Effect of a novel consensus bacterial 6-phytase variant in grower pigs fed corn-soybean meal-based diets formulated with a full nutrient matrix and no added inorganic phosphorus

Abstract

The capacity of a novel consensus bacterial 6-phytase variant (PhyG) to entirely replace dietary inorganic phosphorus (Pi) source in grower pigs fed diets with reduction of calcium (Ca), net energy (NE), and digestible amino acids (AA) was evaluated, using growth performance and apparent total tract digestibility (ATTD) of nutrients as outcome measures. A total of 352 mixed-sex pigs (initial BW 23.4 kg) were randomized to 4 treatments, 8 pigs/pen, and 11 pens/treatment. Diets were corn-soybean meal-based and formulated by phase (grower 1, 25 to 50 and grower 2, 50 to 75 kg BW). The positive control diet (PC) provided adequate nutrients and a negative control diet (NC) was formulated without Pi (1.2 g/kg ATTD P) and reduced in Ca (-0.12 to -0.13 percentage points), NE (-32 kcal/kg), and digestible essential AA (-0.004 to -0.026 percentage points) vs. PC. Two further treatments comprised the NC plus 500 or 1,000 FTU/kg of PhyG. Data were analyzed by ANOVA, mean contrasts and orthogonal polynomial regression. Nutrient reductions in the NC reduced (P < 0.05) average daily gain (ADG) during grower 1 and overall (73 to 136 d of age), increased (P < 0.05) feed conversion ratio (FCR) during grower 1 and overall and tended to reduce (P < 0.1) average daily feed intake (ADFI) during grower 2 and overall, vs. PC. Phytase supplementation improved (P < 0.05) FCR during grower 1, ADG during grower 2 and overall, ATTD of DM and P, and tended to improve DE (P = 0.053) in a linear dose-dependent manner. PhyG at 1,000 FTU/kg resulted in growth performance (all measures, all phases) equivalent to PC. The findings demonstrate that PhyG at 1,000 FTU/kg totally replaced Pi in complex grower pig diets containing industrial co-products, compensated a full nutrient matrix reduction and maintained performance.

Introduction

In commercial pig production, ingredients including inorganic phosphate costs remain high and diets are typically formulated on a least-cost basis (Dubeau et al., 2009). In this context, achieving the correct dietary input of phosphorus (P) that is sufficient to meet animal requirements but is not in excess, is key. Insufficient dietary P impairs bone development (Cromwell et al., 1970, Sørensen et al., 2018) and reduces growth performance (Dersjant-Li et al., 2018), whereas an excess of P is not only costly but environmentally unsustainable, and can contribute to environmental pollution (Sharply et al., 1994).

Pigs lack sufficient endogenous enzyme to release P from plant-based phytate (salt of phytic acid, myo-inositol hexaphosphate; IP6), so inorganic phosphate (Pi) is commonly added to the diet to ensure P requirements are met. Exogenous phytase enzymes of microbial or fungal origin have been added to pig and poultry diets since the 1990s as a means of increasing P-availability and reducing the need to add Pi to the diet (Selle and Ravindran, 2008; Lei et al., 2013). Their efficacy at enhancing P-digestibility and improving growth performance is well proven (Dersjant-Li et al., 2015; Humer et al., 2015; Dersjant-Li et al., 2017a; Grela et al., 2020). Since their introduction, continued advancements in phytase science and technology have resulted in increasingly efficacious phytases becoming available. In addition, recent studies have shown a clear, positive, linear, dose-dependent effect for phytase on nutrient digestibility and utilization in pig diets (Adedokun et al., 2015; Dersjant-Li et al., 2017a; Dersjant-Li and Dusel, 2019), which has led to an upward shift in the dose-levels recommended for phytase application in feed, from 500 FTU/kg to 1,000 FTU/kg or higher. For example, a Buttiauxella sp. phytase was shown to exhibit linear improvements in average daily gain (ADG) of grower pigs compared with a negative control diet (Dersjant-Li et al., 2017a).

