Introduction

The intense genetic selection for rapid growth in broiler chickens has resulted in the inadequate supply of minerals and metabolic imbalances, leading to a series of unintended negative effects, including skeletal deformities (Julian 1998; Plumstead et al. 2008; Leeson and Summers 2009; Shim et al. 2012). Distress and skeletal disorders are a constant concern, not only for production efficiency and profit for producers, but also for the welfare and health of the birds (Julian 1998; Shim et al. 2012). Changes in conformation and ever-increasing body weight on a physically less-mature skeleton induce the greatest skeletal anomalies in modern broilers (Applegate and Lilburn 2002). Together with Ca, available phosphorus (avP) is responsible for bone rigidity and compressive strength (Proszkowiec-Weglarz and Angel 2013). Moreover, phosphorus (P) is necessary for the production of ATP, RNA and deoxyribonucleic acid, enzymes, and phosphoglycerides, and plays an integral role in maintaining acid–base balance and immune function (Campbell and Farrell 2012; Shim et al. 2012).

P is also an important cause of eutrophication, often increasing the amount of plant and algae growth in lakes and reservoirs (Yan et al. 2004; Jeon et al. 2015). The amount of dietary P and the efficiency of its utilization in the chick’s gut determine the amount of P in the excreta. Approximately 50%–85% of P in plants is in the phytate structure that birds cannot utilize, thereby reducing its absorption efficiency (Plumstead et al. 2008). Phytate (myo-inositol hexakisphosphate, IP₆) or phytic acid is the major storage form of P, comprising 1%–5% by weight in cereals, legumes, oilseeds, and nuts (Vats and Banerjee 2004). The bioavailability of phytate-P for broilers ranges from 0% to 80% depending on diet composition, bird age, and metabolic adaptation (Van der Klis and Versteegh 1999). The chemical characteristics of IP₆ influence exogenous and intestinal phytase efficacy in releasing both phytic-P and any minerals it has complexed with (Angel et al. 2002; Lu et al. 2013). In the gut, phytate forms insoluble complexes with other dietary nutrients, limiting micro-flora and endogenous phytase activity, and resulting in up to 70% dietary P excretion (Lei and Stahl 2001; Gupta et al. 2015). Phytate–protein complexes at both acidic and alkaline pH affect changes in protein structure, resulting in reduced enzymatic activity, protein solubility, and proteolytic digestibility (Adeola and Cowieson 2011).

Additive supplementation of microbial phytases (myo-inositol hexakisphosphate phosphohydrolase) in commercial feed manufacturing has become standard to catalyse the breakdown of phytic acid (IP₆) into a series of lower phosphate esters of myo-inositol and inorganic orthophosphate (Dersjant-Li et al. 2015). With advances in biotechnology, it has become possible to improve the phytase enzyme structure by genetic manipulations in coding sequence of genes (Onyango et al. 2005). Furthermore, extra-phosphoric effects from phytase supplementation beyond the standard doses have been reported to enhance nutrient digestibility when marginally deficient diets are fed, thereby improving the performance of broilers (Lei and Stahl 2001; Campasino et al. 2014). Yan et al. (2004) showed that broilers may be grown effectively on a modified feeding program with sufficient P levels with or without phytase supplementation in the starter period, followed by markedly reduced levels during the grower and finisher period. However, the ideal phytase should be active in the stomach, be catalytically efficient, heat stable during feed processing and storage, and proteolysis resistant, and be inexpensive (Lei and Stahl 2001). It is hypothesized that enzyme type will affect weight gain, feed intake (FI), and bone mineralization in broilers fed P-deficient diets supplemented with phytase enzymes at different doses. Therefore, the objective of this study was to evaluate the efficacy of three microbial phytases, namely a modified Escherichia coli phytase, Phytaverse; an enhanced E. coli phytase, Quantum Blue; and a Buttiauxella species bacterium-sourced phytase, Axtra-PHY (AxP) in diets deficient in avP on the growth performance, bone development, and strength of broilers.

