The positive effects of super-dosing phytase on the growth performance of growing pigs are mainly due to increased dietary digestible phosphorus

Abstract

This study investigated the effects of super-dosing phytase (up to 3,000 phytase units/kg—FTU/kg) and supplementary dietary inositol (2 g/kg) on phosphorus (P) digestibility and retention (experiment 1), and the growth performance (experiment 2) of growing pigs fed either P-deficient or P-adequate diets. In experiment 1, a total of 96 Duroc × Landrace × Yorkshire barrows [initial body weight (BW) = 37.2 ± 0.4 kg] were housed individually in 32 stainless steel metabolism cages. The barrows were blocked by BW and randomly allocated to 8 treatment groups, using a randomized complete block design. Experiment 1 was conducted in 3 runs to complete 12 replicate pigs per group. Total feces and urine were collected to determine the digestibility and retention of P. In experiment 2, a total of 2,496 Duroc × Landrace × Yorkshire pigs (initial BW = 24.6 ± 0.3 kg) were blocked by BW and sex and randomly allocated to 8 treatment groups, using a randomized complete block design with 12 replicates of 26 pigs per pen, per group. The treatment setup was the same across both experiments. The 8 experimental treatments comprised 2 total P levels (deficient, total P = 0.45% and adequate, total P = 0.60%). Within each P level, 4 diets were fed: control (without added phytase), control plus inositol (2 g/kg), control plus phytase (500 FTU/kg), and control plus phytase (3,000 FTU/kg). Phytase supplementation significantly enhanced P digestibility and retention across both P levels. Phytase supplementation at 500 and 3,000 FTU/kg improved P digestibility from 50.3% to 74.4% and 81.3%, respectively, in P-deficient diets, and from 59.2% to 73.5% and 78.0%, respectively, in P-adequate diets. Similarly, P retention increased from 36.6% to 62.6% and 69.6% in P-deficient diets and from 46.0% to 61.6% and 66.3% in P-adequate diets. In addition, phytase supplementation significantly improved the growth performance of pigs, resulting in increased final BW, average daily gain, and gain-to-feed ratio over the 42 d experimental period. Supplementary dietary inositol alone had no observable impact on P metabolism or growth performance. The interaction between dietary P level and phytase supplementation suggested that the optimal benefit of phytase supplementation may depend on the baseline dietary P content. These results suggested that phytase super-dosing enhanced P digestibility and utilization, reduced P excretion, and optimized growth performance in P-deficient diets. These benefits were mainly driven by the digestible P released by phytase, rather than the presence of inositol.

The positive effect of super-dosing phytase in pig diets was driven by improved phosphorus digestibility and utilization rather than inositol release. The impact on growth performance can be limited when the dietary P level is adequate.

Introduction

Dietary phosphorus (P) plays a critical role in optimizing growth performance in pigs (Zhai et al., 2022). The P in plant-based feed ingredients has a relatively low digestibility, primarily due to its high phytate-bound P (phytate-P) content that monogastric animals cannot effectively utilize, so inorganic P has traditionally been included in swine diets to fulfill their P requirements (Selle and Ravindran, 2008; Adeola and Cowieson, 2011). Over the past 2 decades, phytase supplementation has become a common practice in swine nutrition, both to reduce P excretion into the environment and minimize feed costs by enhancing the degradation of phytate-P (Cowieson et al., 2017). Extensive research supports the utilization and efficacy of phytase in swine diets, recommending that feed formulations use digestible P release values, or phytase equivalence, to accurately represent the actual amount of P released from phytate-P (Dersjant-Li et al., 2015).

Recent studies have suggested that additional phytase supplementation, termed “super-dosing,” may further enhance pig performance beyond P release alone (Holloway et al., 2019). Investigations have indicated that super-dosing phytase may improve the digestibility of minerals, amino acids, and energy, contributing to enhanced growth performance (Lagos et al., 2022). However, the precise mechanisms underlying these performance improvements remain unclear.

Phytic acid, the primary storage form of P in plants, contains 6 phosphate groups bound to an inositol ring. Previous research has demonstrated incomplete phosphate release from phytic acid at lower phytase supplementation levels (Lu et al., 2019a). Conversely, super-dosing phytase potentially facilitates more complete phosphate liberation, resulting in increased availability of inositol. Inositol is known to provide physiological benefits, including the promotion of gut health and nutrient transporter expression (Ogunribido et al., 2022). Consequently, many recent studies have explored the relationship between the effects of phytase super-dosing and plasma inositol concentrations. Although inositol and phytase supplementation increased plasma myo-inositol levels, inositol alone showed limited performance benefits (Lu et al., 2019a; Moran et al., 2019).

The efficacy of phytase in improving P digestibility can be reduced by dietary mineral levels, notably calcium (Ca) and P (Cao et al., 2023, 2025). High inorganic P levels may reduce phytase efficacy, suggesting that phytase’s P release potential could be overestimated in practical diets where total dietary P typically exceeds the levels used in phytase efficacy studies (Dersjant-Li et al., 2018; Cao et al., 2025). Super-dosing phytase could ensure adequate P release, effectively meeting pigs’ digestible P requirements and potentially yielding superior growth performance compared with standard phytase supplementation (Zeng et al., 2014).

