Application of enzyme matrix values for energy and nutrients to a wheat-corn-soybean meal-based broiler diet supplemented with a novel phytase, with or without a xylanase–β-glucanase, achieved a production benefit over a nutritionally adequate unsupplemented diet

Abstract

The effect of applying an energy and nutrient matrix to a wheat-corn-soybean meal-based diet supplemented with a novel consensus bacterial 6-phytase variant (PhyG) and xylanase–β-glucanase on growth performance, bone mineralization, carcass weights, feed costs, and carbon footprint was evaluated. A randomized complete block design (3,300 Ross 308 mixed-sex birds; 60 pens, 12 pens per treatment) tested 5 treatments: 1) a positive control diet (PC), containing 0.92, 0.84, 0.71% Ca and 0.43, 0.38, 0.30% digestible P during 1 to 10, 11 to 21, and 22 to 32 d of age, respectively; 2) a negative control reduced in Ca, digestible P, digestible AA, ME, and Na by phase based on the PhyG dosing regimen (NC1); 3) NC1 supplemented with PhyG at 2,000, 1,500, and 1,000 FTU/kg by phase (NC1+PhyG); 4) as NC1 but additionally reduced in ME (NC2); and 5) NC2 supplemented with PhyG as in 3) plus 1,220 U/kg of xylanase and 152 U/kg of β-glucanase (NC2+PhyG+XB). Final (d 32) BW, overall (0–32 d of age) ADFI, FCR, d 10 and 32 tibia ash and carcass part weights were reduced or impaired (P < 0.05) in NC1 and NC2 vs. PC (d 32 BW −477 g/bird (23.4%) and −422 g/bird (20.7%), respectively). Growth performance (all measures, all phases) was improved and tibia ash (at 10 and 32 d of age), total carcass thigh, breast and leg weights were increased (P < 0.05) in NC1+PhyG vs. NC1, and NC2+PhyG+XB vs. NC2. Overall growth performance outcomes in NC1+PhyG and NC2+PhyG+XB were not different (P > 0.05) from the PC. Total feed cost and carbon footprint per kilogram BW gain (BWG) were reduced (P < 0.05) vs. PC in NC2+PhyG+XB [−0.052 € and −376 g CO₂ eq./kg BWG, respectively] and NC1+PhyG [−0.038 € and −260 g CO₂ eq./kg BWG, respectively]. The results validated the nutrient matrices in the test diets and highlighted a potential feed cost and environmental sustainability benefit which was greatest when the enzymes were applied in combination.

INTRODUCTION

European broiler diets contain wheat in addition to corn and soybean meal (SBM) as the conventional raw materials. Wheat is rich in starch (a source of energy) but also higher in fiber than corn (Nyman et al., 1984) and contains antinutritional compounds (ANF), including high concentrations of nonstarch polysaccharides (NSP) that reside in fibrous plant cell walls. A high dietary NSP content can increase digesta viscosity and reduce nutrient absorption in the small intestine and negatively affect gut microbial populations and gut health (Choct et al., 1999; Ravindran and Son, 2011; Bedford and Apajalahti, 2022). Broiler diets also increasingly contain industrial by-products such as rapeseed meal and sunflower meal, as alternative, cheaper, sources of key nutrients. These are substantially higher in NSP (de Vries and Lannuzel, 2021) and also tend to be higher in another ANF, phytate (Selle et al., 2011), than conventional cereal and oilseed ingredients.

Microbial phytase is used almost ubiquitously in commercial broiler diets because of its high efficacy to improve the availability and utilization of P in feed. It catalyzes the stepwise hydrolysis of phytate (salt of phytic acid, myoinositol hexakisphosphtate; IP₆), the major source of P in plant-based feed ingredients, releasing inorganic phosphate (iP) to the animal for use in growth and maintenance (Selle and Ravindran, 2007; Greiner and Konietzny, 2011). Phytases can also improve the digestibility of other nutrients beyond P. The phytase tested in the present study has been shown to improve the digestibility of Ca (Babatunde et al., 2021; Espinosa et al., 2021; Bello et al., 2022), protein and amino acids (AA; Babatunde et al., 2022; Dersjant-Li et al., 2022a,b), energy (Dersjant-Li et al., 2022b), and sodium (Espinosa et al., 2021). These “extra-phosphoric” effects result from a reduction in the presence of phytate in the digesta to interact with and impair the digestibility of these nutrients due to its degradation by phytase (Selle et al., 2000,2009). Responses vary across phytases (Bello et al., 2019; Dersjant-Li and Kwakernaak, 2019), at different phytase dose levels (Dersjant-Li et al., 2022a,b,c) and in diets of differing composition (in particular depending on the concentration and inherent digestibility of the affected nutrient and of the contained phytate; Ravindran et al., 1999; Li et al., 2017). For PhyG, multiple studies have shown a consistency of improvement in growth performance and in the digestibility of Ca, P, AA, and energy in birds supplemented with the phytase and these studies have enabled the derivation of matrix values for each of these nutrients, that are linked to the diet composition. For example, the PhyG digestible AA matrix was generated based on meta-analysis of 13 datasets from in vivo studies that evaluated the dose-response effect of the phytase on the ileal digestibility of AA when added to diets of different composition (Babatunde et al., 2021, 2022; Dersjant-Li et al., 2022a).

The application of matrix values with phytase can reduce the overall cost of the diet, enable more flexibility in ingredient quality and choice as well as reduce nutrient excretion and thereby contribute to environmental goals. However, although use of a mineral matrix [reduction in dietary Ca and digestible P content] is well accepted among nutritionists and producers, the implementation of a full matrix, that additionally includes a reduction in dietary digestible AA, energy, and sodium (Na), is less widespread, primarily because of greater variability in the bird response for these nutrients across the phytase literature (Dersjant-Li et al., 2019). Part of this variability is due to a substantial difference between individual phytases in their biochemical and enzymatic properties (Menezes-Blackburn et al., 2015) which, along with other factors, affects the degree of bird response in vivo. Hence, studies to validate the use of a full nutrient matrix have to be conducted on an individual phytase basis and using the diet composition for which their use is intended.

