for monogastric animals it is considered as an antinutrient due to its chelating capacity with minerals, carbohydrates, proteins³ and amino acids.⁴ The fact that poultry species have limited ability to hydrolysis phytic acid⁵ and release P from its structure, addition of exogenous microbial phytase in diets is a common practice nowadays. This strategy allows the reduction of inorganic P supplementation in the diet, decreasing P excretion and therefore to mitigating environmental pollution.⁶ This phosphoric effect of phytase is embraced worldwide in poultry and swine production; however, the consequences of phytase in the release of other nutrients, known as the extra-phosphoric effect,⁷ has been under research in recent years.⁸

Phytic acid can negatively affect the solubility and digestion of proteins, likely through two paths of action. One mechanism is related to the chelation of protein by phytic acid and depends on isoelectric point of the protein and the pH of the digesta. In an acidic pH, phytic acid can directly chelate positive charges in the a-NH₂ group or to some basic amino acids, such as arginine, histidine, and lysine, forming binary complexes. In a basic pH, negative charges on the carboxyl group of all amino acids bind with divalent cations, mainly minerals, which are chelated by phytic acid, ending up in the formation of tertiary complexes.⁹ The other mechanism is based on the capacity of phosphate groups to act as a kosmotropic substance.¹⁰ In this case, phosphate groups are able to establish hydrogen bonds with water surrounding proteins, changing protein structure to a rigid status. Thus, protein solubility and digestion is reduced in the digestive tract. Bye et al.¹¹ concluded that sodium phytate decreased solubility and stability of lysozyme in vitro, acting as a kosmotropic molecule at concentrations higher than 5 mM, whereas at concentrations lower than 5 mM, direct binding of sodium phytate was responsible for the destabilization of the protein.

Lysine is often considered as the reference amino acid in the ideal amino acid profile concept applied to animal feed formulation,¹² despite methionine is the first limiting amino acid in poultry. Lysine was chosen taking into account that laboratory analysis is easier, lysine’s only role is in body accretion and egg deposition, and also because it is not turned to any other amino acid.¹⁰ Formulating diets with the ideal profile concept sets lysine requirements first, expecting that the remaining amino acid requirements can be adjusted in a fixed manner relative to lysine.¹³ In the case of laying hens, requirement for digestible lysine (dLys) depends on factors such as bird age, productive period, and genetic line, which could affect the response in egg mass and body weight.¹⁴

Taking into consideration the effect that phytic acid has on protein solubility, it is possible that phytase can affect dietary amino acid requirements of laying hens, although the response could be influenced by enzyme dose. It is expected that higher doses of phytase could improve utilization of protein content in the diet in comparison to the standard 300 FTU/kg dose,¹⁵ related to greater phytic acid hydrolysis in the digestive tract.¹⁶ Implementation of these high doses, known as superdosing, in broiler has led to addition of 1 500 FTU/kg in order to obtain hydrolysis of more than 85 % of the phytic acid present in the diet,¹⁷ resulting in better animal performance.⁷ In the case of standard doses, Selle et al.¹⁸ indicated that inclusion of 766 FTU/kg would result in 42 % phytic acid degradation, and that the P equivalency for 500 FTU/kg is 0.12 % available P (avP) in the case of broilers and pigs, which establishes hydrolyzed capacity and P supply for standard doses.

In laying hens, most of published phytase studies have included doses of 300 to 400 FTU/kg,^19-21 and there is lack of information about use of higher doses. No differences were observed by Mellef et al.²² when evaluating 400, 800, and 1 200 FTU/kg in 30 week-old Shaver 2 000 layers. Similarly, Silva et al. did not observe any differences with 300, 600 or 1 200 FTU/kg in 44 week-old Lohmann Brown layers. With the same strain, Agbede et al.²³ tested 0 and 1000 FTU/kg between 18^th and 22^th week of age, with no change in the response to phytase addition. These studies suggest that, although diverse experimental conditions were employed, higher doses of phytase did not exhibit positive results in laying hens.

Studies that evaluate the extra-phosphoric effect of phytase are not currently available in laying hens. Additionally, interactive effects of phytase doses and dLys levels in diet have only reported in broilers.²⁴ In the current study, it was hypothesized that incremental doses of a 6-phytase derived from E.coli would increase dietary lysine availability and consequently would enhance production in Bovans White hens from 40 to 63 weeks of age. Therefore, the aim of the present study was to evaluate the effect of four doses of phytase (0, 300, 1 200, and 4 800 FTU/kg) and three levels of dLys (0.67, 0.77, and 0.87 %) on production and bone parameters of laying hens fed a corn and soybean meal based diet deficient in avP (0.12 %). Enzyme effect was also compared with inorganic P addition by increasing avP level to 0.25 % in a positive control.