However, previous studies on the effect of super-dosing phytase supplementation on growth performance in pigs have yielded inconsistent results. While some research has demonstrated linear growth-promotion effects due to phytase super-dosing (Holloway et al., 2019; Moran et al., 2019), other studies have indicated that adding phytase above the normal dosage range results in no further improvement. Zeng et al. (2014, 2015) reported that adding phytase at 20,000 phytase units/kg (FTU/kg) improved the digestion of P and other minerals and that pig weight gain improved quadratically. Similar results were reported by Knapp et al. (2018), who observed an interaction between dietary P and Ca levels, demonstrating a lack of improvement in growth performance due to super-dosing phytase when dietary mineral levels were adequate.

Super-dosing phytase may enhance growth performance through 2 primary mechanisms: first, through the degradation of phytate to release inositol, which could regulate glucose metabolism and thus influence energy metabolism (Lu et al., 2019a); and second, by increasing available P through phytate degradation (Dersjant-Li et al., 2018), thereby improving growth. Analysis of previous research suggests that dietary digestible P levels may critically influence the growth-promoting effects of phytase supplementation (Zeng et al., 2014, 2015; Knapp et al., 2018). Specifically, when dietary digestible P content is below the requirement for growing pigs, phytase supplementation can enhance P digestibility and consequently improve growth performance. It is possible that when dietary digestible P levels meet or exceed the pigs’ nutritional requirements, additional phytase supplementation tends to provide no further beneficial effects. Ogunribido et al. (2022) and others reported that while dietary myo-inositol improved gut health markers such as epithelial barrier integrity and nutrient transporter mRNA expression in pigs. However, no effect on growth performance or phosphorus retention was observed. Similarly, studies by Lu et al. (2019a) and Moran et al. (2019) showed that inositol supplementation elevated plasma inositol levels but had a limited impact on performance in growing pigs. Based on these observations, we hypothesized that dietary P levels play a decisive role in determining whether phytase super-dosing will yield growth benefits. In addition, there are still very few studies on the effect of super-dosing phytase in post-nursery-phase commercial settings.

The objective of the present study was to investigate the effects of super-dosing phytase (up to 3,000 phytase units/kg—FTU/kg) and supplementary dietary inositol (2 g/kg) on P digestibility and retention (experiment 1), and the growth performance (experiment 2) of growing pigs fed either P-deficient or P-adequate diets. This helped to elucidate whether improved growth performance due to phytase supplementation was primarily attributable to increased digestible P supply.

Materials and Methods

The procedures for pig handling and collection were approved by the Animal Care and Use Committee of the Southwest University of Science and Technology (L2024023) and were in accordance with the Chinese animal welfare guidelines.

Animals, dietary treatments, and sample collection

In experiment 1, a total of 96 Duroc × Landrace × Yorkshire barrows [initial body weight (BW) = 37.2 ± 0.4 kg] were individually housed in stainless steel metabolism cages, each equipped with a feeder and a nipple drinker. The barrows were blocked by BW and randomly allocated to eight treatment groups, using a randomized complete block design. The experiment was conducted with 3 runs to complete 12 replicate pigs per group. Total collection of feces and urine was performed to determine the digestion and retention of P. Room temperature was maintained at 20 ± 2 °C. The daily feed allowance was calculated at the beginning of the experiment and divided into 2 equal-sized meals, fed at 0800 and 1800 hours. The pigs had free access to water at all times. The pigs were given 5 d to adapt to the diets, followed by total collection of feces and urine from days 6 to 12, following Liu et al. (2014). Chromic oxide was added to the morning meal on days 6 and 11 to indicate the initiation and the termination of fecal collection. Fecal collection started with the first appearance of the green chromic oxide marker and ceased with the appearance of chromium-labeled feces after day 11. The fecal samples collected from each pig were mixed at the end of the experiment. The diet formulation used in experiment 1 was the same as in days 0 to 21 of experiment 2.

In experiment 2, a total of 2,496 Duroc × Landrace × Yorkshire pigs [initial body weight (IBW) = 24.6 ± 0.3 kg]. The same number of barrows and gilts were used in this study. Pigs were blocked by BW and sex and then randomly allocated to 8 treatment groups, using a randomized complete block design with 12 replicates of 26 pigs per pen, per group. The pigs were housed in concrete-floored pens with a single-sided 4-hole feeder and 4-cup waterers. The stocking density for the experiment was 0.72 m² per pig. The relative humidity inside the animal house for all the experiments ranged from 40% to 70%, and the environmental temperature inside the swine confinement building was controlled at 20 ± 2 °C. All pigs received ad libitum feed and water throughout the experiment. The experimental period lasted for 42 d. Individual BW and the overall feed usage were measured on days 0, 21, and 42 to calculate the average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed ratios.

The feed treatment setup was the same across both experiments. The 8 experimental treatments comprised 2 total P levels (deficient, total P = 0.45% and adequate, total P = 0.60%). Within each P level group, there were 2 diets: control (without added phytase), control plus inositol (2 g/kg), control plus phytase (500 FTU/kg), and control plus phytase (3,000 FTU/kg). The level of inositol inclusion in the experimental diet was selected to approximate the maximum potential inositol release from dietary phytate (Lu et al., 2019a). Phytase (RONOZYME HiPhos, 20,000 FTU/g product) was purchased from DSM-Firmenich. The phytase activity was determined at 25,916 FTU/g. As shown in Tables 1 and 2, the experimental base diet contained corn, soybean meal, and monocalcium phosphate, formulated to meet the nutrient requirements of growing pigs (NRC, 2012).