Carbohydrases, including xylanase and β-glucanase are increasingly used in combination with phytase, to maximize nutrient availability and utilization from feed, especially in high fiber diets and those containing added by-products. These enzymes hydrolyze indigestible NSPs, specifically, xylans and β-glucans, in the fibrous plant cell walls of wheat and other fibrous cereals and derived ingredients to release encapsulated nutrients and energy, leading to improved BW and feed conversion (Gilani et al., 2021). In diets containing wheat, Zhang et al. (2014) reported xylanase-related improvements in the apparent ileal digestibility (AID) of crude protein (CP; +3.5%), starch (+9.3%), soluble NSP (+43.9%), and insoluble NSP (+42.2) as well as apparent total tract digestibility of DM (+5.7%), alongside improved feed conversion ratio [measured as gain-to-feed ratio (G:F)] and BW gain (+5.8%) at 21 d of age in broilers. In theory, phytase and carbohydrases may act synergistically in diets containing wheat and fibrous by-products if the hydrolysis of NSPs in the cell walls of these ingredients increases accessibility of phytase to the phytic acid contained therein (Woyengo and Nyachoti, 2011). In practice, additive, subadditive, or synergistic effects of carbohydrases with phytase have been observed (Cowieson and Adeola, 2005; Juanpere et al., 2005; Romero et al., 2013, 2014; Singh et al., 2017) and it is necessary to validate nutrient matrix values for each specific enzyme combination separately.

The principal aim of this study was to evaluate whether the application of a full nutrient matrix to a wheat-corn-SBM-based diet containing by-products supplemented with a commercially available novel consensus bacterial 6-phytase variant, with or without a xylanase and β-glucanase combination, could maintain growth performance, bone mineralization and carcass characteristics equivalent to a nutritionally adequate diet. A secondary aim was to quantify any feed cost and production benefit of the enzyme-supplemented diet.

MATERIALS AND METHODS

Experimental Design, Birds, and Housing

All animal care procedures were approved by the Neuro Livestock Research (NLR) ethics committee prior to the start of the research and conformed to the South African Poultry Association's code of practice.

A total of 3,300 one-day-old straight-run Ross 308 broilers were obtained from a commercial hatchery and assigned to 60 floor pens (55 birds/pen, stocking density 22 birds/m²) with 12 replicate pens per treatment, in a randomized complete block design, with every consecutive 5 pens containing all 5 treatments in random order considered as block. Hatchlings were weighed and assigned to pens so that each pen contained birds of approximately equal BW. Pens were located in a commercial broiler house in which the ambient temperature was maintained initially at 35°C and then gradually reduced to 24°C by 28 d of age. The lighting regime was LD 18:6 h. Pine wood shavings were used as litter. Diets and water were provided ad libitum for the duration of the trial (1–32 d of age).

Dietary Treatments

The 5 dietary treatments included a nutritionally adequate positive control (PC) diet, 2 negative control (NC) diets (NC1 and NC2), NC1 supplemented with a commercial phytase (PhyG) dosed at 2,000 phytase units (FTU) per kilogram of feed in starter phase (1–10 d of age), 1,500 FTU/kg in grower phase (11–21 d of age) and 1,000 FTU/kg in finisher phase (22–32 d of age) (NC1+PhyG), and NC2 supplemented with PhyG as in NC1 but also with a commercial xylanase–β-glucanase combination that supplied 1,220 U of xylanase and 152 U of β-glucanase per kilogram of final feed (NC2+PhyG+XB). The PC diet was formulated in 3 feeding phases according to Dutch industry standards for nutrient contents in broiler diets (CVB, 2018) which are slightly below the breeder recommended levels (Aviagen Inc., 2019b). The ingredient content of the diets is presented in Table 1 and the calculated nutrient content is given in Table 2. The energy and nutrient reductions in the NC1 and NC2 vs. PC (Table 2) were provided by the supplier of the enzymes and were derived based on the dietary substrate levels. The reductions were achieved by optimizing the feed formulation, taking account of the constraints imposed by the applied nutrient reductions. The phytase was a novel consensus bacterial 6-phytase variant (Axtra PHY GOLD, Danisco Animal Nutrition & Health (IFF)) produced in Trichoderma reesei. The xylanase-β-glucanase combination comprised of a 1,4-β-xylanase (EC 3.2.1.9) produced in T. reesei and an endo-1,3(4)-β-glucanase (EC 3.2.1.6) produced in T. reesei (Axtra XB, Danisco Animal Nutrition & Health (IFF)). The diets were manufactured at the NLR feed mill in 3 separate batches according to the formulation for PC, NC1 and NC2. The NC1 and NC2 batches were then each divided into 2 equal portions and the enzymes added to one portion, according to treatment. Enzymes were mixed with a small amount of the basal diet before adding to the main diet and then diets were thoroughly mixed to ensure a homogeneous distribution of the enzymes. Starter phase diets were prepared as crumbled diets whereas grower and finisher diets were fed as pelleted diets (pelleting temperature 80°C).

Table 1.

Ingredient composition (as fed basis) of the starter (1–10 d of age), grower (11–21 d of age), and finisher (22–32 d of age) phase basal diets.