Materials and methods

Results and discussion

Analysed enzyme activity in all treatments was above the expected, but they were present in accordance with target dose, which confirmed that the enzyme was present in diets, in gradually increasing levels (Table 2). Diets without addition of exogenous enzyme were not analyzed because the assay technique is specific for the exogenous microbial phytase. There are several factors that can affect the exogenous phytase quantification in feeds, including sampling conditions in the farm or laboratory processing when the enzyme activity is determined.²⁷ Despite assay, values being higher than expected, they confirmed that the interpretation of the results in this study could be based on the initially proposed doses. In the case of dLys dietary, contents were similar to expected levels (see footnote in Table 3).

A positive effect on egg production was seen when phytase was included in the diet, although the extent of the response was linked to enzyme dose employed. Layers fed 1 200 FTU/kg phytase had higher egg production percentage than those fed no phytase in 0.12 % avP diets, F_3,169= 2.01, p = 0.03. Furthermore, layers fed 300 and 4 800 FTU/kg phytase in 0.12 % avP diets or with 0.25 % avP in the phytase-free diet did not show statistical differences from those fed 1 200 FTU/kg phytase. The response in percent egg production to incremental phytase dose was quadratic, showing evidence of maximum production at 1 200 FTU/kg F_2,179= 6.39, p = 0.01 (Table 3). Mellef et al.²² reported that inclusion of 1 200 FTU/kg of a 6-phytase from A. oryzae produced a greater number of eggs laid in comparison to 800 FTU/kg, and likewise 800 FTU/kg was better than 400 FTU/kg in Hy-Line W36 hens. In contrast, Silva et al.²⁸ did not find differences between 300, 600, and 1 200 FTU/kg of a 3-phytase from A. niger. Additionally, Silva et al.²⁸ recommended using 300 FTU/kg phytase in order to improve FCR in Lohman Brown hens. The inconsistency in the response to different doses of phytase could be explained by differences due to the origin of the enzyme. Studies in vitro have found that phytases can differ considerably in intrinsic catalytic properties.^27,29 Onyango et al.³⁰ reported a higher residual activity of a 6- phytase from E. coli in vivo compared with a similar phytase from P. lycii when 1000 FTU/kg dose was evaluated in the digesta obtained from gizzard, jejunum, and ileum of seven day-old chicks.

In the present reseach, unlike egg production, egg weight remained unchanged when phytase was added or even when dLys content varied F_12,169= 1.44, p = 0.15, although both factors have been stated to positively influence egg weight.^29,31 Only, egg weight was higher in hens fed the phytase-free diet containing 0.25 % avP than the average of those fed diets with 0.12 % avP F_12,169= 3.70, p = 0.06, as also reported by Ceylan et al.³² and Englmaierová et al.³³, but in contrast from studies by Augspurger et al,³⁴ Lim et al.,²¹ and Viana et al.³⁵ (Table 3).

Despite no influence of phytase and dLys on egg weight, an interaction was evident in egg mass. Thus, at 0.77 % dLys, phytase at 1 200 and 4 800 FTU/kg gave better production than 300 FTU/kg. In contrast, in the others two levels of dLys (0.67 % and 0.87 %), 4 800 FTU/kg affected negatively egg mass F_12,169= 1.86, p = 0.04. In spite of the interaction, the quadratic response to phytase doses, with a maximum at 1 200 FTU/kg in egg production highlights the adverse response in production at 4 800 FTU/kg in this trial F_2,179= 2.56, p = 0.07. Furthermore, incremental dLys levels resulted in positively linear responses in egg mass, highlighting the influential effect of dLys on production, where 0.87 % dLys maximized egg mass F_1,180= 3.55, p = 0.06. Egg mass average from hens fed 0.12 % avP diets was similar to the control diet F_12,169= 0.57, p = 0.45 (Table 3).

Feed consumption was lower in hens fed diets with 0.67 % dLys compared with those fed 0.77 %; meanwhile, 0.87 % was similar to 0.67 and 0.77 % levels F_12,169 = 1.85, p = 0.04. Additionally, a linear effect of feed intake was obtained to increasing dLys concentration, indicating an insufficient dietary supply with 0.67 % F_1,180= 4.79, p = 0.03 (Table 3). Laying hens can adjust feed consumption to nutrient content of the diet, particularly energy,³⁶ and to reduce intake of mineral deficient diets,³⁷ although regarding lysine it is not clear. Torii et al.³⁸ found that the restriction of L-lysine in the diet caused low feed consumption in rats, leading to anorexia and growth retardation, and it was remedied by blood lysine infusion. Torii’s study showed that the ventromedial portion of the hypothalamus and the lateral hypothalamic area in the brain are responsible for the regulation of lysine levels in rats, demonstrated a role of central nervous system in lysine appetite. In the current study, low consumption of the 0.67 % dLys diets probably resulted in low metabolic activity and reduced egg production, which was partially offset by phytase addition.