In addition to P-digestibility and retention, exogenous phytase can also improve the digestibility of calcium (Ca) (Almeida et al., 2013), protein, and amino acids (AA) (Adedokun et al., 2015; Dersjant-Li and Dusel, 2019). The mechanisms by which these “extra-phosphoric” effects are achieved are thought to be linked to the hydrolysis of phytate by phytase in the upper gastrointestinal tract (GIT) (stomach of pigs) which reduces phytate availability to form insoluble complexes with these nutrients further along the GIT, or otherwise to the disruption of nutrient absorption as a result of phytate adverse effects on normal digestive processes (Cowieson et al., 2004; Selle et al., 2012; Truong et al., 2015). Determination of the magnitude of phosphoric and extra-phosphoric effects for individual phytases has enabled the derivation of dose level specific matrix values for digestible P, Ca, essential AA, and energy in pig diets, specifying the reductions in these nutrients that can be applied to account for the activity of the phytase. Such reductions in the presence of phytase have the potential to reduce feed costs for producers if less Pi needs to be added to meet P-requirements and if harder-to-digest alternatives to corn such as distillers’ dried grains with solubles (DDGS) can be included to meet some of the energy and protein requirements at lower overall feed cost. Evidence from phytase studies applying a full nutrient matrix (i.e., reduction in digestible P, Ca, essential AA, and energy) in pig diets is currently limited. However, a Buttiauxella sp. phytase dosed at 1,000 FTU/kg in a commercial broiler diet reduced in digestible P, Ca, AA, energy, and Na was recently shown to be effective in maintaining growth performance equivalent to a nutritionally adequate diet while conferring a reduction in feed costs, under a semi-commercial setting (Dersjant-Li et al., 2020a).

Given the increasing efficacy of available phytases in pig and poultry diets, the question of whether an exogenous phytase could entirely replace Pi in commercial diets is now receiving attention. Most recently, a novel consensus bacterial 6-phytase variant (PhyG) with high activity at low pH (Christensen et al., 2020) was evaluated in weaned piglets [~10 to 30 kg body weight (BW)] without added Pi and shown to maintain growth performance and bone parameters equivalent to that of a nutritionally adequate diet when dosed at 1,000 FTU/kg (Dersjant-Li et al., 2020b). Based on the average P-equivalence across metacarpi bone ash, ADG and feed conversion ratio (FCR), it was estimated that PhyG could replace 1.83 g/kg digestible P from monocalcium phosphate (MCP, a source of Pi) in the diet of weaned piglets when dosed at 1,000 FTU/kg (Dersjant-Li et al., 2020b).

This study tested the hypothesis that the novel consensus bacterial 6-phytase, PhyG, could maintain a similar growth performance and mineral digestibility in grower pigs fed a corn, soybean meal, DDGS, and wheat middlings-based diet formulated with a full nutrient matrix including reduction of Ca, net energy (NE), and digestible AA, and no added Pi when compared with those fed nutritionally adequate diet without phytase. In addition, feed cost analysis was conducted to evaluate the extent of any feed-cost reduction that might be enabled by the novel phytase when applied in conjunction with a full nutrient matrix to grower pig diets.

Materials and Methods

Pigs, housing, and experimental design

The study was carried out by Imasde Agroalimentaria, S. L. (Madrid, Spain) at the SAT Carraturégano experimental farm, in Segovia, Spain. All animals used in the research were raised and treated in accordance with Spanish guidelines for the care and protection of pigs as set out in Real Decreto 1135/2002 (B.O.E. number 278) as amended by Real Decreto 1392/2012 (B.O.E number 241). The experimental procedures were reviewed and approved by the Imasde Agroalimentaria Animal Care and Use Committee, Madrid, Spain.

A total of 352 [(Piétrain × Duroc) × (Large White × Landrace)] male and female pigs, initial BW 23.4 ± 1.3 kg (73 d of age), were assigned to 44-floor pens (each pen 11.25 m²) on the basis of initial BW, with 8 pigs per pen (4 male, 4 female, approximately equal initial BW per pen), and 11 pens per treatment, in a completely randomized design. Pens were located in 6 environmentally controlled animal rooms with a lighting regime of LD16:8h and average room temperature of 25 °C controlled through a mechanical ventilation system. Diets and water were provided ad libitum for the duration of the study (63 d).

Experimental diets

The experimental diets were based on corn, soybean meal, corn-DDGS, and wheat middlings, and were formulated by phase [Grower 1, 25 to 50 kg (73 to 112 d of age); Grower 2, 50 to 75 kg (112 to 136 d of age]. Diet composition is given in Table 1. The positive control (PC) diet was formulated to meet the nutritional requirements of the pigs during grower phase in accordance with recommendations set by the NRC (2012). A negative control (NC) diet was formulated to be the same as the PC but without Pi (MCP) and with reductions of 32 kcal/kg NE, 0.12 to 0.13 percentage points Ca, and 0.004 to 0.026 percentage points digestible AA, in accordance with the nutrient matrix for an existing commercial Buttiauxella sp. phytase at 1,000 FTU/kg. Two further treatment diets comprised the NC plus 500 FTU/kg or 1,000 FTU/kg of a novel consensus bacterial 6-phytase variant, PhyG, produced in Trichoderma reesei (Danisco Animal Nutrition, IFF, The Netherlands). Titanium dioxide (0.5%) was added to all diets as an indigestible marker during the last 2 wk of the grower 2 phase of the study. Diets were fed to pigs in pelleted form (pelleting temperature of 65 °C).

