Application of a minerals, digestible amino acids and energy matrix to a broiler diet supplemented with a bacterial 6-phytase maintained growth performance and reduced feed costs compared to a minerals matrix alone

Abstract

The effect of stepwise application of a nutrient and energy matrix for a consensus bacterial 6-phytase variant (PhyG) to a corn-based diet, without or with xylanase-amylase-protease (XAP) supplementation, was evaluated in broilers. Day-old, Ross 308 males (2,400) were assigned to 8 treatments (10 replicate floor pens per treatment) in a randomized controlled block design. Diets were formulated in 4 phases and comprised: 1) nutritionally adequate control (PC); 2) negative control (NC1) reduced in Ca, digestible P and Na (by 0.22, 0.23 and 0.04 % points, respectively, vs. PC); 3) as NC1 but reduced in ME (by 72 to 51 kcal/kg; NC2); 4) as 3) reduced in digestible amino acids (AA) (by ≤ 0.06 % points vs. PC; NC3); 5), 6) and 7), as 2), 3) and 4) supplemented with PhyG at 1,250 phytase units (FTU)/kg (NC1+PhyG, NC2+PhyG and NC3+PhyG, respectively), and; 8) as 7) reduced in ME (by 61 kcal/kg), supplemented with XAP (2,000 xylanase units/kg, 200 amylase units/kg and 4,000 protease units/kg; NC4+PhyG+XAP). Orthogonal polynomial contrasts revealed linear and quadratic relationships (P < 0.05) between the degree of matrix applied and final (d 42) BW, overall ADG, average daily feed intake (ADFI), BW-corrected feed conversion ratio, d 21 tibia ash and tibia breaking strength. These measures were incrementally impaired with increasing matrix severity through PC, NC1, NC2 and NC3. Supplemental PhyG increased (P < 0.05) overall ADG and ADFI above the level of the respective NC, and maintained growth performance, tibia ash, breaking strength and carcass weights comparable to PC. Treatments NC3+PhyG and NC4+PhyG+XAP maintained all outcome measures at levels not different from the PC but with a lower estimated feed cost per kilogram BW gain than PC or NC1+PhyG (-0.039 USD/kg BW gain in NC3+PhyG vs. NC1+PhyG; P < 0.05). Application of a full nutrient and energy matrix with phytase can maintain growth performance and reduce feed costs compared with a minerals matrix alone.

Introduction

Since their commercialization in the early 1990s, microbial phytases have become an almost ubiquitous addition to broiler diets for the main purpose of increasing P availability and reducing its excretion into the environment. The primary mode of action of exogenous phytase is to dephosphorylate plant-derived phytate (myo-inositol hexakisphosphate, IP₆) in the low pH environment of the upper gastrointestinal tract (principally the gizzard; Truong et al., 2016), releasing inorganic phosphate (iP) which can be absorbed and utilized by the bird. The positive impact of exogenous phytase on P digestibility and utilization in poultry has been extensively demonstrated and reviewed, most recently by Selle et al. (2023). Phytate degradation also leads to a reduction in Ca-phytate complexing in the digesta which increases the availability of soluble Ca for absorption and utilization (Selle et al., 2009). Further, a marked and consistent ‘sodium sparing’ effect from phytase has been observed (Cowieson et al., 2004; Ravindran et al., 2006) which may be due to a reduction in endogenous Na secretion (as NaHCO₃) that is needed to buffer HCl in the presence of phytate (Selle et al., 2023).

As a consequence of the compelling data available for P, Ca and Na, prescribed reductions in these minerals (‘matrix values’) are commonly applied when formulating diets with phytase to account for the expected contributions of the enzyme when administered at a specified dose. Matrix values vary from one phytase to another dependent on their unique properties, specific activity and in vivo efficacy, and must take into account variation in the diet composition and changing nutrient requirements as birds age. Matrix values are derived from the conduct of multiple digestibility studies in different settings, such as those by Dersjant-Li et al., 2020 and Babatunde et al. (2021, 2022). Values for digestible amino acids (AA) and energy are less widely accepted than those for digestible P, Ca and Na, even though there is now substantial evidence of a beneficial effect of phytase on the digestibility of these nutrients (Selle et al., 2023; Dersjant-Li et al., 2022). Application of a ‘full matrix’ [meaning the application of prescribed reductions in the formulated content of digestible P, Ca, digestible AA, Na and metabolizable energy (ME)] is being applied more frequently to diets containing phytase and could serve to reduce feed costs and deliver production and sustainability benefits if reduced nutrient inputs are needed. However, as part of decision making about whether to apply a full matrix, there is a need to systematically evaluate the effect of stepwise reduction of minerals, minerals plus energy, and minerals plus energy plus digestible AA, on growth performance and production outcomes, to determine the extent of benefits. This has not previously been investigated within a single study. As part of this, there is a need to understand whether a full matrix can achieve any additional production benefit compared with a minerals only matrix. It also needs to be clarified whether adding other types of exogenous enzymes on top of phytase could achieve any further growth performance or other production benefit, which may be of interest to producers.

Studies of a consensus bacterial 6-phytase variant known as PhyG have demonstrated its mode of action in broilers in effecting the stepwise degradation of phytate in the upper digestive tract, to release iP (Christensen et al., 2020). In vivo data support that this phytase can improve the digestibility and retention of energy, Ca, P, protein, AA and Na (Dersjant-Li et al., 2020; Dersjant-Li et al., 2022a; Espinosa et al., 2021). The study by Dersjant-Li et al. (2020) reported that 250 to 1,000 FTU/kg of the phytase added to a corn-soybean meal-based diet was effective in replacing an ∼1.8 g/kg total P in starter diets and 2.2 g/kg total P in finisher diets, whereas the separate study published in 2022 (Dersjant-Li et al., 2022a) reported a 5.8 % increase in the AID coefficient of gross energy (GE) in young broilers supplemented with 500 to 1,000 FTU/kg of the phytase. Other studies have shown that this phytase can be effective in maintaining growth performance when applied to diets with a full matrix (Marchal et al., 2021; Bello et al., 2023). However, even when dosed at 4,000 FTU/kg, some undigested ME and protein remains in the digesta (Babatunde et al., 2021; 2022) that could, in theory, be reduced by additional protease, starch or fiber-digesting enzymes.

The objective of this study was to test the stepwise effect of reductions in minerals (Ca, digestible P and Na), minerals plus energy, or a full matrix (minerals plus energy plus digestible AA), on growth performance, bone mineralization, carcass characteristics, feed costs and production sustainability in broilers fed a corn-soybean meal-based diet supplemented with PhyG. The further effect of adding in a xylanase-amylase-protease (XAP) combination and greater ME and digestible AA reduction to the diet, was also studied.

Materials and methods

Birds, housing and experimental design

The protocol was reviewed by the Animal Ethics Committee of Agrivet Research and Advisory Pvt. Ltd. to ensure that the stipulations of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Fisheries, Animal Husbandry and Dairying, Department of Animal Husbandry and Dairying, CPCSEA guidelines for poultry/ birds facility (2020) were followed.

The experimental design was a randomized complete block design with 8 treatments, 10 replicate floor pens per treatment and 30 birds per pen (stocking density 0.074 to 0.093 m² /bird). Pens were supplied with clean litter. Ross 308 males (n = 2,400) were obtained on day-of-hatch from a commercial hatchery, individually weighed and assigned to pens so that each pen contained birds of approximately equal average BW. Pens were located in an environmentally controlled 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.