In the literature, there is no common recommendation for dLys level in the diet for white laying hen; the Bovans guide suggests 903 mg/d of consumption for optimal production,³⁹ NRC goes down to 580 mg/d, or 690 mg/d when using 2 900 kcal in the diet.²⁵ Rostagno et al.⁴⁰ recommended intermediate values of 756 mg/d for 40 week-old hens and 708 mg/d for 65 week-old. Silva et al.¹⁴ estimated that dLys consumption of 707, 660, and 669 mg/hen/d could optimize egg mass in Dekalb White hens of 37-40, 41-44, and 45-48 week-old, respectively. The levels recommended by Silva´s study were close to the averages in this study of 692 mg/d in the lowest dLys diets tested and taken as deficient.

Despite that dLys influence was significant on feed consumption, phytase impact present in other parameters like egg production, remained in the FCR. Thus, phytase reduced FCR relative to no addition of the enzyme in diets with 0.12 % avP F_12,169= 2.43, p = 0.07, contrasting with previous reports (Table 3). Augspurger et al.³⁴ found that there was no change in FCR when 150, 300, and 10 000 FTU/kg doses from a 6-phytase, produced by E. coli and expressed in A. niger, was evaluated in White Leghorn hens fed 0.10 % avP. Silversides et al.³¹ reported similar results with 300, 500, and 700 FTU/kg doses of a 6-phytase from E. coli expressed in S. pombe in 34 to 40 week-old ISA-white hens. In this analysis, maximum feed consumption allowed by hen, could have resulted in the lack of an effect on egg weight. Phytase inclusion was able to increase nutrient availability, since, even with the same quantity of feed eaten, better productive performance was obtained for all doses tested (300, 1 200, and 4 800 FTU/kg), improving FCR in those hens. Layers fed diets containing 0.25 % avP gave an intermediate FCR F_12,169= 2.56, p = 0.63, and it is important to highlight that the extra-phosphoric effect was obtained even with standard doses, although the effectiveness of phytase was relating to dose rate.

At the beginning of the experiment the average BW was similar among hens F_12,169= 0.81, p > 0.07, but at the end of experimental period, highly statistical significances were found F_12,169= 2.24, p = 0.01. Layers fed low avP diets with 300 and 1 200 FTU/kg phytase showed a higher final BW than those fed without phytase with the same avP level. Inclusion of 4 800 FTU/kg phytase or increasing avP to 0.25 % showed intermediate body weights at the end of the experimental period. Layers fed diets without phytase had more weight loss; those fed diets containing 0.12 % avP lost 5.4 %, while the phytase-free 0.25 % avP diet gave losses of 7.4 %. Hens fed phytase at 300, 1 200, and 4 800 FTU/kg showed BW losses of 2.5, 1.3, and 2.2 %, respectively. Final BW and BW loss showed highly significant quadratic responses to increasing phytase dose, with the maximum at 1 200 FTU/kg, F2,179 = 17.79, p < 0.01 (Table 3).

A reduction in the anti-nutritional effect of phytic acid from the diet by exogenous phytase addition was evident with all three doses evaluated, however, at 1 200 FTU/kg the response was greater. A higher phytic acid hydrolysis by the highest phytase dose could have released more P,^41-43 and reduced the chelating capacity of intact phytic acid and high inositol esters. Thus, nutrients such as protein⁴⁴ and carbohydrates⁴⁵ could have been more available, leading to an improvement in production and less use of body reserves by the layers. Silversides, et al.³¹ found that only 700 FTU/kg phytase gave positive changes in BW when 300, 500, and 700 FTU/kg doses were evaluated. Scott et al.⁴⁶ demonstrated that inclusion of 250 and 500 FTU/kg of 3-phytase from A. niger prevented BW loss in a proportional manner, without being affected by changes in dietary P and Ca levels, demonstrating that phytase has the capacity to maintain egg production without use of body reserves. Nevertheless, contrary results have been published where any significant improvements in hen BW have been evident with incremental phytase doses.^34,47 Francesch et al.¹⁹ observed greater BW gain with the use of 300 and 500 FTU/kg phytase than with increasing inorganic P level in the diet (1.1 vs 3.2 g/kg of non-phytic P), although 150 FTU/kg addition did not provide the same effect. Furthermore, the majority of layers lost BW during the experimental period, as to reported by Keshavarz⁴⁸ with the evaluation of vitamin D3, 25-OH-D3 and 300 FTU/kg of phytase.