Table 1.

Ingredient composition (as-fed basis) and calculated nutrient content of the experimental diets1

Item	Grower 1 (73 to 112 d of age)		Grower 2 (112 to 136 d of age)
	PC	NC	PC	NC
Ingredient, %
Corn	55.86	57.89	58.11	59.97
DDGS	6.00	6.00	6.00	6.00
Wheat middlings	10.00	10.00	10.00	10.00
Soybean meal (45.5% CP)	20.55	20.36	18.40	18.23
Animal fat	4.49	3.47	4.40	3.43
Calcium carbonate	0.907	0.913	0.805	0.774
Monocalcium phosphate	0.745	-	0.607	-
Salt	0.418	0.419	0.393	0.394
DL-Methionine	0.082	0.063	0.031	0.011
L-Lysine HCL	0.555	0.527	0.409	0.380
L-Threonine	0.082	0.054	0.039	0.011
L-Tryptophan	0.012	0.004	0.001	-
Vitamin-mineral premix2	0.300	0.300	0.300	0.300
Titanium dioxide	-	-	0.500	0.500
Calculated analysis, %
Dry matter	88.05	87.81	88.01	87.79
Ash	4.60	4.04	4.26	3.77
Crude protein	17.00	17.00	16.00	16.00
Ether extract	7.59	6.63	7.54	6.63
Crude fiber	3.71	3.74	3.64	3.67
Neutral detergent fiber	12.85	13.01	12.81	12.96
Starch	38.13	39.42	39.56	40.75
Calcium	0.660	0.530	0.590	0.470
Total phosphorus	0.588	0.424	0.548	0.415
Calcium: total phosphorus	1.122	1.251	1.076	1.134
Phytate-phosphorus	0.289	0.293	0.285	0.288
Digestible phosphorus	0.260	0.119	0.230	0.116
Sodium	0.180	0.180	0.170	0.170
DE, kcal/kg	3,507	3,485	3,490	3,467
NE, kcal/kg	2,475	2,443	2,475	2,443
SID2 Lysine	0.980	0.963	0.850	0.833
SID Methionine	0.313	0.295	0.252	0.234
SID Methionine+Cysteine	0.550	0.534	0.480	0.464
SID Threonine	0.590	0.564	0.520	0.494
SID Tryptophan	0.170	0.163	0.150	0.149

¹PC, positive control diet; NC, negative control diet; SID, standardized ileal digestible.

²Provided per kilogram of diet: Vitamin A: 6,500 IU; Vitamin D3: 1,500 IU; Vitamin E: 25.0 mg; Vitamin K3: 1.2 mg; Vitamin B1: 1.6 mg; Vitamin B2: 3.0 mg; Calcium pantothenate: 9.0 mg; Vitamin B6: 1.6 mg; Vitamin B12: 20.0 µg; Nicotinic acid: 17.0 mg; Folic acid: 0.10 mg; Biotin: 0.02 mg; Betain: 100 mg; Fe (as FeSO₄.H₂O): 80.0 mg; Cu (as CuSO₄·5H₂O): 10.0 mg; Mn (as MnO): 30.0 mg; Zn (as ZnO): 80.0 mg; I (as Ca(IO₃)₂: 1.0 mg; Se (as Na₂SeO₃: 0.3 mg.

Measurements and sampling

Pigs were weighed individually at the start of the study and at the end of each phase (112 and 136 d of age). Feed disappearance was monitored per phase. Pens were inspected daily for pig mortality and dead animals were removed and individually weighed. These data were used to calculate mortality-corrected average daily feed intake (ADFI), ADG and FCR on per pen basis for each phase and overall (73 to 136 d of age).

On the last 3 d of phase 2 (133 to 136 d of age), fresh fecal samples were collected by rectal palpation from 4 pigs per pen and stored at -20 °C for later determination of apparent total tract digestibility (ATTD) of nutrients.

Samples of the diets were analyzed for dry matter (DM), crude protein (CP), crude fiber, ash, starch, ether extract, Ca, and P by Finca Mouriscade Laboratory (Vilanova, Lalín, Spain). Fecal samples were analyzed for DM, Ca, P, and gross energy (GE) by Agrolab Ibérica S.L.U (Tarragona, Spain). Phytase activities in the final diets were analyzed by Danisco Animal Nutrition Research Centre, Brabrand, Denmark.