Treatment diets and enzymes

All diets were fed in 4 phases: 1 to 10 d of age (starter), 10 to 21 d of age (grower), 21 to 35 d of age (finisher 1) and 35 to 42 d of age (finisher 2). Diets were fed as a crumble in starter phase and pelleted during grower and finisher phases (pelleting temperature 80±2 °C). There were 8 treatment diets. Treatment details are summarized in Table 1. The full ingredient and calculated nutrient composition of the basal diets is shown in Table 2, Table 3. Treatment 1 was a positive control (PC) diet based on corn and soybean-meal with a small amount (∼5 %) of rapeseed meal. It provided adequate nutrients for broilers (CVB, 2018) and had a formulated phytate content of 0.27 to 0.28 %. Treatments 2, 3, and 4 comprised unsupplemented negative control (NC) diets that were reduced in either: minerals (Ca, digestible P, Na; treatment 2; NC1), minerals and ME (treatment 3; NC2), or minerals, ME and digestible AA (treatment 4; NC3), respectively, vs. PC. The magnitude of the nutrient and energy reductions were derived in accordance with the manufacturer’s recommended matrix PhyG when used at 1,250 phytase units (FTU)/kg (no PhyG was added to treatment diets 2, 3 or 4). The digestible AA reductions in NC3 were applied for all 18 AA (essential and non-essential), although only the essential AA are listed in Table 2, Table 3. In each case, the degree of the AA reduction was in accordance with the manufacturer’s recommendations for use of the supplemental phytase at 1,250 FTU/kg.) Across phases, the nutrient reductions were: 0.23 % points digestible P (where dicalcium phosphate was used as the inorganic P source); 0.22 % points Ca; 0.04 % points Na; 72 to 51 kcal/kg ME, and up to 0.06 % points digestible AA, according to treatment. These reductions were achieved by optimizing the feed formulation within the constraints of the applied nutrient reductions. Treatments 5, 6 and 7 were as treatments 2, 3 and 4 but supplemented during all phases with 1,250 FTU/kg of a commercial consensus bacterial 6-phytase variant [PhyG, Danisco Animal nutrition & Health (IFF)] produced in Trichoderma reesei. Treatments 5, 6 and 7 are herein referred to as NC1+PhyG, NC2+PhyG and NC3+PhyG, respectively. Finally, treatment 8 was as treatment 7 but with a further 61 kcal/kg reduction in ME applied to the diet and an additional digestible AA reduction of up to 0.02 % points, supplemented with a commercial xylanase-amylase-protease combination [XAP, Danisco Animal Nutrition & Health (IFF)], herein referred to as NC4+PhyG+XAP. The XAP contained a combination of an endo- 1,4-beta-xylanase (EC 3.2.1.8), alpha-amylase (EC 3.2.1.1) and an alkaline serine protease (EC 3.4.21.62). The XAP was added to the diet to supply xylanase at 2,000 xylanase units (XU)/kg, amylase at 200 units (U)/kg and protease at 4,000 U/kg. No unsupplemented NC4 diet was tested because the severity of the combined nutrient and energy reductions in this diet would have been expected to produce growth problems and animal welfare issues among birds and would not have been in compliance with European Guidelines on the protection of animals for scientific purposes (European Council, 2010).

Table 1.

Treatment details.

	PhyG, FTU/kg	Xylanase/amylase/protease (XAP), U/kg	Nutrient and energy reductions vs. PC
			Digestible P, % points1	Ca, % points	Na, % points	ME, kcal/kg	Digestible AA, % points
PC	-	-	-	-	-	-	-
NC1	-	-	-0.23	-0.22	-0.04	-	-
NC2	-	-	-0.23	-0.22	-0.04	-72 to -51	-
NC3	-	-	-0.23	-0.22	-0.04	-72 to -51	Up to -0.06
NC1+PhyG	1,250	-	-0.23	-0.22	-0.04	-	-
NC2+PhyG	1,250	-	-0.23	-0.22	-0.04	-72 to -51	-
NC3+PhyG	1,250	-	-0.23	-0.22	-0.04	-72 to -51	Up to -0.06
NC4+PhyG+XAP	1,250	2,000/200/4,000	-0.23	-0.22	-0.04	-132 to -112	Up to -0.08

^IIn finisher 2 phase, all inorganic phosphate was removed and the digestible P reduction achieved was 0.21%.

AA, amino acids; NC, negative control; PC, positive control; FTU, phytase units; PhyG, a consensus bacterial 6-phytase variant; XAP, xylanase-amylase-protease combination.

Table 2.

Ingredient and calculated nutrient composition of the basal diets during starter and grower phase.

	Starter (1 to 10 d of age)					Grower (10 to 21 d of age)
	PC	NC1	NC2	NC3	NC41	PC	NC1	NC2	NC3	NC41
Ingredients, % as fed
Corn	59.37	62.19	61.70	62.76	60.85	60.78	63.22	64.62	65.33	66.38
Soybean meal	31.83	31.71	28.14	26.64	26.10	26.22	25.80	25.71	24.57	24.55
Full fat soya	1.20	1.20	2.03	1.71	2.27	2.10	2.01	1.99	1.99	1.85
Rapeseed meal	0.97	0.48	4.68	5.58	5.76	2.82	2.97	2.86	3.45	2.88
Rice hulls	-	-	-	-	1.84	-	-	-	-	0.60
Soy oil	2.28	1.34	0.45	0.40	0.32	3.58	2.76	1.60	1.53	0.62
Limestone	1.04	1.31	1.28	1.27	1.25	0.94	1.20	1.20	1.20	1.20
Dicalcium phosphate	1.73	0.36	0.31	0.32	0.31	1.55	0.18	0.17	0.17	0.18
L-Lys HCl	0.23	0.23	0.25	0.24	0.22	0.22	0.23	0.23	0.20	0.20
DL- Met	0.30	0.30	0.28	0.24	0.23	0.27	0.27	0.26	0.22	0.22
L- Thr	0.12	0.12	0.12	0.09	0.08	0.10	0.10	0.10	0.07	0.06
Vitamin-mineral premix2	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15
Sodium bicarbonate	0.40	0.25	0.26	0.25	0.24	0.40	0.24	0.25	0.24	0.24
Salt	0.19	0.19	0.18	0.19	0.19	0.20	0.19	0.19	0.20	0.20
Nutrient composition, % unless otherwise stated
ME, kcal/kg	2,950	2,950	2,878	2,878	2,818	3,050	3,050	2,989	2,989	2,928
Crude fat	4.95	4.10	3.37	3.29	3.27	6.41	5.66	4.54	4.50	3.60
Crude fiber	2.63	2.60	2.95	2.98	3.61	2.61	2.64	2.64	2.66	2.80
Ash	6.56	5.30	5.30	5.24	5.28	6.53	5.28	5.28	5.23	5.23
Starch	38.95	40.73	40.70	41.41	40.22	39.98	41.55	42.44	42.93	43.56
Calcium	0.92	0.70	0.70	0.70	0.70	0.84	0.62	0.62	0.62	0.62
Digestible phosphorus	0.42	0.20	0.20	0.20	0.20	0.37	0.16	0.16	0.16	0.16
Total phosphorus	0.71	0.48	0.50	0.50	0.50	0.67	0.45	0.45	0.45	0.45
Phytate phosphorus	0.27	0.27	0.29	0.29	0.29	0.28	0.28	0.28	0.28	0.28
Sodium	0.19	0.15	0.15	0.15	0.15	0.19	0.15	0.15	0.15	0.15
CP	22.19	22.18	22.27	21.75	21.78	20.50	20.52	20.53	20.17	20.01
Digestible Lys	1.22	1.22	1.22	1.18	1.16	1.11	1.11	1.11	1.07	1.06
Digestible Met & Cys	0.90	0.90	0.90	0.85	0.84	0.84	0.84	0.84	0.79	0.78
Digestible Thr	0.81	0.81	0.81	0.76	0.75	0.74	0.74	0.74	0.70	0.68
Digestible Trp	0.22	0.22	0.22	0.22	0.21	0.20	0.20	0.20	0.20	0.19
Digestible Arg	1.34	1.34	1.32	1.28	1.28	1.22	1.21	1.21	1.19	1.18
Digestible Ile	0.82	0.82	0.81	0.79	0.78	0.75	0.75	0.75	0.73	0.72
Digestible Val	0.91	0.91	0.91	0.89	0.88	0.84	0.84	0.84	0.82	0.82
Acid detergent fiber	3.42	3.40	4.03	4.11	4.11	0.26	0.27	0.27	0.27	0.27
Neutral detergent fiber	8.75	8.89	9.63	9.78	10.76	3.58	3.64	3.65	3.72	3.63
Total NSPs	13.18	13.31	13.90	14.02	13.76	12.82	13.06	13.16	13.21	13.13
CFP total, CO2 eq. g/ton	1,974	1,940	1,816	1,744	1,734	1,834	1,791	1,749	1,700	1,660

¹NC4 was not fed as a stand-alone diet, only when supplemented with phytase (PhyG) and xylanase-amylase-protease (XAP).