Table 1.

Diet formulations and their calculated nutrients used in experiment 1 (days 0– to 21)

	Insufficient P				Adequate P
Ingredients, g/kg	Control	Control + Inositol	Control + 500 FTU/kg Phytase	Control + 3,000 FTU/kg Phytase	Control	Control + Inositol	Control + 500 FTU/kg Phytase	Control + 3,000 FTU/kg Phytase
Corn	71.2	69.2	69.7	68.2	69.7	67.7	69.2	66.7
SBM	23.0	23.0	23.0	23.0	23.0	23.0	23.0	23.0
Soy oil	3.0	3.0	3.0	3.0	3.7	3.7	3.7	3.7
Monocalcium phosphate	0.4	0.4	0.4	0.4	1.1	1.1	1.1	1.1
Limestone	0.9	0.9	0.9	0.9	1.1	1.1	1.1	1.1
Salt	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3
Vitamin premixa	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
Inositol premixb	0.0	2.0	0.0	0.0	0.0	2.0	0.0	0.0
Phytase premixc	0.0	0.0	0.5	3.0	0.0	0.0	0.5	3.0
Lys	0.4	0.4	0.4	0.4	0.4	0.4	0.4	0.4
Met	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Thr	0.4	0.4	0.4	0.4	0.4	0.4	0.4	0.4
Total	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0
Calculated nutrients and energy d
Protein, g/kg	176.5	176.5	176.5	176.5	175.8	175.8	175.8	175.8
ME, kcal/kg	3901.0	3901.0	3901.0	3901.0	3901.0	3901.0	3901.0	3901.0
Ca, g/kg	4.99	4.99	4.99	4.99	6.56	6.56	6.56	6.56
P, g/kg	4.54	4.54	4.54	4.54	5.99	5.99	5.99	5.99
ATTD P, g/kg	1.98	1.98	1.98	1.98	3.44	3.44	3.44	3.44
Phytate P, g/kg	2.49	2.49	2.49	2.49	2.46	2.46	2.46	2.46
Ca : P	1.10	1.10	1.10	1.10	1.10	1.10	1.10	1.10
NDF, %	10.6	10.6	10.6	10.6	10.6	10.6	10.6	10.6
ADF, %	4.2	4.2	4.2	4.2	4.1	4.1	4.1	4.1
SID amino acids, g/kg
Ile	6.3	6.3	6.3	6.3	6.3	6.3	6.3	6.3
Lys	11.3	11.3	11.3	11.3	11.3	11.3	11.3	11.3
Met	3.6	3.6	3.6	3.6	3.6	3.6	3.6	3.6
Met + Cys	6.6	6.6	6.6	6.6	6.6	6.6	6.6	6.6
Thr	7.8	7.8	7.8	7.8	7.8	7.8	7.8	7.8
Trp	1.8	1.8	1.8	1.8	1.8	1.8	1.8	1.8
Val	7.1	7.1	7.1	7.1	7.1	7.1	7.1	7.1

^aProvided per kilogram of diet: 8,800 IU of vitamin A; 880 IU of vitamin D; 64 IU of vitamin E; 4 mg of vitamin K (menadione sodium bisulfite); 70 μg of vitamin B_12; 14 mg of riboflavin; 60 mg of D-pantothenic acid; 30 mg of niacin; 6 mg of vitamin B₆; 200 μg of biotin; 1.2 mg of folic acid; 120 mg of Fe (as iron carbonate); 25 mg of Mn (as manganese oxide); 17 mg of Cu (as copper chloride); 0.3 mg of I (as ethylenediamine dihydroiodide); 0.2 mg of Se (as sodium selenite); and 120 mg of Zn (as zinc oxide). Inositol premix made to 0.1 g/kg, which, when added at 20 g/kg, provided 2 g inositol/kg diet.

^bInositol premix was made to contain 10 g inositol/kg premix, which, when added at 20 g/kg provided 2 g inositol/kg of the complete diet.

^cPhytase (RONOZYME HiPhos; DSM Nutritional Products) premix was made with 20,000 FTU/g product with the same corn ingredient (after grinding) to a 0.5% diluted premix to be included as 5 and 30 g/kg of experimental diets to provide 500 and 3,000 FTU/kg of phytase in the completed feed. The analyzed phytase activity was 580 and 3,155 FTU/kg for the 2 phytase treatments in P-deficient diets, and 521, 3,237 FTU/kg for the 2 phytase treatments in P-adequate diets, respectively.

^dThe analytical results of the CP, NDF, ADF, Ca, P, and AA profiles were close to the formulated values.

Table 2.