Chemical analysis

Frozen fecal samples were thawed, pooled per pen and dried at 60 °C in a forced-air oven for 72 h, and ground to pass through a 1-mm screen using a Retsch grinder (Model ZM1; Retsch Inc., Newton, PA). Nutrients in diet and fecal samples were analyzed according to the following methods: Dry matter was determined according to the moisture method (AOAC method 930.15), protein by the Kjeldah method (AOAC method 984.13), ash by the incineration method (AOAC 942.05), ether extract by Soxhlet fat analysis (AOAC method 920.30), Ca by flame atomic absorption spectrophotometry (AOAC method 968.08), and total phosphorus by the photometric method (AOAC method 965.17) (AOAC, 2000). Titanium dioxide was determined according to ISO method 17294-2 (ISO, 2016). Gross energy was determined using an oxygen bomb calorimeter (Model 1261, Parr Instrument Co., IL, USA). Starch was analyzed by the polarimetric method (ISO 10520: 1997) and crude fiber by the Weende method (Mertens, 2003). Phytase activities were determined using an internally validated method adapted from ISO 30024:2009 (Gizzi et al., 2008), where one FTU (phytase unit) was defined as the quantity of enzyme that released 1 μmol of inorganic phosphate per minute from 5.0 mmol/L sodium phytate substrate at pH5.5 at 37 °C. Phytate phosphorus concentrations in diets were determined by Danisco Innovation Laboratories (Brabrand, Denmark) using a modified version of the HPLC method described by Skoglund et al. (1997)

Calculations

The ATTD of nutrients and digestible energy (DE) were calculated based on the following formulae:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{A}\mathrm{T}\mathrm{T}\mathrm{D}(\mathrm{\%})=[1-\left({\mathrm{X}}_{\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{s}}/{\mathrm{X}}_{\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}}\right)\times \left(\mathrm{T}{\mathrm{i}}_{\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}}/\mathrm{T}{\mathrm{i}}_{\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{s}}\right)]\times 100}\end{array}}$$

where X_feces is the concentration of the nutrient in the feces (mg/kg DM), X_diet is the concentration of the nutrient in the diet (mg/kg DM), Ti_diet is the titanium content in the diet (mg/kg DM) and Ti_feces is the titanium content in the feces (mg/kg DM), and;

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{D}\mathrm{E}\left(\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}/\mathrm{k}\mathrm{g}\right)=\mathrm{A}\mathrm{T}\mathrm{T}\mathrm{D}\mathrm{G}\mathrm{E}\times \mathrm{G}{\mathrm{E}}_{\mathrm{d}\mathrm{i}\mathrm{e}\mathrm{t}}\mathrm{i}\mathrm{n}\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}/\mathrm{k}\mathrm{g}\mathrm{D}\mathrm{M}}\end{array}}$$

Statistical analysis

For all outcome measures, data were analyzed on a per pen basis. Data were analyzed by ANOVA to determine differences among treatments in a randomized design with treatment included as a fixed effect and experimental room included as a random effect, using the Fit Model platform of JMP 14.0 (JMP, 2019). Separation of means between pairs of treatments was achieved using Least Squares means contrast (non-adjusted). The linear response to increasing PhyG dose from 0 (NC) to 1,000 FTU/kg was performed using orthogonal polynomial contrasts. Differences were considered significant at P < 0.05. Where 0.05 < P < 0.1, this was considered a tendency.

Results

Analyzed nutrient levels and phytase activities in the final diets are presented in Table 2. Analyzed Ca was slightly higher than calculated, resulting in analyzed Ca:P_(total) ratios of 1.4:1 to 1.8:1 across all diets. After accounting for endogenous phytase activity in the NC diets, analyzed phytase activity in the PhyG-supplemented treatments confirmed dose levels.

Table 2.

Analyzed nutrient concentrations (as-fed basis) and enzyme activities in the experimental diets¹

	Grower 1 (73 to 112 d of age)				Grower 2 (112 to 136 d of age)
Item	PC	NC	NC+500 FTU/kg Phy G	NC+1000 FTU/kg Phy G	PC	NC	NC+500 FTU/kg Phy G	NC+1000 FTU/kg Phy G
Dry matter	87.70	87.80	87.80	88.00	87.90	87.80	87.60	87.60
Crude protein	17.30	17.10	17.20	17.70	15.70	16.20	15.90	16.20
Crude fiber	3.40	3.50	3.30	3.60	3.50	3.20	3.50	3.60
Ash	4.39	3.94	4.08	4.04	4.67	4.28	4.25	4.34
Starch	40.70	42.20	42.30	41.40	41.80	39.60	43.90	40.40
Ether extract	7.50	6.60	6.40	6.40	7.40	6.50	6.70	6.60
Calcium	1.11	0.85	0.96	0.82	0.68	0.56	0.56	0.56
Total phosphorus	0.62	0.49	0.52	0.51	0.54	0.41	0.41	0.41
Phytate phosphorus	-	0.26	-	-	-	0.26	-	-
Phytase, FTU/kg	313	271	891	1,733	322	310	1116	1426

¹PC, positive control diet; NC, negative control diet.