²Vitamin-mineral premix provided per kg feed: vitamin A 20250 IU, vitamin D3 6750 IU, vitamin E 90 mg, vitamin K3 5.25 mg, vitamin B1 5.25 mg, vitamin B2 12 mg, vitamin B6 5.25 mg, vitamin B 12 0.03 mg, biotin 0.22 mg, pantothenic acid 21.75 mg, folic acid 3.38 mg, niacin 90 mg, manganese 90 mg as yeast protein chelate, zinc 90 mg as yeast protein chelate, iron 45 mg as yeast protein chelate, copper 15 mg as yeast protein chelate, selenium 0.9 mg as yeast protein chelate, iodine 6 mg as sodium iodide; chromium 1.5 mg as yeast protein chelate.

CFP, carbon footprint; NC, negative control; NSP, non-starch polysaccharides

Table 3.

Ingredient and calculated nutrient composition of the basal diets during finisher 1 and finisher 2 phase.

	Finisher 1 (21 to 35 d of age)					Finisher 2 (35 to 42 d of age)
	PC	NC1	NC2	NC3	NC41	PC	NC1	NC2	NC3	NC41
Ingredients, % as fed
Corn	64.65	67.61	69.00	69.18	69.68	66.45	68.73	69.83	70.57	71.83
Soybean meal	19.53	19.63	20.10	18.78	18.12	17.30	16.96	17.03	16.46	16.11
Full fat soya	3.17	3.17	2.62	2.82	2.70	3.52	3.52	3.24	3.24	3.18
Rapeseed meal	5.59	4.76	4.38	5.43	6.01	5.59	5.68	5.70	5.73	5.62
Rice hulls	-	-	-	-	0.50	-	-	-	-	0.30
Soy oil	3.42	2.43	1.50	1.52	0.74	3.74	2.94	2.03	1.93	0.90
Limestone	0.84	1.10	1.11	1.10	1.09	0.78	0.96	0.96	0.96	0.96
Dicalcium phosphate	1.40	0.04	0.04	0.03	0.02	1.26	-	-	-	-
L-Lys HCl	0.20	0.20	0.21	0.18	0.18	0.19	0.19	0.20	0.17	0.17
DL-Met	0.22	0.22	0.23	0.18	0.17	0.20	0.19	0.19	0.15	0.15
L-Thr	0.07	0.07	0.07	0.04	0.03	0.06	0.06	0.06	0.03	0.02
Vitamin-mineral premix2	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15
Sodium bicarbonate	0.40	0.24	0.24	0.23	0.23	0.40	0.24	0.24	0.23	0.23
Salt	0.20	0.20	0.20	0.21	0.21	0.21	0.20	0.20	0.21	0.21
Nutrient composition, % unless otherwise stated
ME, kcal/kg	3,100	3,100	3,049	3,049	2,988	3,150	3,150	3,099	3,099	3,038
Crude fat	6.54	5.65	4.66	4.72	3.95	6.96	6.24	5.32	5.23	4.24
Crude fiber	2.69	2.63	2.61	2.67	2.88	2.61	2.63	2.64	2.62	2.71
Ash	5.60	4.34	4.35	4.31	4.31	5.28	4.07	4.08	4.04	4.03
Starch	42.66	44.51	45.35	45.53	45.88	43.82	45.29	45.98	46.45	47.25
Ca	0.77	0.55	0.55	0.55	0.55	0.71	0.49	0.49	0.49	0.49
Digestible P	0.35	0.14	0.14	0.14	0.14	0.32	0.13	0.13	0.13	0.13
Total P	0.65	0.42	0.42	0.43	0.43	0.62	0.42	0.42	0.42	0.42
Phytate phosphorus	0.28	0.28	0.28	0.28	0.29	0.28	0.28	0.28	0.28	0.28
Na	0.19	0.15	0.15	0.15	0.15	0.19	0.15	0.15	0.15	0.15
CP	18.85	18.84	18.82	18.57	18.49	17.99	18.04	18.06	17.76	17.64
Digestible Lys	0.99	0.99	0.99	0.95	0.94	0.93	0.93	0.93	0.90	0.88
Digestible Met & Cys	0.77	0.77	0.77	0.73	0.72	0.73	0.73	0.73	0.68	0.68
Digestible Thr	0.65	0.65	0.65	0.61	0.60	0.62	0.62	0.62	0.58	0.57
Digestible Trp	0.18	0.18	0.18	0.18	0.17	0.17	0.17	0.17	0.17	0.16
Digestible Arg	1.09	1.08	1.08	1.06	1.05	1.03	1.02	1.02	1.01	1.00
Digestible Ile	0.67	0.67	0.67	0.66	0.65	0.64	0.64	0.64	0.63	0.62
Digestible Val	0.77	0.77	0.77	0.76	0.76	0.73	0.74	0.74	0.73	0.72
Acid detergent fiber	0.27	0.27	0.27	0.27	0.27	0.26	0.27	0.27	0.27	0.27
Neutral detergent fiber	3.93	3.86	3.81	3.95	4.02	3.89	3.95	3.96	3.96	3.95
Total NSPs	12.83	12.91	13.03	13.13	13.25	12.59	12.81	12.95	12.93	12.96
CFP total, CO2 eq. g/ton	1,618	1,590	1,556	1,512	1,456	1,554	1,518	1,480	1,451	1,400

¹NC4 was not fed as a stand-alone diet, only when supplemented with phytase (PhyG) and xylanase-amylase-protease (XAP), as detailed in Table 1.

²Composition given in Table 2.

CFP, carbon footprint; NC, negative control; NSP, non-starch polysaccharides

The diets were manufactured in 5 batches according to the formulation for PC, NC1, NC2, NC3 and NC4. The main batches of NC1, NC2 and NC3 were each divided into 2 equal portions and the phytase (premixed with 10 kg of basal diet) added. Diets were thoroughly mixed to ensure a homogenous distribution of the phytase. The XAP was added directly to NC4, and the diet mixed as for the other diets.

Measurements and sampling

Samples of all final diets were collected for proximate and enzyme analysis. Body weight (BW) was measured on a per pen basis on each of d 1, 10, 21, 35 and 42 and used to calculate ADG. Feed supplied to birds was weighed on d 1, 10, 21 and 35 and residuals were measured on d 10, 21, 35 and 42 for the calculation of ADFI (corrected for mortality), per phase. Birds were checked daily for mortality and dead birds removed, weighed and recorded. Mortality-corrected feed conversion ratios (FCR) were calculated from values of ADG and ADFI for each individual phase and for cumulative periods (1 to 21, 1 to 35 and 1 to 42 d of age). Mortality and BW-corrected FCR (FCRc) was also calculated for these periods.

At 21 d of age, 2 birds per pen were euthanized by mechanical stunning followed by exsanguination. The left and right tibias were extracted and each pooled per pen. Left tibias were used for bone ash analysis and right tibias for breaking strength determination. At 42 d of age, 4 birds per pen were euthanized for carcass weight, carcass part weight and yield analysis. The weights of the eviscerated carcass, heart, abdominal fat, breast, legs and thighs were recorded, and the yields of these components (as a percentage of carcass weight) were calculated (carcass yield was calculated as a percentage of final liveweight).