Diet formulations and their calculated nutrients used in experiment 2 (days 22 to 42)

	Insufficient P				Adequate P
Ingredients, g/kg	Control	Control + Inositol	Control + 500 FTU/kg Phytase	Control + 3,000 FTU/kg Phytase	Control	Control + Inositol	Control + 500 FTU/kg Phytase	Control + 3,000 FTU/kg Phytase
Corn	75.2	73.2	74.7	72.2	74.0	72.0	73.5	71.0
SBM	20.0	20.0	20.0	20.0	20.0	20.0	20.0	20.0
Soy oil	2.5	2.5	2.5	2.5	3.2	3.2	3.2	3.2
Monocalcium phosphate (A)	0.4	0.4	0.4	0.4	1.1	1.1	1.1	1.1
Limestone (38% Ca; B)	0.9	0.9	0.9	0.9	1.0	1.0	1.0	1.0
Salt	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3
Vitamin premix (C)	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
Inositol premix	0.0	2.0	0.0	0.0		2.0	0.0	0.0
Phyase premix	0.0	0.0	0.5	3.0		0.0	0.5	3.0
Lys	0.4	0.4	0.4	0.4	0.4	0.4	0.4	0.4
Met	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Thr	0.4	0.4	0.4	0.4	0.4	0.4	0.4	0.4
Total	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0

Calculated nutrients and energy
Protein, g/kg	165.4	165.4	165.4	165.4	164.0	164.0	164.0	164.0
ME, kcal/kg	3873	3873	3873	3873	3873	3873	3873	3873
Ca, g/kg	4.82	4.82	4.82	4.82	6.43	6.43	6.43	6.43
P, g/kg	4.41	4.41	4.41	4.41	5.83	5.83	5.83	5.83
ATTD P, g/kg	1.90	1.90	1.90	1.90	3.36	3.36	3.36	3.36
Phytate P, g/kg	2.44	2.44	2.44	2.44	2.41	2.41	2.41	2.41
Ca : P	1.10	1.10	1.10	1.10	1.10	1.10	1.10	1.10
NDF, %	10.7	10.7	10.7	10.7	10.6	10.6	10.6	10.6
ADF, %	4.0	4.0	4.0	4.0	4.0	4.0	4.0	4.0

SID amino acids, g/kg
Ile	5.8	5.8	5.8	5.8	5.8	5.8	5.8	5.8
Lys	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0
Met	3.1	3.1	3.1	3.1	3.1	3.1	3.1	3.1
Met + Cys	5.8	5.8	5.8	5.8	5.8	5.8	5.8	5.8
Thr	6.9	6.9	6.9	6.9	6.9	6.9	6.9	6.9
Trp	1.9	1.9	1.9	1.9	1.9	1.9	1.9	1.9
Val	6.7	6.7	6.7	6.7	6.7	6.7	6.7	6.7

^aProvided per kilogram of diet: 8,800 IU of vitamin A; 880 IU of vitamin D; 64 IU of vitamin E; 4 mg of vitamin K (menadione sodium bisulfite); 70 μg of vitamin B_12; 14 mg of riboflavin; 60 mg of D-pantothenic acid; 30 mg of niacin; 6 mg of vitamin B₆; 200 μg of biotin; 1.2 mg of folic acid; 120 mg of Fe (as iron carbonate); 25 mg of Mn (as manganese oxide); 17 mg of Cu (as copper chloride); 0.3 mg of I (as ethylenediamine dihydroiodide); 0.2 mg of Se (as sodium selenite); and 120 mg of Zn (as zinc oxide). Inositol premix made to 0.1 g/kg, which, when added at 20 g/kg, provided 2 g inositol/kg.

^bInositol premix was made to contain 10 g inositol/kg premix, which, when added at 20 g/kg, provided 2 g/kg of the complete diet.

^cPhytase (RONOZYME HiPhos; DSM Nutritional Products) premix was made with 20,000 FTU/g product with the same corn ingredient (after grinding) to a 0.5% diluted premix to be included as 5 and 30 g/kg of experimental diets to provide 500 and 3,000 FTU/kg of phytase in the complete feed. The analyzed phytase activity was 580 and 3,155 FTU/kg for the 2 phytase treatments in the P-deficient diets, and 521, 3,237 FTU/kg for the 2 phytase treatments in the P-adequate diets, respectively.

^dThe analytical results of the CP, NDF, ADF, Ca, P, and AA profiles were close to the formulated values.

Chemical analyses

Samples of the diets and feces were dried in a forced-air oven and ground through a 1-mm screen before conducting analyses. Diets and feces were analyzed for dry matter content by oven drying at 135 °C for 2 h (method 930.15; AOAC, 2007). Dietary crude protein concentrations were determined using the combustion procedure (method 990.03; AOAC, 2007) in a Leco CHNS-932 Analyzer (Leco Corp., St. Joseph, MI, USA). Diets and feces were analyzed for P content using the inductively coupled plasma spectroscopy (method 985.01; AOAC, 2007) after wet-ash sample digestion (method 975.03; AOAC, 2007). Diet samples were analyzed for phytate-P concentrations following the protocol described by Aureli et al. (2017). Approximately 0.5 g of finely ground samples were extracted with 10 mL of 0.5 M HCl under agitation at 40 °C for 2 h, frozen overnight, thawed, and re-extracted under the same conditions. After centrifugation, inositol phosphates (IP)s were filtered at 10 °C for 1 h. Aliquots were analyzed using HPLC.

Calculations and statistical analyses

The analyzed total P values were used in the calculations. Feed refusals of each pig in experiment 1 were recorded to calculate daily dry matter and P intake.

The apparent total tract digestibility (ATTD) of P was calculated as described by Akinmusire and Adeola (2009):

ATTD (%) = 100 × (P_I - P_F)/P_I;

P_D = P_I - P_F;

where: ATTD is the ATTD; P_I is the daily P intake of each pig (mg/d); P_D is the daily digested P of each pig (mg/d); and P_F is the daily fecal P of each pig (mg/d).

