A time-series effect of phytase supplementation on phosphorus utilization in growing and finishing pigs fed a low-phosphorus diet

Abstract

Two experiments were carried out to determine a time-series effect of phytase on phosphorus (P) utilization in growing and finishing pigs using growth performance, apparent total tract digestibility (ATTD) of nutrients, P excretion, and plasma concentrations of minerals as the response criteria for evaluation. In both experiments, treatments were arranged as a 3 × 4 factorial in a randomized complete block design with 3 corn–soybean meal-based diets including a P-adequate positive control (PC), a low-P negative control (NC; no inorganic P), and NC supplemented with phytase at 1,000 FYT/kg (NC + 1,000); and 4 sampling time points at days 7, 14, 21, and 28 in experiment 1, and days 14, 26, 42, and 55 in experiment 2. In both trials, 96 growing pigs with average body weight (BW) of 19.8 ± 1.16 and 49.8 ± 3.21 kg, respectively, were allocated to the 3 diets with 8 replicates pens (4 barrows and 4 gilts) and 4 pigs per pen. In experiment 1, pigs fed the PC had higher (P < 0.01) BW, average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed ratio (G:F) when compared with pigs fed the NC. There was an interaction (P < 0.01) between time and diet on the BW and ADG of pigs while a linear and quadratic increase (P < 0.01) was observed with the ADFI and G:F, respectively, over time. Phytase supplementation improved (P < 0.01) all growth performance responses. Pigs fed the PC had greater (P < 0.01) ATTD of P and Ca than pigs fed the NC. There was no interaction effect on the ATTD of nutrients. Phytase addition improved the ATTD of P and Ca over pigs fed the NC. There was an interaction (P < 0.01) between diet and time on the total and water-soluble P (WSP) excreted. There was a quadratic decrease (P < 0.01) in plasma concentration of Ca in pigs over time. In experiment 2, there was a quadratic increase (P < 0.01) in BW, ADG, and G:F of pigs over time. Similarly, the inclusion of phytase improved (P < 0.05) all growth performance parameters except ADFI. A linear increase (P < 0.05) in the ATTD of DM, P, and Ca occurred over time. Phytase inclusion improved (P < 0.01) the ATTD of P and Ca. Plasma concentrations of P were improved by phytase addition. Phytase supplementation of the NC reduced WSP excretion by 45%, 32%, and 35% over the growing, finishing, and entire grow-finish period, respectively. In conclusion, phytase improves the utilization of P in growing and finishing pigs; however, the magnitude of effect on responses may vary over time.

Introduction

Phosphorus (P) remains one of the most widely researched nutrients in monogastric nutrition due to its importance in several biochemical reactions and its debilitating effects on the environment if not properly managed. The swine industry, which is one of the fastest growing areas in the meat industry, produced over 100 million metric tons of pork products worldwide in 2020 (Shahbandeh, 2021). This was accompanied by vast amounts of waste in the form of manure that while beneficial for agronomic purposes, could contain high amounts of nutrients such as P and nitrogen with environmental consequences when improperly managed. The use of phytase in the diets of pigs is common as it has been proven to improve growth performance, nutrient utilization, and bone mineralization of pigs at different physiological stages, while reducing the amount of P lost into the environment (Harper et al., 1997; Jendza et al., 2005; Kim et al., 2017). This is because phytase can hydrolyze the phytic bonds from phytate present in most feed ingredients from the cereals and oilseeds families thus, releasing P and other nutrients, and increasing its utilization in pigs or poultry (Adedokun et al., 2015; Babatunde et al., 2019a).

The growth phase of pigs is usually differentiated into weanling, growing, and finishing phases, and each phase is peculiar when the growth curve of pigs and the utilization of nutrients are considered. The growing and finishing phases are characterized by the high consumption of feed by pigs which results in rapid growth and muscle deposition. Thus, investigating the utilization of nutrients such as P in these phases are of paramount importance. Although several studies have investigated the effects of phytase in growing and finishing pigs (Kemme at al., 1999; Kim et al., 2017; Wensley et al., 2020), there is little information on the effects of phytase on P utilization at different time points within each growth phase of pigs. It is important to have this information because it helps researchers and commercial producers pinpoint the times at which phytase is most efficacious during each growth phase. This information could also play a role in deciding when to alter phytase doses in swine diets during a particular time point in the growing or finishing phases due to the objectives of the producer or researcher. Finally, the loss of P into the environment from swine production, particularly in its soluble form, has been a cause of concern to researchers. Some studies have investigated the effects of phytase on the loss of soluble and insoluble P in the manure of pigs (Angel et al., 2005; Powers et al., 2006; Jendza et al., 2009). However, there is little or no information on how phytase may affect the form or quantity of P being lost into the environment at different time points within the growing and finishing phases of pigs.