Chemical analysis

Multiple samples of each diet were collected, pooled, and analyzed for DM, nitrogen and CP, ether extract, crude fiber, total ash, organic matter, Ca and total P, using AOAC methods 934.01 (AOAC, 1998), 954.01 (AOAC, 1996a), 920.39 (AOAC, 2005a), 978.10 (AOAC, 2005b), 942.05 (AOAC 2012), 942.05 (AOAC, 2012), 927.02 (AOAC, 1990a) and 965.17 (AOAC, 1996b), respectively. 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 and 37 °C. Xylanase analysis was conducted in duplicate at the Danisco Animal Nutrition Research Centre (Brabrand, Denmark), using the method described by Romero et al. (2013). Xylanase activity was reported as activity units (U) as described by Romero et al. (2013). One xylanase unit was defined as the amount of enzyme that released 0.48 μmol of the reducing sugar xylose from wheat arabinoxylan per min at pH 4.2 and 50 °C. Amylase and protease were not analyzed. The enzymes in the XAP were added as co-granule in which the activities of each individual enzyme had previously been verified. The activity of the xylanase was used as a proxy to indicate the presence of the amylase and protease.

Tibia ash was determined by oven-drying whole bones in ceramic crucibles in a muffle furnace at 605°C for 12 h according to the method of Singh et al. (2013). The percentage of ash was determined relative to dry weight of the tibia. Tibia breaking strength was measured by the 3-point bending test using Material Tester (Burton et al., 2020).

Feed costs and Carbon footprint calculations

Feed intake and ADG determinations were used to calculate the total cost of feed (in USD) per kilogram of body weight gain during the entire wean-to-finish period, based on feed ingredient prices in the US during February 2021, including the cost of the enzymes. An estimate of the carbon footprint (CFP) of each of the treatment diets per kilogram of BW gain (BWG) during the experimental period was made using the FeedPrint database (FeedPrint NL, 2020). This tool calculates the CFP of feed raw materials during their complete life cycle, including crop production, processing of crop and animal products, compound feed production and utilization by the animal, including transport and storage between all steps of the production chain. It includes the CFP from land use and land-use change.

Calculations and statistical analysis

Pen was the experimental unit. All data were analyzed by one-way ANOVA, with treatment included as a fixed effect. Tukey’s HSD test was used to determine significant differences between treatment means. Orthogonal polynomial contrasts were performed on data from treatments PC, NC1, NC2 and NC3 to evaluate relationships between increasing the severity of the applied dietary matrix and bird growth performance and bone mineralization responses. Data were tested for normality prior to analysis using the Distribution function in JMP 16.0 (JMP, 2022). All analyses were conducted in the Fit Model Platform of JMP 16.0 (JMP, 2022). Effects were considered statistically significant at P < 0.05.

Results

Diet analysis

The analyzed nutrient composition in the treatment diets is presented in Table 4, by phase. Analyzed nutrients matched closely with formulated values (maximum variation 15 %) except for crude fiber. Crude fiber levels were up to 26 % higher than formulated in all diets except for NC4+PhyG+XAP in which they were close to target values (within 10 %). Phytase and xylanase activities confirmed the supplementation of these enzymes in the supplemented diets (Table 4).

Table 4.

Analyzed nutrient composition and phytase activity of the treatment diets (%, unless otherwise stated).

Treatment1	CP	Crude fat	Moisture	Ash	Crude fiber	Ca	P	Phytase, FTU/kg2
Starter, d 1 to 10
PC	22.37	4.85	11.29	4.75	3.26	1.01	0.70	279
NC1	22.42	4.25	11.26	4.58	3.27	0.76	0.53	268
NC2	22.39	3.56	11.11	4.53	3.30	0.76	0.53	189
NC3	21.55	3.66	11.30	4.49	3.34	0.76	0.54	185
NC1+PhyG	22.24	4.29	11.14	4.88	3.28	0.75	0.53	1,273
NC2+PhyG	22.51	3.52	11.08	4.77	3.18	0.74	0.51	1,552
NC3+PhyG	21.87	3.47	11.20	4.59	3.33	0.73	0.52	1,397
NC4+PhyG+XAP	21.75	3.32	11.17	4.87	3.68	0.73	0.54	1,262
Grower, d 10 to 21
PC	21.22	6.43	10.51	5.80	3.12	0.90	0.68	160
NC1	21.22	5.96	10.48	5.44	3.17	0.64	0.53	174
NC2	21.28	4.60	10.36	5.60	3.16	0.64	0.54	120
NC3	20.40	4.78	10.52	5.32	3.20	0.67	0.54	215
NC1+PhyG	20.49	5.55	10.63	5.47	3.11	0.65	0.53	1,452
NC2+PhyG	20.54	4.66	10.66	5.24	3.28	0.65	0.52	2,066
NC3+PhyG	20.56	4.70	10.68	5.41	3.17	0.67	0.52	2,147
NC4+PhyG+XAP	20.43	4.14	10.42	5.37	3.09	0.67	0.52	1,505
Finisher 1, d 21 to 35
PC	18.97	6.84	11.10	4.82	3.19	0.85	0.67	193
NC1	18.85	5.58	11.05	4.84	3.11	0.58	0.44	136
NC2	18.84	4.65	11.34	4.78	3.17	0.58	0.45	152
NC3	18.59	4.88	11.27	4.92	3.09	0.58	0.45	140
NC1+PhyG	18.86	4.84	11.28	4.88	3.12	0.59	0.45	1,925
NC2+PhyG	18.85	5.08	11.15	4.85	3.04	0.58	0.42	1,783
NC3+PhyG	18.85	4.78	11.30	4.88	3.16	0.57	0.48	1,841
NC4+PhyG+XAP	18.54	4.48	11.36	4.87	3.02	0.57	0.43	1,524
Finisher 2, d 35 to 42
PC	18.14	7.48	11.51	4.77	2.96	0.74	0.64	NA
NC1	18.32	6.40	11.26	4.76	2.91	0.52	0.45	NA
NC2	18.15	5.38	11.32	4.81	2.83	0.49	0.45	NA
NC3	17.82	5.20	11.64	4.90	2.89	0.51	0.41	NA
NC1+PhyG	18.14	6.49	11.18	4.94	2.94	0.60	0.45	NA
NC2+PhyG	17.80	5.56	11.20	4.88	2.98	0.52	0.44	NA
NC3+PhyG	17.77	5.55	11.17	4.49	3.02	0.52	0.43	NA
NC4+PhyG+XAP	17.93	4.15	11.22	4.92	2.92	0.52	0.45	NA

¹Treatment details are given in Table 1.

²Analyzed xylanase activity in NC4+PhyG + XAP was in the range of 2,839 to 4,226 U/kg across phases.

NA: not analyzed; NC, negative control; FTU, phytase units; PC, positive control; PhyG, a consensus bacterial 6-phytase variant; XAP, a xylanase-amylase-protease combination.

Growth performance

The effect of treatment on growth performance is shown in Table 5 (by phase) and Table 6 (cumulatively). During starter phase (d 1 to 10; Table 5), ADG, ADFI and d 10 BW were all reduced in NC1 (mineral reduction), NC2 (mineral plus ME reduction) and NC3 (mineral plus ME plus AA reduction), compared with the PC (P < 0.05); the size of the reductions did not differ significantly among NC treatments but were substantial in percentage terms (-15.8 to -21.3 % for ADG and -11.4 to -15.2 % for ADFI, across treatments, vs. PC). The FCR was also increased in NC1 and NC3 compared with the PC (P < 0.05) but not in NC2. Compared with the respective NC, the addition of PhyG increased (P < 0.05) ADG, ADFI and d 10 BW in NC1+PhyG, NC2+PhyG and NC3+PhyG, to a similar level that in all cases did not differ significantly from the level achieved by the PC, whereas FCR was unaffected by phytase addition. Treatment NC4+PhyG+XAP maintained BW and ADG at levels that were not significantly different from the PC and increased (P < 0.05) ADFI above the level of the PC but FCR did not reach the level of the PC (1.132 vs. 1.073; P < 0.05).

Table 5.

Effect of treatment on growth performance, by phase.