	Starter (1–10 d of age)			Grower (11–21 d of age)			Finisher (22–32 d of age)
Diet	PC	NC1	NC2	PC	NC1	NC2	PC	NC1	NC2
Ingredient, %
Wheat	28.32	36.07	36.98	28.28	34.10	35.85	34.27	40.78	42.84
Corn	25.00	25.00	25.00	30.00	30.00	30.00	30.00	30.00	30.00
Soybean meal	30.32	26.82	27.04	24.41	22.30	22.05	18.00	14.76	14.23
Sunflower meal	4.00	4.00	4.27	4.50	4.50	4.50	5.00	5.00	5.00
Rapeseed meal	3.00	3.00	3.00	3.50	3.50	3.50	4.00	4.00	4.00
Soy oil	3.88	1.48	0.50	4.03	2.19	0.74	4.17	2.54	1.05
Palm oil	1.00	0.50	0.20	1.00	0.50	0.50	1.00	0.50	0.50
Limestone	1.13	1.20	1.20	1.08	1.13	1.14	1.01	1.06	1.06
Monodicalcium phosphate	1.68	0.52	0.51	1.44	0.35	0.34	1.02	−	−
L-Lysine HCl	0.31	0.32	0.29	0.34	0.32	0.31	0.32	0.35	0.34
DL-Met	0.29	0.22	0.20	0.27	0.21	0.19	0.21	0.16	0.15
L-Thr	0.14	0.12	0.10	0.13	0.11	0.10	0.09	0.09	0.08
L-Arg	−	0.03	0.01	−	−	−	−	0.01	0.01
L-Val	0.02	0.01	−	0.04	0.02	0.01	−	−	−
L-Ile	−	−	−	0.03	0.01	0.01	0.01	0.02	0.02
Sodium bicarbonate	0.45	0.27	0.26	0.47	0.27	0.27	0.46	0.29	0.29
Sodium chloride	0.16	0.15	0.16	0.15	0.15	0.15	0.15	0.14	0.14
Vitamin-mineral premix1	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30
Coccidiostat2	0.05	0.05	0.05	0.05	0.05	0.05	−	−	−
Phytase3	−	−/+	−/+	−	−/+	−/+	−	−/+	−/+
Xylanase and β-glucanase4	−	−	−/+	−	−	−/+	−	−	−/+

¹Supplied per kilogram of diet: 4,800 IU of vitamin A; 2,000 IU of vitamin D3; 24 mg of vitamin E; 0.8 mg of vitamin K3; 0.8 mg of vitamin B1; 2 mg of vitamin B2; 20 mg of B3; 4.8 mg of vitamin B5; 1.2 mg of vitamin B6; 10 mcg of vitamin B12; 140 mg of choline chloride; 0.8 mg of folic acid; 0.1 mg of Biotin; 15 mg of Fe; 4 mg of Cu; 44 mg of Mn; 40 mg of Zn; 0.8 mg of I; 0.12 mg of Se; 0.2 mg of Co; and 50 mg of antioxidant.

²Cycostat (Zoetis Australia Pty Ltd., NSW, Australia).

³A novel consensus bacterial 6-phytase variant (PhyG), added at 2,000, 1,500, and 1,000 FTU/kg during starter, grower, and finisher phase, respectively.

⁴A combination of xylanase and β-glucanase that supplied 1,220 U of xylanase and 152 U of β-glucanase per kilogram of final feed.

Table 2.

Calculated nutrient composition and estimated carbon footprint (CFP) content of the starter (1–10 d of age), grower (11–21 d of age), and finisher (22–32 d of age) phase basal diets.

Diet	Starter (1–10 d of age)			Grower (11–21 d of age)			Finisher (22–32 d of age)
	PC	NC1	NC2	PC	NC1	NC2	PC	NC1	NC2
Calculated nutrients, % unless stated
GE, kcal/kg
ME, kcal/kg	2,950	2,871	2,793	3,025	2,959	2,881	3,100	3,056	2,978
CP	22.19	21.45	21.67	20.26	19.88	19.93	19.36	18.85	18.89
SID Lys	1.22	1.16	1.15	1.11	1.06	1.05	0.99	0.95	0.94
SID Met and Cys	0.90	0.81	0.80	0.84	0.77	0.75	0.77	0.72	0.70
SID Met	0.60	0.52	0.50	0.56	0.49	0.47	0.49	0.44	0.42
SID Thr	0.81	0.75	0.74	0.74	0.69	0.68	0.65	0.62	0.61
SID Trp	0.23	0.22	0.23	0.21	0.20	0.20	0.19	0.18	0.18
SID Arg	1.34	1.29	1.29	1.20	1.16	1.16	1.09	1.03	1.03
SID Ile	0.81	0.76	0.78	0.75	0.71	0.71	0.67	0.64	0.64
SID Leu	1.49	1.43	1.45	1.37	1.34	1.34	1.28	1.23	1.23
SID Val	0.91	0.86	0.86	0.84	0.80	0.80	0.75	0.72	0.72
Calcium	0.92	0.68	0.68	0.84	0.62	0.62	0.71	0.50	0.50
Total P	0.73	0.48	0.49	0.65	0.43	0.44	0.59	0.37	0.38
Digestible phosphorus1	0.43	0.23	0.23	0.38	0.19	0.19	0.30	0.12	0.12
Phytate phosphorus	0.29	0.29	0.30	0.28	0.29	0.29	0.28	0.28	0.28
Sodium	0.19	0.14	0.14	0.19	0.14	0.14	0.19	0.14	0.14
Total arabinoxylans	4.27	4.61	4.69	4.31	4.58	4.67	4.50	4.77	4.88
Total beta-glucans	3.12	3.21	3.27	3.09	3.18	3.22	3.11	3.18	3.22
Total NSP	14.11	14.22	14.46	13.84	14.03	14.16	13.54	13.58	13.68
CFP, g CO2 eq./kg feed2	1,912	1,680	1,572	1,695	1,532	1,471	1,440	1,236	1,128

Abbreviations: CFP, carbon footprint; SID, standardized ileal digestible.

¹Excluding the contribution of the added phytase.

²Total carbon footprint, including the carbon footprint from fossil fuels and from land use change. Calculations made using Wageningen Feedprint NL software (Feedprint NL, 2020).