Thus, the objective of this study was to investigate a time-series effect of phytase supplementation on P utilization in growing and finishing pigs fed a low-P diet. Two independent trials were conducted and growth performance, nutrient utilization, P excretion, and plasma concentration of minerals were the evaluation responses. We hypothesized that there will be no impact of time on the effects of phytase on growing and finishing pig responses.

Materials and Methods

All protocols used in this study were approved by the Purdue University Animal Care and Use Committee.

Experiment 1

A total of 96 growing pigs (48 barrows and 48 gilts) with an average initial body weight (BW) of 19.8 ± 1.16 kg were assigned to 3 treatments. Four replicate pens (1.7 × 3.0 m) each of barrows and gilts with 4 pigs per pen were used in a randomized complete block design. Treatments were arranged as a 3 × 4 factorial with 3 diets and 4 sampling time points. Dietary treatments consisted of a positive control (PC) with adequate supply of all nutrients including total calcium (Ca) and available P at 7.05 and 3.3 g/kg, respectively; a negative control (NC), similar to the PC but with all the inorganic P (IP) removed, hence a reduced available P of 1.5 g/kg; and an NC supplemented with phytase at 1,000 phytase units (FYT)/kg (RONOZYME HiPhos, DSM Nutritional Products, Switzerland). All diets were in mash form and formulated to meet the nutrient requirements of 25- to 50-kg growing pigs as recommended by NRC (2012) except for P and Ca in the NC diets (Table 1). Calcium to available P ratio was maintained at 2:1 in all diets. Titanium dioxide was included in all diets at 5 g/kg as an indigestible marker. Pigs were blocked by BW and sex and assigned to pens such that the average BW across all treatments were similar. Pigs had ad libitum access to water and experimental diets for 28 d with BW and feed intake (FI) recorded at 4 time points (days 7, 14, 21, and 28) to determine the average daily gain (ADG) and gain-to-feed ratio (G:F). Blood was collected via the anterior vena cava into EDTA tubes at day 0 from one median BW pig per pen, and at each time point (i.e., days 7, 14, 21, and 28) from the same pig. Plasma, used in analyzing the concentrations of P and Ca, was obtained by centrifugation of blood samples at 3,000 × g for 15 min at 4 °C (Babatunde et al., 2019a) and stored at −80 °C until further analyses. Similarly, fecal samples were collected at the same time points via rectal palpations from the same pig that had blood drawn. Fecal samples were stored in −20 ° C until further analysis and determination of the apparent total tract digestibility (ATTD) of nutrients.

Table 1.

Ingredients and nutrient composition of experimental diets fed to growing pigs (20 kg) in experiment 1 and finishing pigs (50 kg) in 2 phases of experiment 2

Item			Experiment 21,2
	Experiment 11,2		Phase 1		Phase 2
	PC	NC	PC	NC	PC	NC
Ingredients, g/kg
Corn	641.0	670.9	717.7	717.7	780.2	780.6
Soybean meal	270.0	265.0	210.0	210.0	151.0	151.0
Soybean oil	27.0	17.5	20.0	20.0	18.0	18.0
Monocalcium phosphate	9.5	—	7.2	—	6.2	—
Limestone	12.3	6.4	10.7	5.9	9.7	5.5
Salt	4.0	4.0	4.0	4.0	4.0	4.0
Solka-floc3	—	—	—	12.0	—	10.0
Vitamin premix4	1.5	1.5	1.5	1.5	1.5	1.5
Mineral premix5	0.8	0.8	0.8	0.8	0.8	0.8
DL-Methionine	1.2	1.2	0.2	0.2	0.1	0.1
L-Lysine·HCl	5.2	5.2	2.1	2.1	2.4	2.4
L-Threonine	1.7	1.7	0.3	0.3	0.5	0.5
L-Tryptophan	0.3	0.3	—	—	0.1	0.1
Selenium premix6	0.5	0.5	0.5	0.5	0.5	0.5
Titanium dioxide premix7	25.0	25.0	25.0	25.0	25.0	25.0
Total	1,000.0	1,000.0	1,000.0	1,000.0	1,000.0	1,000.0
Calculated nutrients and energy, g/kg
Crude protein	190.4	190.5	163.3	163.3	140.7	140.8
Metabolizable energy, kcal/kg	3,402.0	3,406.0	3,379.0	3,379.0	3,382.0	3,383.0
Ca	7.1	3.3	5.9	3.0	5.2	2.7
P	5.7	3.7	5.0	3.4	4.5	3.2
Standardized total tract digestibility of P	3.3	1.5	2.7	1.4	2.4	1.2
Analyzed nutrients and energy, g/kg
DM	885	890	882	890	876	879
Gross energy, kcal/kg	4,019	4,037	4,055	4,062	4,015	4,003
Crude protein	182.1	183.0	158.2	157.8	134.2	135.1
Ca	7.2	3.5	5.4	2.8	5.0	2.4
P	5.6	3.8	4.4	3.0	4.0	2.8

¹PC, positive control; NC, negative control.