Growth performance

Effects of treatment on growth performance are presented in Table 3. Compared with PC, pigs fed the nutrient reduced (NC) diets exhibited reduced ADG (-8.1%, P < 0.05) and increased FCR (+5.75%, P < 0.05) during 73 to 112 d of age, and reduced BW at age 112 d (-4.6%). During 112 to 136 d of age, ADFI tended to be reduced in NC vs. PC (P < 0.1), but FCR and ADG during this phase were equivalent. Moreover, the final BW at day 136 was -5.3% lower (P < 0.05) in NC vs. PC. Overall (73 to 136 d of age), ADG was reduced (-7.5%, P < 0.05), ADFI tended to be reduced (P < 0.1), and FCR was increased (+3.4%, P < 0.05) in NC vs. PC. Mortality was equivalent in NC and PC.

Table 3.

Effect of novel consensus bacterial 6-phytase on growth performance in pigs during 73 to 136 d of age fed diets formulated with a full nutrient matrix including reduction of calcium, net energy, digestible amino acids, and no added inorganic phosphorus

							P-values
Item1	PC2	NC2	NC+PhyG 500 FTU/kg	NC+PhyG 1,000 FTU/kg	SEM	ANOVA	PC vs. NC	PhyG vs. NC3,4	PhyG 500 FTU/kg vs. PC4	PhyG 1,000 FTU/kg vs. PC4	Phytase dose-response, linear5
Initial BW, 73 d of age, kg/pig	23.37	23.37	23.42	23.42	-	-	-	-	-	-	-
Grower 1, 73 to 112 d of age
112 d of age BW, kg/pig	54.2	51.7	53.1	53.6	0.69	0.085	0.013	0.056	0.338	0.978	0.963
ADG, g/d	791.5	727.5	761.8	777.0	17.72	0.084	0.012	0.056	0.338	0.977	0.198
ADFI, g/d	1507.2	1454.3	1524.9	1495.3	33.71	0.506	0.250	0.195	0.593	0.632	0.591
FCR, g/g	1.91	2.02	2.00	1.92	0.028	0.019	0.009	0.104	0.023	0.381	0.036
Mortality, %	2.3	3.4	1.1	0.1	1.54	0.449	-	-	-	-	-
Grower 2, 112 to 136 d of age
136 d of age BW, kg/pig	77.9	73.9	76.9	78.3	1.233	0.050	0.026	0.017	0.726	0.391	0.164
ADG, g/d	989.5	921.9	994.5	1028.2	32.14	0.143	0.107	0.025	0.461	0.607	0.019
ADFI, g/d	2262.4	2104.3	2276.8	2262.4	61.02	0.165	0.063	0.039	0.857	0.924	0.174
FCR, g/g	2.31	2.3	2.31	2.24	0.034	0.455	0.925	0.387	0.411	0.126	0.341
Mortality, %	1.1	1.1	1.1	2.3	1.24	0.890	-	-	-	-	-
Overall, 73 to 136 d of age
ADG, g/d	866.1	800.8	851.2	872.9	18.76	0.041	0.026	0.013	0.461	0.606	0.040
ADFI, g/d	1788.8	1696.8	1810.2	1785.5	37.40	0.185	0.086	0.041	0.680	0.742	0.327
FCR, g/g	2.07	2.14	2.12	2.06	0.019	0.013	0.032	0.078	0.009	0.095	0.493
Diet cost6, USD/ton	235.7	229.3	229.5	229.7	-	-	-	-	-	-	-
Feed cost/kg live weight, USD	0.351	0.342	0.349	0.338	0.004	0.064	0.097	0.073	0.775	0.047	-

¹All values are corrected for mortality.

²PC, positive control diet; NC, negative control diet.

³Pooled data from 500 and 1,000 FTU/kg treatments.

⁴Probability of contrast, using Least Squares means contrast.

⁵Linear regression analysis was performed with increasing PhyG dose from 0 (NC) to 1,000 FTU/kg excluding PC, using orthogonal polynomial regression (where the P-value is for phytase dose slope).

⁶Diet cost calculated based on current reported ingredient prices (Nov. 2020).