Measurements and Sampling

Body weight was measured on a per pen basis at the end of each dietary phase (10, 21, and 32 d of age) and used to calculate ADG. Feed was weighed at the start and end of each phase and used to calculate ADFI. Feed conversion ratio was calculated from ADFI and ADG (corrected for mortality). Birds were monitored daily for health and mortality and any dead birds were removed. At 10 and 32 d of age, 4 birds per pen were euthanized by cervical dislocation and the right tibias extracted and pooled. Fibula, muscle and connective tissues were removed, and tibias were dried at 80°C for 72 h and de-fatted using refluxing petroleum ether for 16 h according to the method of Li et al. (2015). De-fatted tibias were dried at 80°C for 48 h to obtain DM and then ashed at 600°C for 16 h. The ash content was reported as ash weight (g) and as the percentage of fat-free DM. At 32 d of age, an additional 3 birds per pen were euthanized using the same method as at 10 d of age and eviscerated for the determination of carcass part weights. The absolute weights of the total carcass, breast filet, thigh filet and leg were determined, and the breast, thigh and leg yields (as a percentage of carcass weight) were calculated.

Chemical Analysis

Representative samples of all treatment diets were analyzed by Chemuniqué International (PTY) Ltd. using AOAC (2000). Samples were analyzed for ash content (method 942.05), crude fiber (method 962.09), crude fat (method 920.39), and CP (method 988.05). Calcium and P were analyzed using inductively coupled plasma atomic emission spectroscopy (ICP) based on AOAC method 935.13 (AOAC, 1999). Diets were also analyzed for phytase and xylanase activity by Danisco Animal Nutrition Research Centre, Brabrand, Denmark. Phytase was determined according to a modified version of AOAC method 2000.12 (Engelen et al., 2001) where one FTU was defined as the quantity of phytase that released 1 µmol of inorganic orthophosphate from a 0.0051 mol/L sodium phytate substrate per minute at pH 5.5 at 37°C. Xylanase was analyzed by Chemuniqué International (PTY) Ltd., where one xylanase unit was defined as the amount of enzyme that released 0.48 µmol of xylose from wheat arabinoxylan per min at pH 4.2 and 50°C. Beta-glucanase was not analyzed because it was added together with xylanase as one product and the measured xylanase activity was used to also confirm the activity of β-glucanase. Previous in-house testing has demonstrated that both enzymes within the xylanase-beta-glucanase cogranule exhibit good pelleting stability with high recovery after pelleting.

Statistical Analysis

Data were analyzed on a per pen basis by 1-way ANOVA, with 3,300 experimental units, 5 treatments, and 12 replications. Treatment was included as a fixed effect. Data were tested for normality prior to analysis, using the Distribution function in JMP 16.0 (2022) which is based on the Proc Univariate procedure of SAS. Means were separated by Tukey's HSD test. All analyses were performed using the Fit Model platform of JMP 16.0 (2022). Effects were considered significant at P < 0.05.

RESULTS

Enzyme Recoveries and Analyzed Nutrients

The level of phytase activity recovered from the PC and unsupplemented NC diets was low (<350 FTU/kg; Table 3). The analyzed phytase activity in the phytase-supplemented diets (NC1+PhyG and NC2+PhyG+XB) was close to target levels during starter and finisher phases. During grower phase they were above target levels (by ∼30% after accounting for native phytase present in the basal diets). Xylanase activities were low in the PC and unsupplemented NC diets and close to target levels in the NC2+PhyG+XB diets. Analyzed levels of nutrients in the diets were broadly consistent with expected levels based on the diet formulations and showed good consistency between the NC diets and the respective phytase-supplemented diets (Table 3).

Table 3.

Analyzed nutrient composition (%, as fed basis, unless stated) and enzyme activities of the experimental diets.

Treatments	Moisture	Ash	Fat	Crude fiber	CP	Ca	P	Phytase, FTU/kg	Xylanase, U/kg
1–10 d of age
PC1	9.71	5.08	7.09	3.63	21.70	0.88	0.70	161	0
NC11	10.32	4.02	4.28	3.73	20.30	0.65	0.47	113	42
NC1+PhyG	10.12	4.29	4.30	3.68	21.99	0.65	0.47	1,866	4
NC21	10.33	4.44	3.23	3.55	21.10	0.68	0.48	123	90
NC2+PhyG+XB	10.15	4.39	3.06	3.53	21.66	0.68	0.48	2,010	1,140
11–21 d of age
PC	8.89	5.38	7.05	4.60	20.07	0.80	0.64	188	153
NC1	9.79	4.56	4.44	4.44	19.95	0.61	0.42	167	0
NC1+PhyG	9.60	4.52	4.13	4.55	20.05	0.59	0.42	1,444	0
NC2	9.96	4.59	3.04	4.36	20.14	0.61	0.43	144	0
NC2+PhyG+XB	9.46	4.65	2.92	4.62	19.99	0.61	0.44	1,434	1,106
22–32 d of age
PC	9.62	4.37	7.50	4.13	19.33	0.71	0.60	267	0
NC1	9.72	3.53	5.75	4.12	18.75	0.53	0.40	306	0
NC1+PhyG	9.59	3.56	5.61	3.89	18.96	0.53	0.36	1,021	0
NC2	9.65	3.58	4.23	3.65	19.07	0.48	0.37	207	0
NC2+PhyG+XB	9.95	3.51	4.23	3.69	18.85	0.48	0.39	1,114	1,490

¹PC, nutritionally adequate positive control; NC1, reduced in digestible P, Ca, digestible AA, ME, and Na vs. PC diet, to account for the expected contribution of the phytase, as detailed in Table 1; NC2, reduced in digestible P, Ca, digestible AA, ME, and Na vs. PC to account for the expected contribution of the phytase and the xylanase and β-glucanase preparation (XB), as detailed in Table 1.