Materials and methods

Experimental diets

Birds were fed in two dietary phases, the starter (0–21 days) and the finisher (22–35 days), to reduce diet-induced variations. The ingredient and chemical composition of experimental diets is shown in Table 1. The description of the nine dietary treatments is shown in Table 2 with three control diets that contained no phytase enzymes. The control starter diets (T1–T3) were formulated as follows: T1 – positive control (PC), the recommended breed standard with 0.43% avP; T2 – negative control 1 (NC1), with 0.23% avP; and T3 – negative control 2 (NC2), the industry standard with 0.33% avP. Treatments T4 to T9 were arranged with enzyme supplementation of the NC1 diet in a 3 × 2 factorial with three phytase enzymes (Fig. 1) added at two different levels, 500 and 1000 FTU/kg (1 FTU of phytase is defined as the quantity of enzyme that liberates 1 µmol of inorganic phosphate per minute from sodium phytate at pH 5.5 and 37 °C). The three phytases include Phytaverse (Novus International, St. Charles, MO, USA), Quantum Blue (AB Vista, Marlborough, UK), and AxP (DuPont, Wilmington, DE, USA). For each control, the finisher diets had a 0.11% reduction in avP as compared to the starter diet, and then treatments 4 to 9 consisted of NC1 with Phytaverse, Quantum Blue, or AxP at 500 or 1000 FTU/kg. The diets were formulated on Format Feed Formulation software (Format Solutions Ltd., Hopkins, USA) using the CVB (Dutch Net Energy) system and were mixed at Simple Grow Agricultural Services. Both diets were pelleted at temperatures set and monitored not to exceed 85 °C.

Table 1.

Composition of broiler starter and finisher diets on as fed basis.

Table 2.

Description of the nine dietary treatments.

Fig. 1.

Phytase enzymes that were used in the study. T4 and T5, Phytaverse (PTV), a modified E. coli phytase at 500/1000 FTU; T6 and T7, Quantum Blue (QB), an enhanced E. coli phytase at 500 and 1000 FTU; and T8 and T9, Axtra-PHY (AxP), a Buttiauxella species bacterium-sourced phytase at 500 and 1000 FTU. [Colour online.]

Fig. 2.

Cumulative mortality (%) of broilers consuming diets deficient in available phosphorus with or without supplementation of heat-stable microbial phytases. PC, positive control (starter diet with 0.43% available phosphorus, avP, finisher with 0.32% avP); NC1, negative control 1 (starter diet with 0.23% avP, finisher diet with 0.12% avP); NC2, negative control 2 (starter diet with 0.33% avP, finisher diet with 0.22% avP); and T4 to T9, NC1 with Phytaverse, Quantum Blue, or Axtra-PHY enzymes at 500 and 1000 FTU/kg.

Results

Growth performance

The response of broiler birds fed avP-deficient, enzyme-supplemented diets is shown in Table 3. There was no interaction effect between phytase enzyme type and their inclusion levels, on BWG of birds at days 7 (d7), 21 (d21), and 35 (d35). Furthermore, the simple effects of enzyme inclusion level did not affect the weight gain of the birds across the growth periods. However, while enzyme type affected BWG at d7 (P = 0.032), this effect was not observed at d21 and d35. The cumulative mortality of the birds showed an average flock mortality of 7.78% with a very high mortality occurring in birds consuming the NC1 diet with 12% mortality at d21 and 23% by d35 (Fig. 2).

Table 3.

Body weight gain and feed intake of broilers fed diets deficient in available phosphorus with or without supplementation of heat-stable microbial phytases.

Across the periods, the NC1 birds (without enzyme supplementation) seemed to have the lowest response in terms of BWG and FI. Furthermore, birds on the negative control treatments (NC1 and NC2) showed lower weight gain when compared with birds on the PC treatment (P < 0.05). When compared with birds on the PC diet, the average of the enzyme-supplemented birds had lower BWG at d7 (P = 0.002) but such difference was not significant at d21 and d35. Phytaverse supplemented birds did not differ from birds on the PC diet in terms of BWG but birds fed Quantum Blue recorded lower weight gain at d7 while birds on AxP diet had lower weight gain at d7 and d35 (P < 0.05) as compared to the PC. While FI differed between birds on the negative control (NC1 and NC2) diets and birds on the PC (P < 0.05),  no differences in FI were observed between the PC and the average of the enzyme-supplemented birds or between PC and birds on the respective enzyme diets.

The interaction effect of enzyme type and inclusion level did not affect FI at d7, while the simple effects of enzyme type, as well as inclusion levels, also did not affect FI at d7. In contrast, the interaction effect of enzyme type and level was observed at d21 (P = 0.002) and d35 (P = 0.031), respectively. At d21, while FI decreased as the inclusion level of AxP increased from 500 to 1000 FTU/kg feed, no such effect was observed in birds supplemented with Phytaverse. However, birds supplemented with Quantum Blue showed a significant increase in FI as inclusion levels increased from 500 to 1000 FTU/kg feed at d21 and d35 (P < 0.05). At d35, inclusion level did not affect FI of birds fed Phytaverse and AxP.