All statistical analyses of the data in the 2 experiments were analyzed using the MIXED procedure in the SAS package (https://github.com/yabwon/SAS_PACKAGES) with individual pigs as the experimental unit. In the model, the main effects of P level and inositol, the linear and quadratic effects of phytase, P × inositol, P × phytase linear, and P × phytase quadratic interactions were considered as fixed effects, and block (replicates) was the random effect. The linear and quadratic effects of phytase within each P level were determined by orthogonal polynomial contrasts generated by Proc IML (https://documentation.sas.com/doc/en/pgmsascdc/9.4_3.4/pgmdiff/n1a0k45i0jwwegn14262pyz304w6.htm). The coefficients for linear response for 0, 500, and 3,000 FTU/kg were −0.513, −0.293, and 0.807, respectively, while the counterparts for quadratic effect were 0.635, −0.762, and 0.217, respectively. Statistical significance was determined at P < 0.05.

Results

Experiment 1

The effects of dietary phytase and inositol supplementation on the digestibility and retention of P in growing pigs are shown in Table 3. The dietary P content ranged from 0.419% to 0.571% across the treatments. In pigs fed a deficient dietary P (approximately 0.42%), phytase supplementation significantly improved P digestibility and retention. Increasing phytase from 0 to 500 and 3,000 FTU/kg increased P digestibility linearly from 50.3% to 74.4% and 81.3%, respectively, P retention from 36.6% to 62.6% and 69.6%, respectively, and the ATTD of P from 2.11 to 3.22 and 3.41 g/kg, respectively. Supplementation with phytase also significantly decreased fecal P output linearly (from 2.00 to 1.06 and 0.75 g/d, respectively) and urinary P output linearly (from 0.55 to 0.49 and 0.47 g/d, respectively), thereby linearly increasing the amounts of P digested and retained P from 2.02 to 3.09 and 3.26 g/d, respectively and from 1.47 to 2.60 and 2.79 g/d, respectively.

Table 3.

Effect of phytase and inositol on the digestibility and retention of P in the P-deficient and P-adequate diets fed to growing pigs

		Total P in diet, %	P intake, g/d	Fecal P output, g/d	Urine P output, g/d	P digested, g/d	P retained, g/d	P digestibility, %	P retention, %	ATTD P a, g/kg
Low dietary P	Low P control	0.420	4.02	2.00	0.55	2.02	1.47	50.3%	36.6%	2.11
	Inositol	0.431	4.13	2.05	0.50	2.08	1.58	50.3%	38.2%	2.17
	500 FTU/kg phytase	0.433	4.15	1.06	0.49	3.09	2.60	74.4%	62.6%	3.22
	3,000 FTU/kg phytase	0.419	4.01	0.75	0.47	3.26	2.79	81.3%	69.6%	3.41
Adequate dietary P	Adequate P control	0.565	5.41	2.21	0.71	3.20	2.49	59.2%	46.0%	3.34
	Inositol	0.557	5.33	2.16	0.67	3.17	2.50	59.5%	47.0%	3.31
	500 FTU/kg phytase	0.571	5.47	1.45	0.65	4.02	3.37	73.5%	61.6%	4.20
	3,000 FTU/kg phytase	0.560	5.36	1.18	0.63	4.18	3.55	78.0%	66.3%	4.37
SEM		-	-	0.193	0.044	0.194	0.19	4.05%	3.99%	0.202
P-values
Main effects	P level		-	0.192	0.086	0.002	0.006	0.942	0.780	0.002
	Inositol		-	0.604	0.401	0.580	0.450	0.439	0.300	0.580
Phytase effect	Linear		-	<0.001	0.487	<0.001	<0.001	<0.001	<0.001	<0.001
	Quadratic		-	0.588	0.732	0.429	0.468	0.458	0.457	0.429
Interactions	P × Phytase Linear		-	-	0.362	0.901	0.934	0.478	0.599	0.901
	P × Phytase Quadratic		-	-	0.338	0.474	0.344	0.433	0.305	0.474
	P × Inositol		-	0.136	0.173	0.089	0.042	0.090	0.037	0.089

^aApparent total tract digestible P, g/kg.

In pigs fed diets with adequate dietary P (approximately 0.565%), phytase similarly improved P digestibility and retention. Increasing phytase supplementation from 0 to 500 and 3,000 FTU/kg enhanced P digestibility linearly from 59.2% to 73.5% and 78.0%, respectively, P retention linearly from 46.0% to 61.6% and 66.3%, respectively, and the ATTD of P linearly from 3.34 to 4.20 and 4.37 g/kg, respectively. Concurrently, the fecal P output decreased from 2.21 to 1.45 and 1.18 g/d, respectively, and urinary P output slightly decreased from 0.71 to 0.65 and 0.63 g/d, respectively. Consequently, the quantity of digested and retained P increased from 3.20 to 4.02 and 4.18 g/d, respectively, and from 2.49 to 3.37 and 3.55 g/d, respectively.

Dietary P supplementation significantly improved fecal P output P digested (g/d), P retained (g/d), P digestibility, P retention, and determined dietary ATTD P content (g/kg; P < 0.001). Inositol supplementation did not significantly influence the P digestibility or retention parameters at either dietary P level. The statistical analysis confirmed significant linear effects of phytase supplementation (P < 0.001) across all measures of P metabolism, whereas the quadratic effects of phytase were not significant. The interaction effects between dietary P levels and phytase supplementation were not significant, indicating that phytase’s beneficial effects were consistent regardless of basal dietary P content. An interaction between dietary P level and inositol was observed for P retention, indicating that inositol supplementation may have a different effect on P utilization in P-deficient and P-adequate conditions.