Treatment	BW, g/bird1	ADG, g/bird/day	ADFI, g/bird/day	FCR, g:g
Starter, d 1 to 10
PC	357ab	31.22ab	33.49b	1.073d
NC1	306c	26.14c	29.67c	1.135ab
NC2	308c	26.29c	28.39c	1.081cd
NC3	302c	25.74c	29.67c	1.154a
NC1+PhyG	366a	32.12a	35.74a	1.112bc
NC2+PhyG	362ab	31.69ab	34.95ab	1.104bcd
NC3+PhyG	352b	30.71b	34.97ab	1.139ab
NC4+PhyG+XAP	357ab	31.24ab	35.38a	1.132ab
SEM	2.863	0.286	0.360	0.009
P-value	<0.001	<0.001	<0.001	<0.001
Grower, d 10 to 21
PC	1,106ab	68.10ab	89.67a	1.323bc
NC1	987c	61.94c	83.21b	1.348bc
NC2	975cd	60.68cd	81.99b	1.354abc
NC3	932d	57.26d	80.40b	1.412a
NC1+PhyG	1,144ab	70.72ab	92.53a	1.313c
NC2+PhyG	1,151a	71.76a	93.12a	1.300c
NC3+PhyG	1,113ab	69.23ab	91.63a	1.325bc
NC4+PhyG+XAP	1,101b	67.62b	92.97a	1.379ab
SEM	11.058	0.936	0.900	0.014
P-value	<.0.001	<.0.001	<0.001	<0.001
Finisher 1, d 21 to 35
PC	1,914a	57.70ab	144.49abc	1.764
NC1	1,765b	55.54ab	139.15bc	1.759
NC2	1,733b	54.10b	136.87cd	1.776
NC3	1,686b	53.83b	129.35d	1.684
NC1+PhyG	1,981a	59.79ab	146.19ab	1.726
NC2+PhyG	1,998a	60.54ab	148.66a	1.710
NC3+PhyG	1,968a	61.05a	145.07abc	1.669
NC4+PhyG+XAP	1,939a	59.89ab	146.18ab	1.700
SEM	23.960	1.539	2.103	0.035
P-value	<0.001	0.002	<0.001	0.280
Finisher 2, d 35 to 42
PC	2,844ab	76.77	168.37	2.207
NC1	2,677bc	76.00	168.27	2.245
NC2	2,598c	72.22	166.50	2.371
NC3	2,596c	78.40	175.18	2.287
NC1+PhyG	2,938a	79.19	173.24	2.281
NC2+PhyG	2,923a	75.11	169.37	2.289
NC3+PhyG	2,894a	76.14	174.80	2.310
NC4+PhyG+XAP	2,883a	79.28	174.62	2.235
SEM	44.382	4.151	4.559	0.103
P-value	<0.001	0.941	0.758	0.974

¹Determined on the final day of each phase.

FCR, feed conversion ratio; NC, negative control; PC, positive control; PhyG, a consensus bacterial 6-phytase variant; XAP, a xylanase-amylase-protease combination.

Table 6.

Effect of treatment on growth performance, cumulatively.

Treatment1	ADG, g/bird/day	ADFI, g/bird/day	FCR, g:g	FCRc g:g	Livability, %	Feed cost, USD/kg BWG2	CFP, g CO2 eq./kg BWG3
d 1 to 21
Treatment means:
PC	50.53ab	62.91b	1.248cd	1.248d	99.7	-	-
NC1	44.89c	57.74c	1.288bc	1.324b	99.0	-	-
NC2	44.29cd	56.48c	1.276bcd	1.316bc	99.3	-	-
NC3	42.26d	56.25c	1.335a	1.388a	98.3	-	-
NC1+PhyG	52.35ab	65.48a	1.253cd	1.242d	99.3	-	-
NC2+PhyG	52.69a	65.41a	1.244d	1.230d	99.3	-	-
NC3+PhyG	50.89ab	64.65ab	1.271bcd	1.269cd	99.7	-	-
NC4+PhyG+XAP	50.31b	65.55a	1.306ab	1.307bc	99.0	-	-
SEM	0.529	0.544	0.009	0.011	0.514	-	-
P-value, ANOVA	<0.001	<0.001	<0.001	<0.001	0.664	-	-
Orthogonal polynomial contrasts:4
P-value, ‘linear’ stepwise PhyG matrix	<0.001	<0.001	<0.001	-	0.165	-	-
P-value, ‘quadratic’ stepwise PhyG matrix	<0.001	<0.001	<0.001	-	0.371	-	-
d 1 to 35
Treatment means:
PC	53.40a	95.56a	1.515	1.515ab	99.3	0.672a	2,601a
NC1	49.16b	90.30b	1.541	1.585a	97.0	0.664a	2,591a
NC2	48.23b	88.61bc	1.543	1.598a	97.0	0.645abc	2,525ab
NC3	46.91b	85.49c	1.524	1.592a	97.7	0.616cd	2424bc
NC1+PhyG	55.32a	97.76a	1.495	1.475b	98.7	0.647ab	2,521ab
NC2+PhyG	55.83a	98.71a	1.488	1.462b	98.7	0.625bcd	2,439bc
NC3+PhyG	54.95a	96.81a	1.482	1.466b	99.7	0.602de	2,359c
NC4+PhyG+XAP	54.14a	97.81a	1.515	1.508ab	97.7	0.587e	2,342c
SEM	0.687	0.944	0.016	0.021	0.758	0.007	25.929
P-value, ANOVA	<0.001	<0.001	0.051	<0.001	0.095	<0.001	<0.001
Orthogonal polynomial contrasts:4
P-value, ‘linear’ stepwise PhyG matrix	<0.001	<0.001	0.688	0.016	0.240	<0.001	<0.001
P-value, ‘quadratic’ stepwise PhyG matrix	<0.001	<0.001	0.375	0.012	0.135	<0.001	<0.001
d 1 to 42
Treatment means:
PC	85.88ab	107.67ab	1.647	1.632bc	97.3	0.718a	2,758a
NC1	81.19bc	103.29bc	1.680	1.680abc	95.3	0.711a	2,750ab
NC2	78.82c	101.61c	1.700	1.774a	96.7	0.698ab	2,709abc
NC3	78.70c	100.42c	1.682	1.757ab	96.0	0.669bc	2,605cde
NC1+PhyG	88.81a	110.34a	1.630	1.601c	96.3	0.693ab	2,677abcd
NC2+PhyG	88.70a	110.48a	1.630	1.606c	98.0	0.673bc	2,608bcde
NC3+PhyG	88.17a	109.81a	1.636	1.621c	99.3	0.654cd	2,543de
NC4+PhyG+XAP	87.56a	110.59a	1.652	1.640abc	97.0	0.631d	2,490e
SEM	1.438	1.164	0.020	0.031	1.036	0.008	32.579
P-value, ANOVA	<0.001	<0.001	0.112	<0.001	0.198	<0.001	<0.001
Orthogonal polynomial contrasts:4
P-value, ‘linear’ stepwise PhyG matrix	<0.001	0.001	0.234	0.004	0.611	0.003	0.013
P-value, ‘quadratic’ stepwise PhyG matrix	<0.001	0.001	0.274	0.010	0.752	0.009	0.020

¹Treatment details are given in Table 1.

²Calculated based on feed ingredient prices in February 2021, inclusive of the costs of the exogenous enzymes.

³Total 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).

⁴Performed using data from treatments PC, NC1, NC2 and NC3 only.

BWG, body weight gain; FCR, feed conversion ratio corrected for mortality; FCRc, feed conversion ratio corrected for mortality and BW; NC, negative control; PC, positive control; PhyG, a consensus bacterial 6-phytase variant; XAP, a xylanase-amylase-protease combination

During grower phase (d 10 to 21), ADG, ADFI and d 21 BW were reduced in NC1, NC2 and NC3 compared with the PC (P < 0.05), with greater reductions in BW and ADG in NC2 and NC3 than NC1 (ADG reduced by 15.9 % in NC3, 10.9 % in NC2 and 9.0 % in NC1, respectively, vs. PC; P < 0.05). The FCR was increased in NC3 vs. PC (+6.7 % above the PC; P < 0.05). Compared to the respective NC, the addition of PhyG increased ADG, ADFI and d 21 BW in NC1+PhyG, NC2+PhyG and NC3+PhyG, to a level that in all cases did not differ significantly from the level achieved by the PC. Meanwhile, FCR was reduced (P < 0.05) by PhyG addition to NC1 and NC2 to levels that were not different from the PC. Treatment NC4+PhyG+XAP maintained all performance measures during grower phase to a level that did not differ significantly from the PC.