The effect of feeding diets deficient in avP with or without microbial phytase supplementation on FCR and PEF is presented in Table 4. There was no interaction effect of enzyme type and inclusion level on FCR and PEF at d7, d21, and d35. While the simple effect of enzyme type showed differences in FCR (P = 0.014) and PEF (P = 0.003) of the birds at d7, the differences were not observed at d21 and d35. Furthermore, the simple effect of enzyme inclusion levels did not affect both FCR and PEF.

Table 4.

Feed conversion ratio and performance efficiency factor of broilers fed diets deficient in available phosphorus with or without supplementation of heat-stable microbial phytases.

Table 5.

Tibia breaking strength, bone ash, calcium, and phosphorus of broilers fed diets deficient in available phosphorus with or without supplementation of heat-stable microbial phytases.

The average of birds on the negative control diets (NC1 and NC2) was not different from birds on the PC diet in terms of FCR, but PC birds showed superior PEF at d21 (P = 0.01) and d35 (P < 0.01). Birds fed the PC diet had lower and thus, better FCR, compared to the average of birds receiving avP-deficient, enzyme-supplemented diets at d7 (P = 0.005) but the FCR did not differ at d21 and d35. Furthermore, when compared to the PC treatment, PEF was lower at d7 and d35 in birds receiving the avP-deficient, enzyme-supplemented diets. Phytaverse-supplemented birds did not differ from the PC birds in terms of FCR and PEF except at d7 when PEF was lower (P = 0.03). In contrast, birds on Quantum Blue and AxP treatments had lower PEF across the periods (P < 0.05), while their FCR was higher and inferior at d7.

Discussion

Earlier studies have associated P deficiency with the loss of appetite, rickets, and growth failure (Scott et al. 1982). As expected, the NC treatments with avP-deficient diets resulted in lower performance in the broilers compared to the PC diet formulated based on the breed standard across the growth period and this agrees with Sharma et al. (2016), who reported lower FI and BW in birds fed the NC diet without phytase, as compared to birds consuming the diet with the recommended P concentration. The lower growth performance of the NC diets showed that the diets were indeed nutrient deficient. Enzyme supplementation becomes less sensitive above the nutrient requirement of the animals (Wang et al. 2020) and this justifies the supplementation of the NC diet with the phytase enzyme.

The weight gain in enzyme-supplemented birds consuming avP-deficient diets validates previous studies that adding higher levels of phytase to a low-P diet promotes improved feed efficiency. Julian (1998) showed that supplementation of a low-P diet with the evolved E. coli-derived phytase improved weight gain, FI, and tibia ash of chicks. Exogenous phytases have extra-phosphoric effects such as improvement in Ca and amino acid digestibility (Adeola and Cowieson 2011), thus the associative effect on tissue accretion and BWG. Walk et al. (2013) proposed that phytase helps to reduce the extra-phosphoric antinutrient effects of phytate, rather than the liberation of excess P. Nevertheless, Cowieson et al. (2006) suggested that higher phytase doses can be beneficial in retaining P, mediated through an improved phytate P digestibility.

Within the period 1–7 days of age, PC birds had higher weight gains than birds fed an avP-deficient diet and supplemented with enzymes, but this effect was not observable across 1–21 and 1–35 days of age periods. The improved growth in NC + enzyme groups after the 7-day period could be associated with the reduced negative effects of phytic acid on the digestibility of protein. Phytic acid binds to protein in a pH-dependent manner and impeded protein and Ca digestion, and amino acid absorption, and this may be ameliorated by phytase (). Furthermore, the P requirement of broiler chickens continuously changes during growth (NRC 1994). Nevertheless, Lu et al. (2013) reported a significant increase in ileal P digestibility and overall P utilization with phytase enzyme.

The level of P deficiency may interact with the age at which P becomes limiting, and the extent of such interaction will influence overall broiler performance. Older birds have an increasing ability to utilize P in P-deficient diets compared to younger birds (Wang et al. 2020) and this may account for the higher tibia ash concentration across the growth phase. Besides, broilers are more sensitive to the level of Ca and avP in a diet than laying hens due to differences in their abilities to efficiently absorb and retain dietary Ca and P. Thus, bone disorders and impaired performance in broilers persist even when the optimal Ca:avP ratio is subsequently maintained (Driver et al. 2005).