Increasing dose of PhyG from 0 (NC) to 1,000 FTU/kg improved FCR during 73 to 112 d of age, and increased ADG during 112 to 136 d of age and overall, in a linear manner (P < 0.05). No linear effect of PhyG dose-level on ADFI was observed, although ADFI was increased (P < 0.05) by PhyG (pooled data from 500 and 1,000 FTU/kg treatments) compared with NC, during 112 to 136 d of age and overall. Compared with NC, PhyG (pooled data from 500 and 1,000 FTU/kg treatments) tended to increase ADG during 73 to 112 d of age and BW at 112 d of age (both P = 0.056), improved (P < 0.05) BW at 136 d of age, ADG during 112 to 136 d and overall, and tended (P = 0.078) to improve the overall FCR. Compared with PC, PhyG at 500 FTU/kg resulted in a similar (P > 0.05) growth performance for all measures and during all phases except for FCR during 73 to 112 d of age and overall, which was 4.7 and 2.4% higher (P < 0.05), respectively, compared with PC. PhyG at 1,000 FTU/kg resulted in growth performance that was equivalent (P > 0.05) to PC for all measures during all phases (grower 1, grower 2, and overall) and overall FCR tended (P = 0.095) to reduce vs. PC. Mortality was unaffected by PhyG supplementation compared with PC or NC diets.

Using current reported ingredient prices including the cost of phytase, the cost per ton and cost per kg live weight of 4 diets were evaluated. PhyG supplementation at 1,000 FTU/kg showed a reduction (P < 0.05) in cost per kg of live BW when compared with pigs fed PC.

Total tract digestibility of nutrients

Pigs fed the NC exhibited reduced ATTD of P (-28.4%) compared with PC (P < 0.001, Table 4). Addition of PhyG to the diet improved ATTD of P (P < 0.001) and DM (P < 0.05) and tended to improve ATTD of DE (P < 0.1) in a linear manner. At 1,000 FTU/kg ATTD of P was improved by 68.1% vs. NC (P < 0.001 based on pooled data from 500 and 1,000 FTU/kg treatments). Pigs fed the NC showed a similar ATTD of Ca when compared with those fed the PC diet; however, it tended to improve in a linear manner (P = 0.10) with increasing levels of phytase.

Table 4.

Effect of novel consensus bacterial 6-phytase on apparent total tract digestibility (ATTD, %) of nutrients in pigs at 136 d of age fed diets formulated with a full nutrient matrix including reduction of calcium, net energy, and digestible amino acids and no added inorganic phosphorus

						P-values
Item	PC1	NC1	NC+PhyG 500 FTU/kg	NC+PhyG 1,000 FTU/kg	SEM	ANOVA	PC vs. NC	PhyG vs. NC2,3	PhyG 500 FTU/kg, vs. PC3	PhyG 1,000 FTU/kg, vs. PC3	Phytase dose-response, linear4
DM	83.0	82.6	83.1	83.8	0.44	0.278	0.603	0.120	0.878	0.143	0.037
Ca	43.8	43.7	44.8	50.0	2.68	0.291	0.968	0.285	0.804	0.111	0.100
P	48.2	34.5	53.8	58.0	1.70	<0.001	<0.001	<0.001	0.022	<0.001	<0.001
DE, kcal/kg	3,448.0	3,410.1	3,422.9	3,455.5	17.90	0.253	0.189	0.173	0.289	0.751	0.053

¹PC, positive control diet; NC, negative control diet.

²Pooled data from 500 and 1,000 FTU/kg treatments.

³Probability of contrast, using Least Squares means contrast.

⁴Linear regression analysis was performed with increasing PhyG dose from 0 (NC) to 1,000 FTU/kg excluding PC, using orthogonal polynomial regression (where the P-value is for phytase dose slope).

Discussion

The study by Dersjant-Li et al. (2020b) reported that, in weaned piglets (42 to 70 d of age), PhyG added at 500 FTU/kg or 1,000 FTU/kg to diets containing no added Pi maintained or improved growth performance measures (BW, ADG, FCR) compared with a nutritionally adequate diet containing 1.8 g/kg ATTD P from MCP. Results of the present study have demonstrated a similar effect in older pigs (grower phase, 73 to 136 d of age); PhyG dosed at 1,000 FTU/kg to Pi-free diets maintained all growth performance measures (BW, ADG, ADFI, and FCR) equivalent to a nutritionally adequate diet. Also, a linear dose–response effect on ADG and FCR was observed in both studies, but the minimum dose-level required to maintain FCR equivalence to PC differed. In the younger pigs studied by Dersjant-Li et al. (2020b), 500 FTU/kg PhyG was sufficient to maintain growth performance to the nutritionally adequate comparator diet, whereas in the present study 500 FTU/kg PhyG maintained ADG and ADFI, however, FCR was only improved to the level of the PC with PhyG at the higher dose-level of 1,000 FTU/kg. It should be noted that in the current study there was complete removal of Pi, with reduction of Ca, NE and digestible AA, whereas in the study by Dersjant-Li et al. (2020b) NC was formulated without Pi and reduced in Ca only. The capacity of PhyG to improve digestibility of AA has been demonstrated previously (Dersjant-Li et al., 2020c) and provides evidence for the downspec of AAs in diets containing PhyG. The more extensive nutrient matrix employed in the present study was based on 1,000 FTU/kg dose, hence a lower dose level of 500 FTU/kg of phytase cannot be expected to recover all growth performance back to the level of the PC. This also explains why 1,000 FTU/kg of PhyG was required to maintain a similar FCR to PC. In addition, with the higher nutrient downspec in comparison to the PhyG study by Dersjant-Li et al. (2020b), coupled with a moderate to high dietary phytate-P content (2.85 to 2.93 g/kg), it may be that 500 FTU/kg phytase is not sufficient to maximize degradation of phytate in the upper gut, leaving an opportunity for further improvement at a higher dose of 1,000 FTU/kg. Moreover, the reduced FCR at this dose vs. PC may suggest that the applied energy contribution could be underestimated for this phytase.