Treatment effects were less evident during finisher 1 and 2 phases. During finisher 1, ADG, ADFI and FCR were not statistically significantly different in NC1, NC2 or NC3 compared to the PC, with the exception of a reduced ADFI in NC3 (P < 0.05). During this phase, all growth performance measures in the PhyG-supplemented treatments and in NC4+PhyG+XAP were maintained at a level not significantly different from the PC. During finisher 2, there were no significant differences between treatments for any performance measure.

Cumulatively, during d 1 to 21 (Table 6), effects of treatment were similar to those observed during grower phase (d 10 to 21). All NC treatments exhibited reduced ADG, ADFI and increased FCRc (P < 0.05) compared with the PC and all NC+PhyG treatments recovered these measures to a level not significantly different from the PC. Significant orthogonal polynomial contrasts were identified between increasing severity of the applied matrix (through PC, NC1, NC2 and NC3) and d 1 to 21 ADG, ADFI and FCR, for both linear and quadratic terms (P < 0.001), whereby the response was incrementally impaired (reduced or increased, as appropriate) as the severity of the matrix increased. The response for the period d 1 to 35 was similar. Although the increase in FCR in the NC diets compared with the PC were numerical based on Tukey test, significant linear and quadratic relationships between increasing severity of the applied matrix and ADG and ADFI were again identified (P < 0.001) and these relationships were also identified for FCRc (P < 0.05). All enzyme-supplemented treatments maintained performance responses to a level not significantly different from the PC during 1 to 35 d of age. For the total experimental period (d 1 to 42), BW ADG, ADFI and d 42 BW were all reduced (P < 0.05) in NC2 and NC3 based on ANOVA and Tukey’s HSD testing; the reductions in d 42 BW vs. PC were 246 g/bird (8.6 %) and 248 g/bird (8.7%) for NC2 and NC3, respectively. In addition, FCRc was increased (P < 0.05) in NC2 vs. PC. Significant orthogonal polynomial contrasts were identified for linear and quadratic terms for ADG (P < 0.001 and P < 0.001, respectively), ADFI (P < 0.001 and P < 0.001, respectively) and FCRc (P < 0.01 and P < 0.05, respectively) for the overall period, with increasing severity of the applied matrix (through PC, NC1, NC2 and NC3). The addition of PhyG to the NC diets improved (increased or decreased, as appropriate) each of d 42 BW, overall ADG and ADFI in NC1+PhyG, NC2+PhyG and NC3+PhyG as well as FCRc in NC2+PhyG and NC3+PhyG compared with the respective NC (P < 0.05), in all cases to levels that did not differ significantly from those achieved by the PC. Meanwhile, the addition of XAP on top of PhyG in NC4+PhyG+XAP maintained all overall growth performance measures to levels not significantly different from the PC. Livability was unaffected by treatment during any of the measured cumulative periods.

Feed costs and Carbon footprint

The impact of treatment on total estimated feed costs per kilogram of BWG and on the estimated CFP (in g CO₂ equivalents) of the diet per kilogram of BWG, during d 1 to 35 and d 1 to 42 is presented in Table 6. For the overall period (d 1 to 42), feed costs were reduced significantly in NC2+PhyG, NC3+PhyG and NC4+PhyG+XAP compared with the PC (by 0.045, 0.064 and 0.087 USD/kg BWG, respectively; P < 0.05) and in NC3+PhyG compared with NC1+PhyG (by 0.039 USD/kg BWG; P < 0.05). Carbon footprint estimates were also reduced significantly in PhyG+NC2, PhyG+NC3 and NC4+PhyG+XAP compared with the PC (by 162, 242 and 268 g CO₂ eq./kg BWG; P < 0.05).

Bone ash and breaking strength

The effect of treatment on tibia ash (as a percentage of DM) and tibia breaking strength at 21 d of age is presented in Table 7. Both were reduced in NC1, NC2 and NC3 vs. PC (by 8.6 to 11.8 % and 16.6 to 16.8 %, respectively; P < 0.05). Orthogonal polynomial contrasts were significant for linear and quadratic terms for both measures (P < 0.01 in all cases), whereby the value of these measures reduced with increasing severity of the applied matrix. The addition of phytase in NC1+PhyG, NC2+PhyG and NC3+PhyG increased tibia ash compared with the respective NC (P < 0.05), in all cases to a level not significantly different from that achieved by the PC, whereas tibia breaking strength was increased (P < 0.05) only in NC2+PhyG and NC3+PhyG, again to a level not significantly different from the PC. Treatment NC4+PhyG+XAP maintained both tibia ash and strength to a level not significantly different from the PC.

Table 7.

Effect of treatment on tibia ash and breaking strength at 21 d of age.

Treatment1	Tibia ash, % DM	Tibia breaking strength, kgF
Treatment means:
PC	45.03a	20.84a
NC1	41.15b	17.37b
NC2	39.70c	17.38b
NC3	40.08bc	17.33b
NC1+PhyG	43.71a	20.65ab
NC2+PhyG	43.79a	21.58a
NC3+PhyG	44.53a	21.72a
NC4+PhyG+XAP	43.95a	20.38ab
SEM	0.305	0.763
P-value, ANOVA	<0.001	<0.001
Orthogonal polynomial contrasts:2
P-value, ‘linear’ stepwise PhyG matrix	<0.001	0.004
P-value, ‘quadratic’ stepwise PhyG matrix	<0.001	0.001

¹Treatment details are given in Table 1.

²Performed using data from treatments PC, NC1, NC2 and NC3 only.

NC, negative control; PC, positive control; PhyG, a consensus bacterial 6-phytase variant; XAP, a xylanase-amylase-protease combination.

Carcass characteristics

The effect of treatment on carcass characteristics is shown in Table 8. Eviscerated carcass weights were reduced in NC2 and NC3 compared with the PC (P < 0.05) and increased in NC2+PhyG and NC3+PhyG compared with the respective NC (P < 0.05), to levels that were not significantly different from the PC. Heart weights also were increased in NC2 and NC3 compared with the PC (P < 0.05) and heart yields were increased in NC1, NC2 and NC3 compared with the PC (P < 0.05). Conversely, both heart weights and yields were reduced in NC2+PhyG and NC3+PhyG compared with the respective NC (P < 0.05), to levels comparable to PC. Leg weights and yields were increased in NC3+PhyG vs. NC3 and thigh weights were increased in NC2+PhyG and NC3+PhyG compared with the respective NC treatments (P < 0.05). For all carcass part measures, average values obtained in the PhyG-supplemented treatments (NC1+PhyG, NC2+PhyG and NC3+PhyG) as well as in NC4+PhyG+XAP were maintained at levels that were not significantly different from those achieved by the PC.

Table 8.

Effect of treatment on carcass part weights (g) and yields (% of carcass weight).