Growth Performance

There were significant effects (P < 0.001) of treatment on all growth performance measures during all individual phases and cumulatively (Table 4). On average, birds fed the PC diet achieved a final (d 32) BW of 2,032 g/bird and an overall (d 1–32) FCR of 1.413 (Table 4). Compared to the PC, birds fed the nutrient reduced NC1 and NC2 diets exhibited reduced ADFI (all phases; P < 0.05), BW (all phases; P < 0.05; −9% at 10 d of age, −17 to −20% at 21 d of age, and −21 to −23% at 32 d of age), ADG (all phases) and increased FCR (NC1 increased in grower phase only, NC2 increased in all phases; P < 0.05). Average final BW at 32 d of age was 1,555 g/bird in NC1 (−23.5% vs. PC, P < 0.05) and 1,610 g/bird in NC2 (−20.8% vs. PC, P < 0.05). Supplementation of PhyG to NC1 increased BW, ADG and ADFI during all phases and overall (P < 0.05); final (d 32) BW was 31.5% higher in NC1+PhyG than NC1 (2,045 vs. 1,555 g; P < 0.05). The FCR of birds fed NC1+PhyG was also reduced vs. NC1 and PC during starter phase (by −5.6 and −3.0%, respectively; P < 0.05), reduced vs. NC1 but higher than PC (+3.8%) during grower phase (P < 0.05), and not different from PC in finisher phase and overall. Overall responses (all measures) of birds fed NC1+PhyG were not significantly different to those of birds fed the PC. Supplementation of PhyG and XB to NC2 increased BW, ADFI and ADG and reduced FCR during all individual phases and overall (P < 0.05); final (d 32) BW was 25.8% higher in NC2+PhyG+XB than NC2 (2,025 vs. 1,610 g). Compared to PC, birds fed NC2+PhyG+XB exhibited higher BW, ADFI and ADG during 0 to 10 d of age (+3.5, +3.0, and +4.2%, respectively; P < 0.05), whereas FCR was not different to PC. During 11 to 21 and 22 to 32 d of age, the growth performance of birds fed NC2+PhyG+XB was not different to that of birds fed the PC (all measures). For the overall period, there was no difference in growth performance responses (all measures) of birds fed treatment NC2+PhyG+XB compared to PC. Livability was high (>98% in all phases) in the PC, NC1+PhyG and NC2+PhyG+XB but reduced in NC1 and NC2 vs. PC (P < 0.05) during finisher phase (95.7 and 96.5%, respectively, vs. 99.1%) and overall (94.5 and 95.8%, respectively, vs. 98.8%; in PC P < 0.05).

Table 4.

Effect of phytase alone or together with a xylanase and β-glucanase preparation, on growth performance, feed costs, and total carbon footprint^1,2.

Treatments	BW3, g/bird	ADG, g/bird	ADFI, g/bird	FCR, g:g/bird	Livability, %	Feed cost, €/kg BWG	CFP (g CO2 eq)/kg BWG5
1–10 d of age
PC4	303.2b	26.09b	27.59a	1.079bc	100.0	−	−
NC14	274.9c	23.29c	25.38b	1.109ab	99.0	−	−
NC1+PhyG	315.3a	27.32a	28.01a	1.047d	99.4	−	−
NC24	277.0c	23.48c	26.08b	1.131a	99.4	−	−
NC2+PhyG+XB	313.8a	27.18a	28.42a	1.090cd	99.3	−	−
Pooled SEM	2.026	0.201	0.217	0.008	0.322	−	−
P value	<0.001	<0.001	<0.001	<0.001	0.244	−	−
11–21 d of age
PC	1005.8a	63.70a	82.79b	1.303d	99.5	−	−
NC1	801.2c	47.66c	66.13d	1.390ab	99.1	−	−
NC1+PhyG	1014.5a	63.41a	85.53a	1.353c	99.1	−	−
NC2	833.9b	50.48b	70.46c	1.402a	99.1	−	−
NC2+PhyG+XB	999.5a	62.13a	84.77ab	1.366bc	99.6	−	−
Pooled SEM	7.751	0.592	0.670	0.006	0.345	−	−
P value	<0.001	<0.001	<0.001	<0.001	0.688	−	−
22–32 d of age
PC	2,031.5a	93.23a	146.3a	1.572b	99.08a	−	−
NC1	1,555.4b	68.57b	107.3c	1.594ab	95.65c	−	−
NC1+PhyG	2,045.3a	93.73a	145.3a	1.557b	99.08a	−	−
NC2	1,609.9b	70.53b	113.1b	1.634a	96.54bc	−	−
NC2+PhyG+XB	2,025.0a	93.21a	145.1a	1.567b	98.55ab	−	−
Pooled SEM	14.664	0.956	1.104	0.011	0.519	−	−
P value	<0.001	<0.001	<0.001	<0.001	<0.001	−	−
1–21 d of age
PC	−	45.79a	56.49b	1.243c	99.55	−	−
NC1	−	36.04c	46.73d	1.304a	98.20	−	−
NC1+PhyG	−	46.23a	58.14a	1.268b	98.65	−	−
NC2	−	37.63b	49.33c	1.323a	98.65	−	−
NC2+PhyG+XB	−	45.49a	57.93ab	1.283b	98.94	−	−
Pooled SEM	−	0.366	0.404	0.005	0.408	−	−
P value	−	<0.001	<0.001	<0.001	0.220	−	−
1–32 d of age
PC	−	62.10a	87.37a	1.413c	98.80a	0.529a	2,203a
NC1	−	47.24b	67.53c	1.448b	94.53c	0.497b	1,985b
NC1+PhyG	−	62.54a	88.12a	1.417c	97.89ab	0.491bc	1,943c
NC2	−	48.95b	71.26b	1.476a	95.75bc	0.485c	1,886d
NC2+PhyG+XB	−	61.89a	87.90a	1.430bc	97.73ab	0.477d	1,827e
Pooled SEM	−	0.460	0.589	0.005	0.616	0.002	8.192
P value	−	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

^a–eTreatment means within each column and phase grouping with uncommon superscripts are significantly different at P < 0.05.

¹Values are means of n = 12 observations.

²Differences between means were identified using Tukey's HSD test.

³Determined on the final day of each phase.