P is associated with the modulation of FI and improved synthesis of growth hormones (Dos Santos et al. 2019). Unlike FCR, where lower values indicate greater flock performance efficiency, a higher PEF value reflects a greater production efficiency (). Overall results on the growth rate, FI, FCR, and PEF of birds consuming avP-deficient diets supplemented with 500 FTU microbial phytases improved close to the breed performance standards but increasing enzyme dose to 1000 FTU/kg feed may not result in added advantages on growth performance. According to Walk et al. (2013), 500 FTU of phytase in corn-soy diet with approximately 0.24% phytate P hydrolyses approximately 62% of the phytate, liberating 0.15% phytate P. Although caution not to affect the mechanism through which intestinal Na-dependent phosphate uptake is stimulated, dietary phosphate deprivation is an important physiological regulator of intestinal phosphate absorption (Fang et al. 2012).

Furthermore, a higher Ca:P ratio of the diet may increase the formation of insoluble Ca–phytate complex in the small intestine (Dersjant-Li et al. 2015). The PC, NC1, and NC2 diets in the current study had Ca:avP ratios of 0.50, 0.33, and 0.43 in the starter feed and 0.46, 0.22, and 0.35 in the finisher feed, respectively. The NC diets were thus adequately balanced to allow phytase activity when supplemented with enzymes. All three enzyme products showed improved growth response in broilers, although birds supplemented with Quantum Blue had lower BWG and FI within the 1–7-day age period but this was short-lived as such differences were not observed at d21 and d35. The source of exogenous microbial phytase used may have a significant effect on the response by broilers in terms of growth as well as bone mineralization (Wang et al. 2020). According to Dersjant-Li et al. (2015), various enzyme products have different pH and temperature ranges within which phytase activity is optimized, with phosphoric effects on broiler performance.

The architecture of the bone structure influences bone strength. Consequently, if it is not maintained leg problems lead to poor animal welfare and become the biggest cause of economic losses in a poultry house due to condemned carcasses and (or) reduced meat grades (Almeida Paz and Bruno 2006; Buijs et al. 2012). BBS determines the amount of force the bones can withstand before fractures and breakage occurs, and is a function of its physical, architectural, and matrix characteristics (Proszkowiec-Weglarz and Angel 2013). Tibiae bone is one of the most mineralized bones in the poultry skeleton, and tibiae ash is the primary criterion for the determination of calcium and phosphorous requirements in most commercial poultry species (Skinner and Waldroup 1995; Angel 2007).

Commercially, phytase is supplemented at 500 FTU/kg diet; doses from 1000 FTU/kg diet upwards may have extra-phosphoric benefits due to a 30% additional increase in phytate dephosphorylation (Lei and Stahl 2001; Cowieson et al. 2006; Campasino et al. 2014). In the current study, enzyme type and interactions between enzyme type and levels had no significant effect on BBS at d21 and d35, respectively; however, enzyme level effect of P = 0.040 was obtained at d35. Phytaverse birds had higher BBS at 21 and 35 days, respectively, for both inclusion levels, while no differences were observed between PC and enzyme-supplemented birds. Morgan et al. (2016) showed that phytase supplementation at 500 FTU/kg increased BBS, regardless of phytate susceptibility to phytase, compared to a diet with no phytase. In turkey poults, Applegate et al. (2003) noted that 500 FTU/kg E. coli phytase supplementation provided 68.2% non-phytate P (nPP) sparing effects, with 0.49% of diet P retained towards the upper extreme of the linear curve of the reference diets at 0.23% nPP, and 0.47%–0.7% nPP, respectively, as compared with only 58.9% P being retained from the unsupplemented control diet.