Studies involving other phytases in low/no Pi diets have observed similar positive linear dose–response relationships between phytase and ADG in grower pigs and maintenance of growth performance outcome measures equivalent to PC with a dose level around 1,000 FTU/kg, but have generally not incorporated a full nutrient matrix. Dersjant-Li et al. (2017b) observed a similar positive, linear, dose–response effect (0, 250, 500, or 1,000 FTU/kg) of a Buttiauxella sp. phytase on ADG during grower phase (30 to 85 kg) in pigs fed mixed cereal and soybean diets containing no added Pi and reduced in Ca and P but not in AA or energy. In that study, 1,000 FTU/kg of the phytase maintained BW and FCR equivalent to PC while ADG was improved by 5.3% vs. PC (Dersjant-Li et al., 2017b), while in the present study ADG only tended to be higher than PC with PhyG at 1,000 FTU/kg. A similar study of the same Buttiauxella phytase in complex diets without Pi reported that 1,000 FTU/kg maintained performance vs. PC during grower phase (25 to 75 kg) (Dersjant-Li et al., 2018). Again, that study did not include AA downspec in the matrix applied. The maintenance of all performance measures equivalent to PC at 1,000 FTU/kg of PhyG in grower pigs in the present study appears to compare with these previous results with Buttiauxella sp. phytase.

No impact of the PhyG supplemented treatments on mortality was observed. Feed intake showed a tendency to be reduced during grower 2 phase and overall (but not in grower 1 phase) in response to the nutrient reductions in the NC and was increased back to the level of the PC by PhyG supplementation during these phases. The effect of dietary P on feed intake in pigs has been debatable, varying depending on the stage of growth, and the extent and duration of deficiency (Misiura et al., 2020). Either deficiency or excess of dietary P has shown to negatively affected feed intake, suggesting that pigs are sensitive to inadequate levels of P in diets (Alebrante et al., 2011). Several previous studies have reported reduced feed intake by grower pigs in low-P diets compared with P-adequate diets (Dersjant-Li et al., 2018; Tsai et al., 2020), whereas others found no effect (Dersjant-Li et al., 2017b). Misiura et al. (2020) recently modeled the likely outcomes of how pigs respond to dietary P deficiency based on currently available empirical evidence and concluded that there were no evidence pigs modify their feed intake in response to a P-deficiency in feed, although they noted a decrease in feed intake accompanied by reductions in growth performance. In the present study, PhyG supplementation of the P-reduced diets improved (increased) feed intake back to the level produced by the nutritionally adequate diet, suggesting that addition of the novel phytase to the nutrient-reduced diets stimulated feed consumption.

The observed equivalence of growth performance in pigs supplemented with PhyG at 1,000 FTU/kg to PC, during both phases and overall, is of potential interest to producers and animal nutritionists if this can be achieved at reduced overall feed cost. The total feed cost per kilogram of live weight was estimated for each treatment based on the measured feed intakes and market prices for the feed ingredients in 2020 (USD per ton). According to these calculations, PhyG at 500 and 1,000 FTU/kg, respectively, reduced the total feed cost (inclusive of the cost of the phytase) per kg live weight by 0.60% and 3.70% compared with pigs fed the nutritionally adequate PC.