⁴PC, nutritionally adequate positive control; NC1, reduced in digestible. P, Ca, digestible AA, ME, and Na vs. PC diet, to account for the expected contribution of the phytase, as detailed in Table 1; NC2, reduced in digestible P, Ca, digestible AA, ME, and Na vs. PC to account for the expected activity of the phytase and the xylanase and β-glucanase preparation (XB), as detailed in Table 1.

⁵Total carbon footprint (CFP), including the carbon footprint from fossil fuels and from land use change. Calculations made using Wageningen Feedprint NL software (Feedprint NL, 2020).

Feed Costs and Carbon Footprint of Diets

An estimate of the total cost of the treatment diets per kilogram of BW gain (BWG) based on feed ingredient prices in January 2022, inclusive of the costs of the enzymes, is presented in Table 4. An estimate of the total carbon footprint (CFP: land use change + fossil) per kg BWG for each diet is also shown. Feed costs per kg BWG were highest for the PC diet, significantly lower for all other diets (P < 0.05) and lowest for diet NC2+PhyG+XB. This diet conferred a reduction in feed cost per kg BWG of 0.014 € vs. NC1+PhyG and 0.052 € vs. PC. The CFP of the diets (per kg BWG) followed a similar pattern, reduced in NC2+PhyG+XB by 116 g CO₂ equivalents vs. NC1+PhyG and by 376 g CO₂ equivalents vs. PC.

Bone Ash

The effect of treatment on tibia ash at 10 and 32 d of age is shown in Table 5. When assessed as a percentage of fat-free DM, tibia ash was reduced in NC1 and NC2 vs. PC (by 9.1 and 6.9% points, respectively, at 10 d of age, and by 9.7 and 6.9% points, respectively, at 32 d of age; P < 0.05); the reductions were greater in NC1 than NC2 (P < 0.05). When assessed as total tibia ash weight, tibia ash was reduced by a similar amount in NC1 and NC2 vs. PC at 10 d of age and by a greater amount in NC1 than NC2 at 32 d of age (P < 0.05). The addition of PhyG with or without XB improved tibia ash (vs. the respective NC) both on a percentage and absolute weight basis, at both 10 and 32 d of age (P < 0.05), in all cases not different from the level achieved by birds fed the PC.

Table 5.

Effect of phytase alone or together with a xylanase and β-glucanase combination on tibia ash (percentage and weight) at 10 and 32 d of age^1,2.

	10 d of age		32 d of age
Treatments	Tibia ash, % fat-free DM	Tibia ash weight, g	Tibia ash, % fat-free DM	Tibia ash weight, g
PC3	47.29a	0.232a	47.29a	0.232a
NC13	38.24c	0.149b	37.58c	0.142c
NC1+PhyG	47.20a	0.237a	47.20a	0.237a
NC23	40.43b	0.163b	40.43b	0.163b
NC2+PhyG+XB	47.10a	0.233a	47.04a	0.237a
Pooled SEM	0.53	0.005	0.390	0.002
P value	<0.001	<0.001	<0.001	<0.001

^a–cTreatment means within each column with uncommon superscripts are significantly different at P < 0.05.

¹Values are means of n = 4 observations.

²Differences between means were identified using Tukey's HSD test.

³PC, nutritionally adequate positive control; NC1, reduced in digestible P, Ca, digestible AA, ME, and Na vs. PC diet, to account for expected contribution of the phytase, as detailed in Table 1; NC2, reduced in digestible P, Ca, digestible AA, ME, and Na vs. PC to account for the expected contribution of the phytase and the xylanase and β-glucanase preparation (XB), as detailed in Table 1.

Carcass Characteristics

Treatment had no effect on carcass component yields as a percentage of carcass weight. However, absolute weights differed among treatments (P < 0.001 in all cases, Table 6). Total carcass, breast, thigh and leg weights were all reduced in NC1 and NC2 vs. PC (−19 to −23% vs. PC, P < 0.05. In all cases, these measures were improved in NC1+PhyG vs. NC1 and in NC2+PhyG+XB vs. NC2 (P < 0.05) whereas they were not significantly different to values achieved by birds fed the PC.

Table 6.

Effects of phytase alone or together with a xylanase and β-glucanase combination on carcass characteristics^1,2.

	Absolute weight, g				Yield, % of carcass weight
Diet	Carcass	Breast filet	Thigh filet	Leg	Breast filet	Thigh filet	Leg
PC3	1,473.2a	380.5a	187.3a	180.8a	25.82	12.73	12.29
NC13	1,144.1c	294.9b	144.4b	146.7b	25.77	12.64	12.83
NC1+PhyG	1,501.6a	390.0a	193.8a	198.8a	25.94	12.92	13.22
NC23	1,196.5b	305.2b	151.2b	147.0b	25.62	12.68	12.33
NC2+PhyG+XB	1,501.7a	390.6a	190.2a	187.0a	25.99	12.66	12.48
Pooled SEM	12.445	5.896	3.056	5.84	0.36	0.211	0.378
P value	<0.001	<0.001	<0.001	<0.001	0.956	0.897	0.384

^a–cTreatment means within each column with uncommon superscripts are significantly different at P < 0.05.

¹Values are means of n = 4 observations.

²Differences between means were identified using Tukey's HSD test.

³PC, nutritionally adequate positive control; NC1, reduced in digestible P, Ca, digestible AA, ME, and Na vs. PC diet, to account for expected contribution of the phytase, as detailed in Table 1; NC2, reduced in digestible P, Ca, digestible AA, ME, and Na vs. PC to account for the expected contribution of the xylanase and β-glucanase preparation (XB), as detailed in Table 1.