Layers fed with 0.25 % avP diets without phytase gave lower abdominal fat deposition relative to those fed with 0.12 % avP supplemented with 1 200 FTU/kg, F_12,169= 2.52, p = 0.05. Layers fed diets containing 0.25 % avP had lower abdominal fat content than those fed 0.12 % avP diets F1_2,169= 5.85, p = 0.02 (Table 3). Phytase addition seemed to influence the energy reserves in laying hens, and this could be related with the fact that high phytase doses may release inositol, which can affect the transport of lipoproteins into liver for the synthesis of triacylglycerol.⁴⁹ Furthermore, higher phytase doses could also reduce the chelating capacity of phytic acid for lipids.⁵⁰ The current trial highlights the fact that inorganic P supplementation was unable to promote better performance or maintain body reserves, while supplementation of 1 200 FTU/kg phytase to a low avP diet improved performance and could serve as a tool to prolong laying period and help to improve profitability of egg production. This seems to be possible due to extra-phosphoric effect of the phytase and its greater effect when higher doses of enzyme were employed.

Layers fed 0.12 % avP diets supplemented with either 1 200 or 4 800 FTU/kg phytase laid less broken eggs than those fed with no phytase supplementation. Nevertheless, there were no differences among those treatments and 0.12 % avP diets with 0 or 300 FTU/kg phytase F_3,169= 2.90, p = 0.04. Similar results were obtained for soft-shell eggs, except that hens fed 4 800 FTU/kg did not produce less defect eggs when fed the phytase-free 0.12 % avP diet F_3,169= 2.91, p = 0.04. The responses in percent of broken F_2,179= 3.60, p = 0.03 and soft-eggs F_2,179= 3.58, p = 0.03 were fitted to quadratic models for phytase inclusions, with minimum value at 1 200 FTU/kg dose. Also, increasing dLys levels in the diet resulted in a positive linear reduction for soft-shell eggs F_1,180= 3.55, p = 0.06. The production of dirty eggs was not influenced by treatments, and regression models were not significant F_12,169= 1.09, p = 0.37 (Table 4). Taking into consideration the results obtaining in egg classification, 1 200 and 4 800 FTU/kg of phytase improved eggshell formation, leading to a stronger structure, probably due to more mineral availability, decreasing the incidence of broken eggs. Hassanien and Sanaa⁵¹ found that 1000 FTU/kg phytase resulted in higher eggshell resistance to breakage in comparison to 700 FTU/kg, which in turn showed increases relative to 500 or 0 FTU/kg. In the same study, improved eggshell breaking results with phytase was reflected in higher P, Ca, and Mg levels in blood plasma. In the current study, 4 800 FTU/kg was not as good as 1 200 FTU/kg in reducing soft-shell egg incidence, nor were 300 or 0 FTU/kg. This indicates that phytase dose effects on shell parameters can vary, which in turn can influence the production of marketable eggs.

Lysine is a structural component in the protein matrix of mammillary bodies in the eggshell⁵²; thus, it can alter mineral deposition during shell formation, leading to defects in the microstructure.⁵³ In the present study, lysine influenced the incidence of soft-shell eggs, but not shell thickness, reinforcing the idea that microstructure conformation of eggshell has a greater influence than macro-structural components. This is because size and orientation of mineral crystals in the eggshell can directly affect mechanical properties.⁵⁴ An important point to highlight on the design of the present study is the fact that individual caging system allowed the observation of dietary effects on egg cracking, avoiding those resulting from the limitation of cage space.

When non-sellable egg (broken and soft-shell) was subtracted from egg mass to give “marketable egg mass”, dLys levels influence on the effect of phytase became more apparent F_12,169= 2.41, p = 0.02 (Table 4). Thus, in the current study, lysine shortage (0.67 %) decreased the use of nutrients released from phytase, resulting in a lower response to higher phytase doses. Additionally, for 0.87 % dietary lysine without phytase, P shortage and high lysine content led to a nutritional imbalance that negatively impacted performance; better performance was achieved by phytase addition at 300 and 1 200 FTU/kg, but not at 4 800 FTU/kg. In medium level of dLys (0.77 %), 300 FTU/kg was not able to get egg mass production to the level seen at 1 200 and 4 800 FTU/kg (Figure A). Only one previous study in poultry has been published on the combined implication of phytase dose and lysine level in diet. Selle et al.⁴⁶ in 7-28 day-old broilers, found that the addition of 500 FTU/kg 3-phytase from A. niger performed better with the dLys level considered as deficient (10 g/kg) compare to the adequate level (11.8 g/kg). Although, marketable egg mass was strongly affected in a quadratic manner by increasing of phytase dose, with a positive effect up to 1 200 FTU/kg F_2,179= 5.61, p = 0.01. Dietary 0.25 % avP resulted in less marketable egg mass in comparison to 0.12 % avP diets F_12,169= 7.24, p = 0.01. There was no effect of treatment on shell thickness F_12,169= 0.53, p = 0.89 and yolk pigment F_12,169= 1.10, p = 0.36. Layers fed 0.12 % avP diets gave higher average Haugh units than diets with 0.25 % avP F_12,169= 3.72, p = 0.05 (Table 4).