The tibia has been noted as the primary storage site for P and tibia ash concentration is noted as a reliable indicator of P status in broilers (Wang et al. 2020). Applegate and Lilburn (2002) suggested that the femur may be a more reliable bone to measure breaking strength, rather than the tibia as it is a more precise indicator of the maximum load (weight of the broiler) at any specific age and it is more sensitive to overall skeletal mineralization, and Ca and P turnover. Vieira et al. (2015), however, reported that both the epiphysis of the tibia and femur showed a similar response in bone density (Seedor index) in birds fed P-deficient or enzyme-supplemented diets, although bone strength was generally higher in the tibia than the femur. Cowieson et al. (2006) reported that birds fed a P-deficient diet had significantly lower toe ash percentage compared to birds fed the diets with standard avP concentration, even though the P-deficient treatments had higher coefficients of P, Na, Ca, Cu, Fe, and Mn retention. In the current study, enzyme effect was highly significant at d35 (P < 0.01) for tibia ash, with significant differences between Phytaverse and PC. An increase in avP from NC1 to NC2 showed an increase in BBS and tibia ash at d21 and d35 and this can be associated with the 0.07% and 0.08% increase in Ca in the NC2 starter and finisher feeds, respectively. Dersjant-Li et al. (2015) noted that the addition of phytase to the NC diet at the levels of 1000 or 1500 FTU/kg significantly increased tibia ash weight, but with no effect on the PC (diet with adequate avP). It may be that the addition of higher concentrations of phytase allows complete dephosphorylation of phytate to occur more effectively under relatively constrictive conditions within the chicken gut (Cowieson et al. 2006).

As observed by Wilkinson et al. (2014), the addition of phytase to the NC1 diet eliminates the Ca–phytate complex, restoring the Ca to avP balance in the diet. The degree to which phytase can dephosphorylate phytate correlates to the Ca level and the ratio of Ca:avP in the diet may have a profound effect on bone mineralization (Dersjant-Li et al. 2015). As such, the actual Ca levels in the PC, NC1, and NC2 diets would be 0.85%, 0.70%, and 0.77% Ca in the starter, and 0.71%, 0.55%, and 0.63% Ca in the finisher diet, respectively. However, upon analysis of the final feed offered to birds, the total Ca concentrations were 0.93%, 0.87%, and 0.91% in the starter feed and 0.79%, 0.78%, and 0.85% in the finisher feed, for the PC, NC1, and NC2 treatments, respectively. Consequently, the Ca:avP ratio in the complete starter and finisher diets is expected to change. Although the actual avP content of the final feed was not analysed, we can speculate that the avP content would have increased in the NC2 diet such that the Ca:avP ratio in the starter feed could be in the range: PC (1.84–2.14); NC1 (3.11–3.78); and NC2 (2.46–2.76) and in the finisher feed: PC (1.93–2.47); NC1 (5.20–6.50); and NC2 finisher (3.15–3.86), respectively. The formulated Ca:avP ratio and Ca levels in this study were above the recommended Cobb500 (2018) nutrient specifications, but the avP level was less. In formulating broiler diets, the total Ca is typically included at 8.0 and 10.0 g/kg (Wilkinson et al. 2014). Similar studies on the Ross breed (female Ross 308 and male Ross 344 × 508) suggested a Ca:avP ratio of 2.3–2.5:1 for optimal performance and bone development (Plumstead et al. 2008; Han et al. 2016). According to Amerah et al. (2014), an increased Ca:avP ratio is likely to reduce phytate degradation and P digestibility.

Furthermore, microbial sources of phytases influence their catalytic activity and variations exist in the efficacy of the new generations of E. coli phytases to liberate inorganic phosphate (Dersjant-Li et al. 2015). As Morgan et al. (2016) suggested, the determination of phytase activity based on a standard procedure involving inorganic P release at a pH of 5.5 may lead to the underestimation of activity of phytase sources with low pH optima and overestimation of those with higher pH optima. Specifically, at pH 4, phytate binds to Ca²⁺ to form stable insoluble complexes, and upon hydrolysis by phytase, it releases P, which disrupts the Ca:avP ratio and increases the formation and excretion of Ca–P salts (Onyango et al. 2005; Dersjant-Li et al. 2015; Pieniazek et al. 2016). However, the use of limestone as a dietary Ca source increases the alkalinity of the diet, and thus digesta pH, inhibiting phytase activity (Pieniazek et al. 2016). The ideal phytase is the one that works over a wide range of pH values and is active in the stomach and upper intestine, in addition to being refractory to endogenous enzymes (Dersjant-Li et al. 2015). The analysis of dietary Ca and avP is necessary for ascertaining the optimal ratio for growth performance and bone development when diets marginal in available P are fed to broiler chickens, with phytase supplementation.

Conclusions

The supplementation of microbial phytases in diets deficient in avP resulted in improved growth performance similar to the PC that had optimal avP. For all three microbial phytases, a dose of 500 FTU/kg diet showed similar responses in broiler performance and 1000 FTU/kg dose only improved BBS and ash concentration, but not growth performance.