DISCUSSION

The diet analyses indicated that the nutrient content of the PC diet was consistent with expectations based on the diet formulation. Nutrient levels in this diet were adequate but not excessive. This was confirmed by the overall growth performance of PC birds being very close to the breeder's objectives (Aviagen Inc., 2019b; d 32 BW 98.7% of breeder objective, 2,032 vs. 2,058 g/bird; d 0 to 32 FCR 99.6% of breeder objective, 1.413 vs. 1.406). On this basis, it was considered that the PC formed an appropriate comparator against which to evaluate responses to the nutrient reduced and enzyme-supplemented treatments. The analysis levels of CP, P, and Ca were indicative of the intended reductions in digestible AA, digestible P, and Ca in NC1 and NC2 vs. PC having been approximately achieved. The substantially reduced growth performance during all phases and in livability during finisher phase, in NC1 and NC2 vs. PC, provided further evidence of the negative impact of the nutrient reductions in these diets and confirmed their nutritional inadequacy. It may have been expected that the further reduction in ME content of NC2 vs. NC1 (−78 kcal/kg) would result in a further reduction in growth performance in this treatment. Interestingly, this was not the case. During all individual phases and overall, BW, ADG, and FCR were either not significantly different from, or improved, in NC2 vs. NC1. This may in part be explained by the increased feed intake that was observed in NC2 relative to NC1, which was apparent during grower and finisher phases and which could have been an adaptation response of the birds to the lower energy content of the NC2 diet aimed at maintaining energy intake. A similar effect was observed by Massuquetto et al. (2020) in broilers fed low energy (ME) pelleted diets. The higher ADG of birds fed NC2 than NC1 during grower phase confirms that the increased feed intake was effective in increasing weight gain, but for the overall period no significant difference in ADG or final BW was evident between NC1 and NC2 whereas overall FCR was significantly higher in NC2 than NC2, again indicating that the NC2 diet was more deficient in energy. The lower reduction in tibia ash (expressed either as a percentage of fat-free DM at d 10 or 32 or by absolute weight at d 32) in NC2 compared with NC1, vs. PC, is also consistent with an adaptation response occurring in NC2; higher feed intake would result in higher P and Ca intake by NC2 birds leading to greater potential for their utilization in tissues. In addition, during starter phase, analyzed Ca was higher in NC2 than NC1, which may also have contributed to the higher bone ash content of NC2 birds at 10 d of age.

The positive effect on growth performance of PhyG supplementation in the NC1 diet was substantial; feed intake and weight gain were increased vs. NC1 and feed efficiency was improved at least to the level achieved by the PC during all individual phases and overall. The improvements in live weight gain and body mass were carried through into individual carcass components (breast, thigh, leg) whose absolute weights were not significantly different to those of PC birds throughout the study, demonstrating that production outcomes were improved alongside the general improvements in weight gain and feed efficiency. These findings strongly suggest that the activity of the supplemental phytase at the applied dose levels ameliorated the negative impact of the applied nutrient reductions (matrix) on growth performance, and hence that the applied matrix values were appropriate. During starter phase, but not later growth phases, there was some evidence that the applied matrix values could have been overly conservative, as BW, ADG and FCR during this phase were improved in NC1+PhyG beyond the level achieved by the PC. This could have been linked to a potential for the supplemental phytase to achieve greater benefit in young birds whose digestive systems (and secretion of endogenous enzymes) are not yet fully mature. However, for the overall period responses did not exceed those of the PC for any outcome measure suggesting that the matrix values were appropriate in older birds and over an entire growth cycle.

The improved FCR in NC1+PhyG vs. NC1 during all individual phases indicates that the beneficial effects of the phytase were not solely the result of increased feed (and therefore nutrient) intake, but also of more efficient utilization of nutrients in feed and conversion into body mass. Nutrient digestibility was not assessed in this study but previous studies of PhyG have established its mode of action in effecting the rapid and extensive hydrolysis of IP₆ to low IP-esters and the associated release of iP (Christensen et al., 2020; Dersjant-Li et al., 2022c) and of protein and AA (Babatunde et al., 2021, 2022; Dersjant-Li et al., 2022a), energy (Babatunde et al., 2021) Ca and P (Dersjant-Li et al., 2020; Babatunde et al., 2022) and (in pigs) Na (Espinosa et al., 2021). It is therefore hypothesized that at least part of the improved growth performance in NC1+PhyG vs. NC1 was the result of improved nutrient digestibility and utilization. The improved tibia ash in this treatment that was observed at both 10 and 32 d of age (not significantly different from PC) supports this hypothesis and suggests that PhyG supplementation of NC1 resulted in greater P and Ca utilization in bone.

The improvements in weight gain and feed intake in NC1+PhyG vs. NC1 increased with bird age (greatest during finisher phase), whereas improvements in feed efficiency decreased with age (greatest during starter phase; FCR was reduced by 5.6, 2.7, and 2.3%, in starter, grower, and finisher phase, respectively). This suggests that the younger birds benefited more from the increased availability of dietary nutrients affected by the supplemental phytase, resulting in a greater uplift in the conversion of feed into body mass than was evident in more mature birds. This may be reflective of the high P requirement of young birds (dietary nonphytate-P requirements for broilers set by the NRC are 0.45% during d 0 to 21, 0.35% during d 21 to 42 and 0.30% during d 42 to 56; NRC, 1994) coupled with an immature digestive enzyme secretory capacity (Ravindran and Abdollahi, 2021), which may have made it more likely that the iP and other nutrients “released” by the supplemental phytase were utilized and used to contribute to requirements than in older birds. This was also why a tiered dosing regimen of phytase by phase had been employed in the trial design, in which the highest dose (2,000 FTU/kg) was applied to the starter NC1+PhyG diets. Broiler nutrient digestibility responses to supplemental PhyG, are known to be highly dose-dependent (Babatunde et al., 2021, 2022; Dersjant-Li et al., 2022b,c). Applying a high phytase dose level during starter phase was expected to have the effect of releasing more iP and other nutrients from the diet during a period of rapid growth and high nutrient requirements. A previous study of PhyG by Marchal et al. (2021) adopted a similar (but not identical) tiered dosing regimen (3,000, 2,000 and 1,000 FTU/kg in starter, grower and finisher phases, respectively) and observed an additional degree of improvement in weight gain and feed conversion efficiency from this dosing regimen compared with application of a lower, constant, dose level (1,000 FTU/kg) across all phases, confirming that a higher dose level of PhyG during starter phase is beneficial.