Experiment 2

The effects of dietary phytase and inositol supplementation on growth performance in growing pigs are shown in Tables 4, 5, and 6.

Table 4.

Effect of phytase and inositol on growth performance from days 1 to 21 in the P-deficient and P-adequate diets fed to growing pigs

		IBW, kg	FBW, kg	ADG, kg/d	ADFI, kg/d	Gain:Feed
Low dietary P	Low P control	24.58	37.15	0.59	1.37	0.438
	Inositol	24.67	37.43	0.60	1.36	0.443
	500 FTU/kg phytase	24.61	38.62	0.67	1.43	0.468
	3,000 FTU/kg phytase	24.57	40.22	0.74	1.55	0.481
Adequate dietary P	Adequate P control	24.50	37.90	0.64	1.37	0.467
	Inositol	24.53	38.25	0.65	1.42	0.460
	500 FTU/kg phytase	24.54	39.44	0.71	1.44	0.493
	3,000 FTU/kg phytase	24.58	39.85	0.73	1.50	0.486
SEM		0.08	0.29	0.01	0.03	0.004
P-values
Main effects	P level	0.377	0.391	0.257	0.959	0.001
	Inositol	0.928	0.595	0.615	0.818	0.411
Phytase effect	Linear	0.970	0.001	0.001	0.165	<0.001
	Quadratic	0.262	0.076	0.149	0.771	<0.001
Interactions	P × Phytase Linear	0.392	0.050	0.096	0.290	0.067
	P × Phytase Quadratic	0.340	0.276	0.373	0.590	0.359
	P × Inositol	0.309	0.636	0.799	0.913	0.306

Table 5.

Effect of phytase and inositol on growth performance from day 22 to 42 in the P-deficient and P-adequate diets fed to growing pigs

		IBW, kg	FBW, kg	ADG, kg/d	ADFI, kg/d	Gain:Feed
Low dietary P	Low P control	37.15	53.38	0.78	2.01	0.383
	Inositol	37.43	53.47	0.76	2.02	0.378
	500 FTU/kg phytase	38.62	56.09	0.83	2.04	0.408
	3,000 FTU/kg phytase	40.22	58.38	0.86	2.11	0.410
Adequate dietary P	Adequate P control	37.90	54.69	0.80	2.03	0.393
	Inositol	38.25	55.37	0.82	2.09	0.390
	500 FTU/kg phytase	39.44	57.17	0.84	2.01	0.419
	3,000 FTU/kg phytase	39.85	57.97	0.86	2.12	0.407
SEM		0.29	0.42	0.01	0.03	0.003
P-values
Main effects	P level	0.391	0.180	0.261	0.684	0.043
	Inositol	0.595	0.790	0.891	0.935	0.928
Phytase effect	Linear	0.001	<0.001	0.001	0.008	0.033
	Quadratic	0.076	0.185	0.887	0.090	<0.001
Interactions	P × Phytase Linear	0.050	0.348	0.535	0.328	0.250
	P × Phytase Quadratic	0.276	0.084	0.144	0.234	0.521
	P × Inositol	0.636	0.596	0.756	0.676	0.807

Table 6.

Effect of phytase and inositol on growth performance from day 1 to 42 in the P-deficient and P-adequate diets fed to growing pigs

		IBW, kg	FBW, kg	ADG, kg/d	ADFI, kg/d	Gain:Feed
Low dietary P	Low P control	24.58	53.38	0.70	1.72	0.407
	Inositol	24.67	53.47	0.68	1.69	0.405
	500 FTU/kg phytase	24.61	56.09	0.75	1.73	0.432
	3,000 FTU/kg phytase	24.57	58.38	0.80	1.83	0.440
Adequate dietary P	Adequate P control	24.50	54.69	0.72	1.70	0.423
	Inositol	24.53	55.37	0.73	1.75	0.418
	500 FTU/kg phytase	24.54	57.17	0.78	1.73	0.450
	3,000 FTU/kg phytase	24.58	57.97	0.80	1.81	0.440
SEM		0.08	0.42	0.01	0.02	0.003
P-values
Main effects	P level	0.377	0.180	0.120	0.711	<0.001
	Inositol	0.928	0.790	0.806	0.976	0.588
Phytase effect	Linear	0.970	<0.001	<0.001	0.004	<0.001
	Quadratic	0.262	0.185	0.254	0.279	<0.001
Interactions	P × Phytase Linear	0.392	0.348	0.438	0.985	0.007
	P × Phytase Quadratic	0.340	0.084	0.112	0.218	0.302
	P × Inositol	0.309	0.596	0.728	0.835	0.645

Growth performance parameters, including IBW, final body weight (FBW), ADG, ADFI, and gain-to-feed ratio, were assessed over 2 growth periods: from day 1 to 21 and day 22 to 42. From day 1 to 21, both phytase supplementation at 500 and 3,000 FTU/kg showed quadratically increased FBW, ADG, and gain-to-feed ratio compared with the controls (P < 0.001). The gain-to-feed ratio increased with increasing dietary P level (P < 0.001). Interactions between phytase and dietary P level were observed in FBW at day 21 (P = 0.05), while a tendency towards interaction was observed between ADG and gain-to-feed ratio (P < 0.10).