Treatment1	Eviscerated carcass, g	Heart, g	Abdominal fat, g	Breast, g	Legs, g	Thighs, g
PC	2,173a	9.78bc	46.35ab	778.8ab	573.7ab	314.2abc
NC1	2,144ab	11.38ab	50.33a	749.5ab	574.4ab	316.8abc
NC2	2,097b	11.93a	42.65ab	753.7ab	555.9b	303.1bc
NC3	2,075b	12.38a	40.50b	740.4b	548.6b	297.8c
NC1+PhyG	2,192a	10.20bc	44.90ab	783.0a	586.3a	330.3a
NC2+PhyG	2,190a	9.70c	42.63ab	767.8ab	600.8a	333.2a
NC3+PhyG	2,206a	9.60c	47.00ab	763.2ab	596.1a	328.2ab
NC4+PhyG+XAP	2,212a	10.03bc	49.78a	767.9ab	599.4a	330.1a
SEM	16.507	0.369	1.948	9.712	6.582	0.198
P-value	<0.001	<0.001	0.003	0.035	<0.001	<0.001

	Carcass yield, %	Heart, %	Abdominal fat, %	Breast, %	Legs, %	Thighs, %
PC	73.89abc	0.332c	1.575ab	26.49	19.51bc	10.69ab
NC1	73.09abc	0.388ab	1.715a	25.55	19.59abc	10.80ab
NC2	72.60bc	0.413a	1.478ab	26.09	19.25c	10.50b
NC3	72.16c	0.429a	1.405b	25.73	19.08c	10.35b
NC1+PhyG	74.13abc	0.345bc	1.520ab	26.48	19.83abc	11.16ab
NC2+PhyG	74.79a	0.331c	1.457ab	26.22	20.51a	11.38a
NC3+PhyG	74.91a	0.326c	1.596ab	25.92	20.24ab	11.14ab
NC4+PhyG+XAP	74.70ab	0.339bc	1.681ab	25.92	20.24ab	11.15ab
SEM	0.488	0.012	0.066	0.300	0.219	6.078
P-value	<0.001	<0.001	0.012	0.276	<0.001	0.002

¹Treatment details are given in Table 1.

NC, negative control; PC, positive control; PhyG, a consensus bacterial 6-phytase variant; XAP, a xylanase-amylase-protease combination.

Discussion

The analyzed values of CP, Ca and P in the PC and in diets NC1, NC2 and NC3 indicated that the intended reductions in AA, Ca and digestible P in the NC diets had been approximately met. Analyzed phytase activities in the PC and NC diets were generally low (< 300 FTU/kg) within the range reported by other, similar, studies (Bello et al., 2022; Dersjant-Li and Dusel, 2019), and most likely reflect the presence of some native, vegetal, phytase in the plant ingredients. (Phytase was not a known ingredient of any of the other ingredients in the diet formulations). This native phytase may have effected some P-release in the treatment diets. However, native phytase has a higher pH optimum than microbial phytase and is less effective for increasing P digestibility (Weremko et al., 2001). Hence, the impact on bird outcomes is considered likely to have been limited. The differences among treatments in the analyzed native phytase activities (PC, NC1–NC3) were small (< 100 FTU/kg) and unlikely to have confounded the ability to compare outcomes across treatments. Subtraction of the values obtained in the respective NC from those obtained in the respective PhyG-supplemented diets indicated that the level of supplemented phytase in the final diets was variable (up to ∼50 % above target dose in one isolated case during a single phase) but acceptable to confirm its supplementation. Xylanase activities also varied among the XAP-supplemented diets (by phase). Such variability is not uncommon in supplemental enzyme studies and could have been due to feed mixing, sampling or analytical irregularities. Crude fiber levels tended to be slightly higher than formulated values across all diets except NC+PhyG+XAP in which they were close to formulated levels and within ∼12 % of the analyzed values in the PC diet.

The results of the ANOVA and Tukey’s HSD analyses suggested that the reduced mineral content of NC1 (-0.22 % points Ca, -0.23 % points digestible P and -0.04 % points Na vs. PC) reduced tibia ash and strength at d 21 and growth performance during starter and grower phases. This indicates that minerals were insufficient in NC1 to support normal growth and bone development, as was expected. The significant linear and quadratic relationships between increasing matrix severity (through PC, NC1, NC2 and NC3) and growth performance responses during d 1 to 42 based on orthogonal polynomial contrast testing also suggested that performance was impaired by the minerals reduction in NC1. This implies an extra-phosphoric effect of the phytase, that would be consistent with the findings of an earlier study by Dersjant-Li et al. (2020) in which broilers fed corn-soybean meal-based diets with comparable reductions in Ca and digestible P exhibited reduced tibia ash and impaired growth performance. The lack of significant growth impairment during finisher phases in NC1 relative to the PC (identified by ANOVA) may be explained by age or adaptation effects; a reduced requirement for minerals as birds age can result in older birds appearing more tolerant of reduced mineral availability. Separately, birds can adapt over time to increase their utilization of Ca and P when these minerals are depleted in the diet (Li et al., 2015; Valable et al., 2018). The increased weight gain of birds when PhyG was added to the NC1 diet is consistent with the proven mode of action of PhyG in improving the availability of nutrients in the diet, in particular of P, Ca, Na, AA and protein (Babatunde et al., 2021; Dersjant-Li et al., 2022a; Espinosa et al., 2021), leading to improved growth as a result of increased nutrient utilization. The increase in d 21 tibia ash in NC1+PhyG vs. NC1, comparable to PC, suggests that bone mineralization was improved by the phytase which is consistent with the primary mode of action of the enzyme in releasing inorganic P from phytate. Overall (d 0 to 42), the similar tibia ash and strength of treatment NC1+PhyG to PC confirms the appropriacy of the applied mineral matrix with PhyG dosed at 1,250 FTU/kg.

The significant negative linear and quadratic relationships between matrix severity (through PC, NC1, NC2 and NC3) and overall growth performance responses also suggested that the energy reduction implemented in NC2 had an additional negative effect on performance, on top of that caused by the mineral matrix in NC1. Differences between NC2 and NC1 treatment means were not significantly different when analyzed by ANOVA and Tukey’s HSD test but this may have been due to a lack of statistical power conferred by multiple comparison testing; BW and ADG were consistently lower and FCR was consistently numerically higher in NC2 compared to PC than those in NC1 compared to PC, during grower, finisher and overall phases. The reduced tibia ash at d 21 in NC2 vs. NC1 is suggestive of a greater negative impact of the NC2 diet on bone mineralization, even though the concentrations of Ca and digestible P in these two diets were approximately equal. This effect could have resulted from the reduced feed efficiency (FCRc), in NC2 relative to PC, which was evident during starter, grower and overall phases and appeared to be driven by a reduction in feed intake (-5.6 % relative to the PC for the overall period) coupled with a greater reduction in ADG than in NC1 vs. PC (-8.2 % vs. -4.6 % for the overall period). These impairments were fully compensated for by the addition of PhyG to NC2 which achieved overall growth performance outcomes, d 21 bone quality responses and carcass characteristics that were not significantly different from those of the PC. By reference to the impaired outcomes in NC2 (that contained an ME matrix) compared with NC1 (that did not), this suggests an extra-phosphoric effect of the phytase in NC2+PhyG on energy digestibility and utilization. Improved energy digestibility by PhyG added to corn-soybean meal-based diets has been reported previously by Dersjant-Li et al. (2022a); the authors observed that PhyG dosed within the range 0 to 1,000 FTU/kg linearly increased the AID of GE and nitrogen in young broilers (1 to 10 d of age) fed a diet with a similar ME reduction to that applied in the present study (-68 kcal/kg vs. PC compared with -72 kcal/kg during 1 to 10 d of age in the present study). At 1,000 FTU/kg (close to the 1,250 FTU/kg applied in the present study) the phytase effected a 5.9 % increase in the AID coefficient of GE in the study by Dersjant-Li et al. (2022a). The results relating to growth performance and bone quality confirm the appropriacy of the applied minerals plus energy matrix with PhyG dosed at 1,250 FTU/kg.