The addition of the xylanase–β-glucanase on top of phytase to NC2 also substantially improved growth performance compared with the nonsupplemented NC2 diet. The improvements in feed intake, weight gain and feed efficiency in NC2+XB+PhyG vs. NC2 were again evident in all individual phases and overall and were again sufficient to bring growth performance up to a level that was not significantly different from that achieved by the PC. This demonstrates that the activity of the added phytase and xylanase–β-glucanase combination at the applied dose levels was appropriate and sufficient to ameliorate the negative effect of the applied energy and nutrient reductions in NC2 on growth performance. The study design, that did not include an NC2+PhyG (without XB) treatment, does not enable the effect of the xylanase–β-glucanase combination to be separated out definitively from that of the phytase in NC2+XB+PhyG. However, given that growth performance was maintained in this treatment to a level that was not significantly different from that achieved by the PC despite the extra 78 kcal/kg reduction in ME and up to 0.02% point digestible AA that was applied to NC2 vs. NC1, and further that the NC2+PhyG+XB treatment reduced overall FCR vs. NC2 by a greater amount than NC1+PhyG did vs. NC1 (−4.6 vs. −3.1 points), these results suggest an additional beneficial effect from the XB in NC2+XB+PhyG over and above that delivered by the phytase alone in NC1+PhyG. The documented mode of action of the XB also supports the attribution of this extra degree of beneficial effect on FCR in NC2+PhyG+XB to the XB component. Gilani et al. (2021) previously demonstrated the efficacy of the xylanase–β-glucanase combination, separate from the effect of supplemental phytase, when applied at the same dose level as in the present study to broiler diets reduced in energy, CP and dig AA. In that study, ADG and BW were both increased and FCR tended to be reduced in the XB-supplemented birds during finisher phase (d 22–35; Gilani et al., 2021). Further, in the wider literature, improved growth performance by xylanase and β-glucanase in diets containing wheat has been attributed to improved digestibility and utilization of CP, starch, and fiber (insoluble and soluble NSPs) (Zhang et al., 2014). The associated modes of action of the enzymes are not fully understood but are thought to include a combination of: 1) the release of encapsulated nutrients from cereal endosperm as a result of the destruction of plant cell walls by the action of the enzymes on cell wall xylans and beta-glucans, 2) a reduction in digesta viscosity due to a reduction in the content of (viscous) soluble NSP, and associated improvement in the mixing of enzymes and solutes in the digesta and in the absorption of metabolites, and 3) a possible prebiotic effect in the gut of oligosaccharides generated by xylanase and β-glucanase activity on NSP in plant cell walls (as recently reviewed by Bedford, 2022).

In addition to considering the beneficial effects of the supplemental enzymes on growth performance and production outcomes, it is also relevant to consider the overall cost-effectiveness of the treatments and to compare their calculated CFP. The use of alternative feed ingredients (including by-products) and supplemental enzymes as approaches to improving the sustainability of broiler production are major new areas of current interest and research (El-Deek et al., 2020). As prices of conventional cereals and oilseeds as well as of specialist ingredients such as inorganic phosphates continue to rise and their availability can be inconsistent, strategies to maximize nutrient utilization from feed are increasingly sought. Such strategies have the potential not only to reduce costs through increased feed efficiency, but also to reduce waste (as a result of reduced excretion of undigested nutrients), to open up opportunities for producers to include a wider variety of (less digestible) raw materials and to use locally available ingredients in the diet, thereby contributing to improving the sustainability of production. As yet, there are few published data available from scientific studies on the comparative feed costs per unit BW gain of enzyme-supplemented broiler diets, and even less data comparing the CFP of such diets. In the present study, total feed costs (inclusive of the costs of the supplemental enzymes) based on market prices in January 2022 were reduced per kilogram of BW gain by 0.038 € in the NC1+PhyG diet and by 0.052 € in the NC2+PhyG+XB diet. In addition, the estimated CFP of these diets was reduced by 260 g CO₂ eq./kg BW gain and by 376 g CO₂ eq./kg BW gain, respectively. These reductions clearly demonstrate an additional cost and environmental sustainability benefit of including the xylanase–β-glucanase within the feed formulation which was enabled by the further reduction in the energy content of the NC2 diet over that applied to NC1.

In conclusion, the present study showed that supplementation of a wheat-corn-soybean meal-based broiler diet with a novel phytase applied in a tiered dosing regimen by phase in combination with the application a full nutrient matrix (reduction in Ca, digestible P, digestible AA, energy, and Na) maintained all measures of growth performance during all growth phases to a level that was not different from that achieved by a nutritionally adequate control diet. This confirms the appropriacy of the applied phytase matrix values. The phytase-supplemented diet also conferred a reduction in total feed costs and CFP per kg BWG compared to the nutritionally adequate PC diet. When a xylanase–β-glucanase combination was added on top of the phytase to the same nutrient-reduced diet but with an additional −78 kcal/kg reduction in ME and up to 0.02% point reduction in digestible AA applied, growth performance was similarly maintained to a level that did not differ from that achieved by the PC diet, confirming the appropriacy of the applied matrix values. A greater beneficial effect of this diet on feed efficiency compared with the phytase-supplemented diet implied an extra beneficial effect from the xylanase–β-glucanase on top of that of the phytase in compensating for the additional energy and digestible AA reduction. The xylanase–β-glucanase plus phytase in combination also conferred a greater reduction in total feed costs and feed-associated CFP than the addition of phytase alone. These data highlight the potential feed cost and environmental sustainability benefits of nutrient-reduced wheat-corn-soybean meal-based diets supplemented with phytase or phytase plus xylanase–β-glucanase over a nutritionally adequate, unsupplemented, diet.