The effects of microbial phytase supplementation on P digestibility and retention in pigs in the context of Pi-free diets have received little research attention to date. In the present study, P digestibility was improved from 34.5% in the NC (0.12% apparent total tract digestible P) to 53.8% with 500 and 58.0% with 1,000 FTU/kg PhyG. A recent meta-analysis of 88 microbial phytase studies (phytase source; Aspergillus niger, Buttiauxella sp., Citrobakter braakii, Escherichia coli, and Peniophora lycii) evaluated the effect of increasing phytase concentration on total tract P digestibility and estimated that, across all phytases (dose ranged from 0 to 2500 FTU/kg diet) and studies (mostly conducted with growing pigs), P digestibility was increased by a maximum of 25 percentage points to a plateau at 64.9% (SE 1.82) and the digestible P concentration was similarly influenced with an increase by 1.01 g/kg (SE = 0.102) to a plateau value of 2.62 g/kg (Rosenfelder-Kuon et al., 2020a). Results of the present study with an ATTD P of 58% and digestible P concentration of 2.72 g/kg with PhyG at 1,000 FTU/kg in a Pi-free diet would place it at the upper boundary of the response range at this dose-level plotted by Rosenfelder-Kuon et al. (2020a). This indicates that, relative to other phytases, PhyG at 1,000 FTU/kg has high efficacy to increase P digestibility during the grower phase.

It is likely that the increase in ATTD P with PhyG would have resulted from increased degradation of IP6 to lower myo-inositol esters (IP5, IP4, IP3, or lower) in the stomach and fore-gut, rather than the hindgut. The activity of PhyG is optimal at low pH (pH3.5 to 4.5 with substantial relative activity at pH1.5 to 3.5) (Christensen et al., 2020) making it suited to the acidic conditions of the stomach of pigs. In vitro, PhyG has been observed to affect the complete and rapid hydrolysis of IP₆ to IP₅, IP₄, and IP₃ at low pH levels (pH2.5 to 4.5) and in vivo, 1,000 FTU/kg of the phytase resulted in a high digestibility of IP6 (89.3%) by the end of the ileum (Christensen et al. 2020). Other authors have noted extensive hydrolysis of any remaining IP6 to low IP esters by microbial phytase in the grower pig hindgut (Rosenfelder-Kuon et al., 2020b), but this is presumed to be the result of the activity of phytase of bacterial origin and does not contribute to total tract P digestibility because P that is released in the hindgut is not absorbed (Fan et al. 2000; Rosenfelder-Kuon et al., 2020b).

In the present study, ATTD of Ca tended to improve with increasing phytase supplementation, with 1,000 FTU/kg showing a 14.4% higher digestibility vs. NC, but this was not significant (P > 0.05). Several previous studies have observed improvements in the digestibility of Ca (and other minerals aside from P) in response to microbial phytase supplementation in pigs and poultry (Kühn and Männer, 2012; Almeida et al., 2013; Adedokun et al., 2015). As already mentioned, one principal mechanism for Ca digestibility improvement by phytase is the increased degradation of dietary phytate that reduces its availability to form insoluble complexes with Ca and thereby improves its availability for absorption by the animal (Selle et al., 2009). However, the efficacy of exogenous phytase to improve mineral digestibility is also impacted by dietary Ca level and by available Ca to P ratios which must be narrow in order to prevent antinutritive effects of excess Ca (González-Vega and Stein, 2014). González-Vega et al. (2016) derived an optimal digestible Ca:P ratio for grower pigs of 1.33:1 to 1.67:1 to maximize bone ash, bone Ca, and bone P. In the present study, digestible Ca:P values were not measured, but total Ca:P was 1.4 to ~1.7:1 in the PC diets and ~1.5:1 in the PhyG-supplemented diets (based on analyzed values). The excess of Ca relative to P in the diets did not appear to impact on the efficacy of PhyG, based on the observed linear improvement in ATTD of P with increasing PhyG dose-level, which was accompanied by increased fecal digestibility of DM and DE. The lack of significant effect of PhyG on total tract digestibility of Ca may have been because Ca was not limiting in the diets.

In conclusion, this study has demonstrated that PhyG at a dose-level of 1,000 FTU/kg in complex grower pig diets containing industrial co-products, was effective in totally replacing Pi with a full nutrient matrix applied to the diet (reduction in digestible P, Ca, energy and AA). The phytase at 1,000 FTU/kg maintained body weight gain, feed intake and tended to improve feed efficiency vs. a nutritionally adequate diet, leading to reduced production cost per kg live weight.

Glossary

Abbreviations

AA

amino acids

ADFI

average daily feed intake

ADG

average daily gain

ATTD

apparent total tract digestibility

BW

body weight

DDGS

distillers’ dried grains with solubles

DE

digestible energy

DM

dry matter

FCR

feed conversion ratio

GE

gross energy

GIT

gastrointestinal tract

IP6

myo-inositol hexaphosphate

MCP

monocalcium phosphate

NE

net energy

Pi

inorganic phosphorus