Figure 1 Response of egg marketable mass (A) and ash concentration in bone (B) to incremental digestible lysine and phytase dosing. 

Bone breaking strength F_3,169= 4.29, p < 0.01 and ash weight F_3,169= 3.62, p = 0.01 was higher in tibia of hens fed diets with 1 200 FTU/kg phytase relative to those fed without phytase. Bone breaking strength, representing bone status, is greater when P assimilation from diet is higher.⁴⁹ In this context, 1 200 FTU/kg phytase improved P bioavailability and in this way bone strength, relative to 300 and 4 800 FTU/kg, giving a quadratic effect. The same pattern was seen with ash weight is accord with the correlations mentioned by Kim et al,⁵⁵ indicating that more P is released from diet with higher than standard doses of phytase to support bone status in laying hen. Adeola and Walk⁵⁶ demonstrated that P utilization for bone mineralization from phytase release could be around 87.4 and 92.8 % with 5 or 6 g/kg of highly soluble Ca, respectively. On other hand, addition of inorganic P to diets resulted in similar bone breaking strength F_1,169= 0.92, p = 0.34 and ash weight F_1,169= 1.01, p = 0.31 as inclusions of 300 and 4 800 FTU/kg phytase (Table 5). These bone status results suggest that an extra-phosphoric effect is obtained using 1 200 FTU/kg in laying hens. Additionally, a dLys level and phytase dose interaction was seen in fat-free dry bone weight and ash weight F_12,169= 1.79, p = 0.01 (Table 5) (Figure B). Additionally, changes in bone ash concentration can depend on dLys level in diet, which could be associated with the protein component of bone tissue, or the overall metabolic modification by dLys level of feed intake and laying rate. With 0.67 and 0.77 % dLys, bone ash concentration was reduced with increasing phytase dose, but at 0.87 % dLys, the response was the opposite. Contrary to this result, Ravindran et al.⁵⁷ did not observe changes in toe ash content with incremental levels of lysine in 28 week-old broilers.

It was possible that inclusion of 4 800 FTU/kg phytase would have increased the hydrolysis of phytic acid, and therefore released more nutrients from the diet. Nevertheless, this dose suppressed performance and gave no beneficial effects on bone parameters in hens fed 0.67 and 0.87 % dLys levels, compare to 1 200 FTU/kg phytase. This could be a consequence of unbalanced ratio of nutrients, specially Ca:P, taking into account the constant supply of Ca across treatments. Li et al.⁵⁸ reported in chickens that while dietary avP level increased, total and ionized Ca in plasma decreased, concluding that excess or deficiency of either mineral can interfere directly in the homeostasis of the other and negatively affect animal performance and bone development. Few studies have tested high inclusion of phytase in laying hen, and, contrary to the current study, Meyer and Parsons⁵⁹ did not find changes in productive responses when using 150, 250, and 15 000 FTU/kg of a 6-phytase from E. coli in W-36 Hy-Line layers. Similar results were reported by Augspurger et al.³⁴ with 250, 500, 1000, and 10 000 FTU/kg from a 6-phytase from E.coli in Single-Comb White Leghorn. In this study, 4 800 FTU/kg phytase increased egg production only in 0.77 % dLys diets.

Conclusion

Supplementation with 1 200 FTU/kg of an E.coli derived phytase improved productive performance of laying hens, while maintained body reserves and bone tissue. Similar effects were not observed with 300 or 4 800 FTU/kg of this phytase, or inorganic P supplementation. It seems that phytase supplementation influenced nutrients other than just P, and that higher phytase doses improved the utilization of phosphorus and other nutrients. Furthermore, dietary levels of dLys influenced phytase efficiency, interfering in the response obtained in productive performance and body composition status in laying hens, without equivalence of phytase addition for dLys level in diet.