From days 22 to 42, significant linear responses to phytase supplementation were observed. Increasing phytase supplementation from 500 to 3,000 FTU/kg showed linear increases in FBW, ADG, and ADFI (P < 0.001) compared with the controls, while gain-to-feed ratio showed a quadratic pattern (P < 0.05). Increasing dietary P level increased the gain-to-feed ratio (P < 0.05). No significant interaction between P level and gain-to-feed ratio was observed.

Overall, from day 1 to 42, phytase supplementation significantly improved growth performance. Increased phytase supplementation from 0 to 500 and 3,000 FTU/kg resulted in linear increases in FBW, ADG, and ADFI compared with the controls (P < 0.05). In addition, phytase supplementation increased the gain-to-feed ratio in a quadratic pattern (P < 0.05). Increases in the dietary P level increased the gain-to-feed ratio (P < 0.001). An interaction between the supplementary phytase level and dietary P level was observed in the gain-to-feed ratio from day 1 to 42 (P < 0.01), while there was a tendency for a phytase level/FBW interaction (P < 0.10). The effect of inositol supplementation alone on growth performance was not observed at either dietary P level.

Discussion

The effect on growth performance of super-dosing with phytase

Phytase supplementation is widely used in the monogastric animal industry because of its ability to improve the utilization of plant-originated P in feed (Cowieson et al., 2017). The effect of dietary phytase supplementation in swine feed and its ability to produce growth performance responses similar to the addition of inorganic P is well documented. It can reduce the quantity of total P required in animal feed as well as the amount of P lost to the environment through excretion (Adeola and Cowieson, 2011). The inclusion of phytase in swine diets provides not only environmental advantage, but also economic benefits, primarily by significantly lowering feed costs through reducing the need for more expensive inorganic phosphorus, and improving phosphorus availability.

A number of studies have reported that super-dosing phytase in the diets of weanling and nursery pigs may result in improved growth performance (Laird et al., 2018; Moran et al., 2019; Lee et al., 2021). In the current study, increasing added phytase from 500 to 3,000 FTU/kg resulted in a linear improvement in the growth performance parameters of growing pigs when the dietary P level was 0.45%. A previous study showed that the FBW, ADG, and ADFI increased linearly with rising levels of dietary phytase supplementation, regardless of whether the dietary P level was deficient or adequate (Holloway et al., 2019). Holloway et al. (2019) also reported that super-dosing with phytase improved ADG and the gain-to-feed ratio in nursery pigs, to a greater extent compared with the improvement in grow-finish pigs. These results align with the present findings, suggesting that phytase super-dosing may benefit the growth performance of pigs beyond those achieved by normal dietary phytase supplementation levels.

When dietary P was adequate, Knapp et al. (2018) reported no improvement in the growth performance of nursery pigs as a result of super-dosing phytase. In the current study, FBW improved in a linear way with increasing phytase supplementation, regardless of the dietary P level. However, an interaction between dietary P and phytase supplementation level was observed, showing a quadratic trend of improvement in gain-to-feed ratio when P was adequate, but a linear trend when the diet was P-deficient. Although the ATTDP release due to super-dosing phytase showed a linear trend with increasing phytase dose across both P levels in the current study, the improvement in growth performance by phytase supplementation showed a quadratic pattern when the dietary phytase was increased to 3,000 FTU/kg in the P-adequate diets. Because the quadratic effect on performance was observed by including 3,000 FTU/kg phytase, it is possible that the optimal inclusion rate was between 500 and 3,000 FTU/kg. Since the focus of the current study was on the interaction between super-dosing phytase with dietary P level and inositol supplementation, future studies are encouraged to investigate a titration of super-dosing phytase between 500 and 3,000 FTU/kg.

Inositol effect and super-dosing phytase

It is possible that part of the effect of super-dosing with phytase results from the extra inositol enzymatically released from phytic acid. In a previous study, Ogunribido et al. (2022) investigated the effects of supplemental myo-inositol on porcine intestinal epithelial cells (IPEC-J2) of weanling pigs. While myo-inositol supplementation did not improve to significantly improve their growth performance metrics (ADG, ADFI, gain-to-feed ratio), it improved their gut health parameters, e.g., increasing their intestinal epithelial barrier integrity (higher transepithelial electrical resistance) and reducing the permeability of their IPEC-J2 cells. In addition, myo-inositol elevated the abundance of nutrient transporter mRNA, suggesting potential beneficial effects on intestinal function and health independent of immediate improvements in growth performance. Theoretically, the elevated abundance of nutrient transporter mRNA in the small intestine might translate into greater P digestibility. However, in the current study and a previous study by Ogunribido et al. (2022), dietary supplementation of inositol did not improve the absorption and retention of P in pigs.

An interaction between dietary P level and the effect of inositol supplementation on P retention was observed in the present study. The retention of P in P-deficient diets was numerically greater than in P-adequate diets (by 2 percentage points), while the numerical difference was much smaller in P-adequate diets (0.9 percentage points). This phenomenon may require further investigation to test the potential of improving gut function using dietary inositol in P-deficient situations, as previously suggested by Ogunribido et al. (2022), that inositol may improve the gut barrier function and reduce permeability. Overall, these results indicated that the benefits of inositol on the physiological function of the intestine may have a limited impact on overall gut absorption capacity in healthy pigs. In addition, further studies may be required to investigate the impact of inositol in pigs facing health challenges.