The addition of an AA matrix (up to -0.06 % points vs. PC) on top of the ME and minerals matrix, via diet NC3, reduced certain measures of growth performance to a greater degree than NC2 according to the results of the ANOVA, and contributed to the significant linear and quadratic relationship between matrix severity and growth performance outcomes identified by the orthogonal polynomial contrast testing. For example, FCR during starter phase was increased to a significantly greater degree in NC3 than in NC2 vs. PC. These observations suggest that the digestible AA reductions in NC3 had a negative effect separate to those of the mineral and ME reductions. As (formulated) levels of limiting essential AA including Lys and Thr in NC3 were well below breeder recommendations (-0.18 to -0.09 % points; Aviagen Inc., 2022), a negative impact on AA utilization affecting growth in NC3 was expected. The addition of PhyG in NC3+PhyG fully compensated for these effects during all phases, as well as those on d 21 bone mineralization, breaking strength and carcass characteristics. This was evidenced by overall growth performance outcomes (all measures), bone quality and carcass characteristics (all measures) in NC3+PhyG achieving levels that did not differ significantly from those achieved by the nutritionally adequate, unsupplemented, PC diet. These results indicate that the applied digestible AA matrix (reduction of up to 0.06 % point content vs. PC), together with the minerals matrix (reduction of 0.22, 0.23 and 0.04 % point content vs. PC for Ca, digestible P and Na, respectively) and energy matrix (reduction of 72 to 51 kcal/kg vs. PC) was appropriate in the NC3 diet with PhyG dosed at 1,250 FTU/kg.

It was interesting that heart weight and yield at 42 d of age were both increased in NC2 and NC3 compared with the PC, and that the addition of PhyG to these treatments reversed this effect. The mechanisms involved here are unclear. Broilers can adapt to diets of different nutrient densities by altering the size and weight of digestive and non-digestive organs in order to maximize efficiency of nutrient use to meet requirements (Lamot et al., 2019). It is possible that the increased heart weight was the result of a metabolic adaptation to the nutrient poor NC2 and NC3 diets to enable the delivery of more blood to the gastrointestinal system to assist with nutrient digestion and absorption. However, evidence to support this in the literature is weak. Khan et al. (2011) and Yang et al. (2015) both reported no effect of low protein and energy diets on heart weight, whereas Bundur et al. (2024) reported reduced heart weights in broilers fed a low- compared with a high-nutrient density diet (in opposition to the present findings) and Sousa et al. (2015) reported increased relative heart (and liver) weights in broilers fed Ca and P-reduced diets, which were ameliorated by phytase (similar to the present study findings). An alternative explanation may involve the Lys-to-energy ratio of the diets. Several studies have observed an interaction between dietary Lys and energy content on carcass characteristic responses (Tang et al., 2007; Mansilla et al., 2022), from which it has been recommended that dietary Lys must be proportionately increased when energy is reduced to maintain muscle (especially breast muscle) deposition and minimize fat deposition (Mansilla et al., 2022). The Lys-to-energy ratio was higher in NC2 and NC3 than PC, and it is possible that this assisted birds with maintaining muscle deposition in the heart.

The efficacy of the XAP enzyme combination added to NC4+PhyG+XAP has been proven in several previous broiler studies (Romero et al., 2013; Romero et al., 2014; Wealleans et al., 2017). The xylanase component is used to reduce the antinutritive effect of non-starch polysaccharides (NSP) on digesta viscosity, nutrient digestibility and growth performance (Choct et al., 1999: Kiarie et al., 2014) while the amylase component is used to increase the digestion of starch, the primary source of energy in broiler diets. When used in combination with protease that effects protein hydrolysis, XAP has been shown to increase the AID of starch, fat, AA, protein and energy and improve growth performance relative to an unsuppplemented control diet (Romero et al., 2013; Romero et al., 2014; Wealleans et al., 2017). Xylanase and amylase are more efficacious in wheat- than corn-based diets (Romero et al., 2014) due to their higher viscosity and content of soluble NSP. However, xylanase is increasingly being used in corn-based diets where high-fiber ingredients or by-products have been added, to enhance fiber digestion. Exogenous amylase can also be effective in such diets for improving starch and energy utilization (Stefanello et al., 2015; Aderibigbe et al., 2020). Based on the existing literature, it was expected that the XAP in NC4+PhyG+XAP would contribute to compensating for the negative effect of the extra 61 kcal/kg ME reduction in NC4 (vs. NC3) on growth performance, by increasing energy availability from fiber and starch and AA availability from protein. This could not be demonstrated conclusively from the results because of the lack of a standalone NC4 treatment which precluded the ability to observe directly whether the extra reduction in ME and digestible AA vs. NC3 had any negative effect. However, it was inferred by the significant negative linear relationship identified between increasing matrix severity and overall growth performance (ADG, ADFI and FCRc) and bone quality outcomes. It is also consistent with published studies showing that a 3 to 5 % reduction in ME during grower phase reduces weight gain and increases FCR (Massuquetto et al., 2020; Ko et al., 2023). The comparable (non-significantly different) overall growth responses, d 21 tibia ash, breaking strength and d 42 carcass characteristics of birds fed NC4+PhyG+XAP with those achieved by birds fed the PC, implies that the enzymes in NC4+PhyG+XAP fully compensated for the nutrient an energy reductions applied to this treatment, including the extra 61 kcal/kg reduction in ME digestible AA reduction of up to 0.02 % points vs. NC3. The relative contributions of the xylanase, amylase, protease and phytase to this effect cannot be ascertained and it should not be assumed that these were additive. Additive, sub-additive and synergi or protease in combination with phytase (Cowieson and Adeola 2005; Singh et al., 2017; Romero et al., 2013).

A further benefit of applying a full matrix with PhyG (NC3+PhyG) or a full matrix plus further ME reduction with PhyG and XAP (NC4+PhyG+XAP) was that these treatments conferred a significant reduction in total estimated feed costs and CFP per kilogram of BW gain over an entire growth cycle (42 d) compared with either a nutritionally adequate unsupplemented diet (PC) or a diet with minerals matrix only applied plus PhyG. The calculated reductions were greatest for the diet supplemented with XAP compared with the PC (equivalent to ∼0.244 USD/bird for diet NC4+PhyG+XAP). The NC4+PhyG+XAP diet also conferred a reduced CFP per kilogram of BW gain compared with the PC (equivalent to ∼750 g CO₂ equivalents/bird). These findings highlight the potential benefits to feed costs and sustainability that can be gleaned through the targeted use of phytase, with or without XAP, that can be greater when energy and AA matrix values are applied on top of the minerals matrix. These benefits are accrued primarily through the ability to reduce the inclusion level of expensive ingredients raw materials (cereals), inorganic phosphate and synthetic crystalline AA. Feed cost or feed cost per kilogram BW gain savings from PhyG ‘full matrix’ application have been reported in previous studies (Bello et al., 2023; Dersjant-Li et al., 2020: Marchal et al., 2021) but these have all been in mixed-cereal diets (containing wheat and corn). To the authors’ knowledge, this is the first report of feed cost savings from the application of a ‘full matrix’ for phytase in corn-based diets.

In conclusion, the application of dose-dependent reductions in the dietary content of digestible AA and ME on top of a reductions in minerals (Ca, digestible P and Na) to corn-soybean meal-based diets supplemented with PhyG phytase at 1,250 FTU/kg maintained broiler growth performance, bone and carcass characteristics at levels that were not different to those achieved by a nutritionally adequate, unsupplemented, diet, over an entire growth cycle. Application of this ‘full matrix’ with PhyG also enabled a feed cost reduction compared with application of a minerals matrix alone. Supplementing XAP on top of the phytase with an additional reduction in ME (and digestible AA) similarly maintained bird responses comparable to the nutritionally adequate diet but conferred numerically greater feed cost and sustainability savings compared to PhyG alone. These findings provide confidence over the application of a ‘full matrix’ for PhyG phytase, with or without XAP, in corn-soybean meal-based diets.

Disclosures

Abiodun Bello, A. E. Ghane, A. de Kreij and Yueming Dersjant-Li are employees of Danisco Animal Nutrition & Health (IFF), a global supplier of enzymes.

Declaration of competing interest

We wish to confirm that there are no known conflicts of interest with this publication and there is no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship was left out. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing, we confirm that we have followed the regulations of our institutions concerning intellectual property. We further confirm that any aspect of the work covered in this manuscript that has involved experimental animals has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. We understand that the corresponding author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the corresponding author, and which has been configured to accept email from abiodun.bello@iff.com