Studies on the effect of dietary inositol supplementation on the growth performance of pigs are rare because inositol is not widely used as a feed additive. In this study, inositol supplementation had little impact on the pigs’ growth performance. Similarly, Lu et al. (2019a) reported that super-dosing phytase (3,000 FYT/kg) significantly improved growth performance in pigs, specifically increasing FBW, ADG, and gain-to-feed ratio compared with the controls. Although Lu et al. (2019a) found that plasma myo-inositol concentrations were significantly elevated by inositol and phytase supplementation, inositol supplementation alone did not show beneficial effects on performance. These results are consistent with the current study. Similar results were reported by Moran et al. (2019); super-dosing phytase and dietary inositol supplementation improved the plasma inositol concentration in nursery pigs, while the performance benefits of dietary inositol were limited to the first 10 d after weaning.

The contributions of P and nutrient digestion to the effect of super-dosing phytase

The effects of super-dosing phytase are shown by the linear and quadratic responses of increased P digestibility and bone mineralization as phytase supplementation levels increase (Adedokun et al., 2015; Kim et al., 2024). High-level phytase supplementation in the current study linearly increased P digestion and retention in growing pigs, regardless of whether the basal diet was P-deficient or P-adequate. Kim et al. (2024) reported that phytase supplementation with increasing doses up to 2,000 FTU/kg led to linearly increased ADG and quadratically improved gain-to-feed ratios in nursery pigs, similar to the results of the current study. Kim et al. (2024) also reported that the higher phytase levels tested (up to 2,000 FTU/kg) might not achieve the maximum potential P release, suggesting the potential for even greater benefits at higher rates of phytase supplementation. In Experiment 1 of the current study, when phytase supplementation was increased from 500 to 3,000 FTU/kg of diet, the observed ATTDP increased from 3.22 to 3.41 g/kg and 4.20 to 4.37 g/kg in the P-deficient and P-adequate diets compared with the controls, respectively, although the increase in ATTDP due to phytase was most pronounced from 0 to 500 FTU/kg (0.9 and 1.2 g/kg ATTDP increase in the P-adequate and P-deficient diets, respectively). Lu et al. (2019b) reported lower levels of the isomers IP4 and IP5 of IP in the small intestine of pigs when supplementary phytase was fed. Adding 2,000 FTU/kg phytase further reduced IP 4, IP 5 levels and resulted in the total disappearance of IP in the small intestine, compared with 500 FTU/kg of dietary supplementation. As a strong antinutritional factor, IP, especially IP 4, IP 5, and IP 6, can not only reduce P digestibility, but can also bond with other minerals (Adeola and Cowieson, 2011).

Elevated ATTDP by super-dosing phytase might have contributed to the further performance improvement observed in experiment 2. The ATTDP requirement of the pigs used in this study was tested in previous studies (Cao et al., 2023, 2025). Vier et al. (2019) reported that ATTDP levels greater than those recommended by the NRC (2012) might be beneficial to growth performance. Thus, the greater ATTDP levels observed could partially explain the improvement of growth performance.

The formulation of the diet in experiment 2 did not overemphasize the nutrient and energy release from phytase in the iso-caloric and nutrient diet formulations, except for the P-deficient and P-adequate diets. It is also possible that the nutrients, such as amino acids and other minerals, and energy that were released by the phytase super-dosing, may have contributed to the growth performance benefits observed in this study. Wensley et al. (2020) reported that inclusion of 1,500 FYT/kg phytase in growing-finishing pig diets improved growth performance, while the extent of this improvement varied depending on how nutrient release values were assigned to phytase. Therefore, it is necessary to evaluate the actual nutrient and energy release from phytase supplementation to avoid overestimation of released nutrients and energy.

Conclusion

In conclusion, super-dosing phytase supplementation (3,000 FTU/kg) further enhanced growth performance in pigs beyond conventional phytase usage levels in P-deficient diets. This positive effect appeared to be driven by improved P digestibility and utilization. The contribution of supplementary inositol to growth performance improvement was limited, with no significant benefit observed from supplemental dietary inositol alone. Further studies are recommended to validate nutrient release profiles and fully elucidate the benefits of phytase super-dosing under various dietary and physiological conditions.

Glossary

Abbreviations

AA

amino acids

ADFI

average daily feed intake

ADG

average daily gain

ATTD

apparent total tract digestibility

ATTDP

apparent total tract digestible phosphorus

BW

body weight

Ca

calcium

DM

dry matter

FBW

final body weight

FTU

phytase unit

IBW

initial body weight

P

phosphorus

Phytate-P

phytate-bound phosphorus

Conflict of Interest Statement

The authors declare that they have no conflict of interest.

Author Contributions

Jianfei Zhao (Methodology, Writing - original draft), Shanchuan Cao (Investigation, Methodology), Heng Yin (Conceptualization), Pengcheng Xue (Methodology), Hengxiao Zhai (Data curation, Formal analysis), Liang Chen (Conceptualization, Data curation), Hongfu Zhang (Writing - review & editing), and Jingbo Liu (Funding acquisition, Supervision, Writing - review & editing).

Data Availability

The supporting data of this study are available within the article.