²NC diets had phytase included at 1,000 phytase units (FYT)/kg as the third dietary treatment. Phytase was prepared as a premix with ground corn to contain 50 FYT per g corn and included into at diets at 20 g/kg at the expense of corn.

³International Fiber Corporation, North Tonawanda, NY.

⁴Provided the following quantities per kg of complete diet: vitamin A, 3,960 IU; vitamin D₃, 396 IU; vitamin E, 26.4 IU; menadione, 1.32 mg; riboflavin, 5.28 mg; _D-pantothenic acid, 13.2 mg; niacin, 19.8 mg; and vitamin B₁₂, 0.02 mg.

⁵Provided the following quantities per kg of complete diet: I, 0.26 mg; Mn, 12.0 mg; Cu, 6.33 mg; Fe, 136 mg; and Zn, 104 mg.

⁶Provided 0.24 mg Se/kg of complete diet.

⁷Prepared as 1 g titanium dioxide added to 4 g corn.

Experiment 2

A total of 96 finishing pigs (48 barrows and 48 gilts) with an average initial BW of 49.8 ± 3.21 kg were assigned to 3 treatments in a randomized complete block design with 8 replicate pens (split evenly between barrows and gilts) and 4 pigs per pen. Treatments were arranged as a 3 × 4 factorial with 3 diets and 4 sampling time points. Experimental diets were similar to those in experiment 1 but were fed in 2 phases as determined by the nutrient requirements of pigs (NRC, 2012). Diets in phases 1 and 2 were formulated to meet the nutrient requirements of 50 to 75 kg and 75 to 100 kg growing pigs, respectively, except for the NC diets with reduced available P at 1.37 and 1.22 g/kg, respectively (Table 1). Calcium to available P ratio was maintained at 2:1 in all diets. Titanium dioxide was included in all diets at 5 g/kg as an indigestible marker. Pigs had ad libitum access to water and experimental diets until day 26 in phase 1 and then phase 2 diets were introduced and fed until day 55. Samples were collected using the same methodology as experiment 1. Body weight and feed consumption were recorded at 4 time points (days 14, 26, 42, and 55) to determine the ADG and feed efficiency. Fecal samples, used to determine the ATTD of nutrients, was collected at the same time points from 1 pig per pen previously assigned at day 0. Blood samples, used to determine the plasma concentration of P and Ca, were collected from the same pig per pen at days 0 and at 2 time points (days 26 and 55).

Chemical analyses

Fecal samples were dried in a forced air oven at 56 ° C for 7 d. Diet and dried fecal samples were finely ground through a 0.5-mm screen in a centrifugal grinder (Retsch ZM 200; Retsch GmbH, Haan, Germany). Dry matter (DM) concentration was determined in the diets and fecal samples by drying in a force-air drying oven for 24 h at 105 °C (Precision Scientific Co., Chicago, IL; method 934.01; AOAC, 2006). Gross energy of diets samples was determined by an isoperibol bomb calorimeter using benzoic acid as the calibration standard (Parr 1261; Parr 105 Instrument Co., Moline, IL). Nitrogen concentrations in the diet samples were determined using the combustion method (TruMac N; LECO Corp., St. Joseph, MI; method 984.13A-D; AOAC, 2006), using EDTA as a calibration standard and values were multiplied by a factor of 6.25 to estimate the CP contents. Titanium concentrations in the diet and fecal samples were determined using methods previously described by Short et al. (1996). Calcium and P concentrations in diets and fecal samples were determined using methods previously described by Babatunde et al. (2021). Water-soluble P (WSP) in feces was determined as described by Jendza and Adeola, (2009). Plasma concentrations of P and Ca were determined in methods previously described by Sands et al. (2001).

Calculations and statistical analysis

The ATTD (%) of nutrients in the experimental diets were determined using the following equations (Adeola, 2001):

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{A}\mathrm{T}\mathrm{T}\mathrm{D},\mathrm{\%}=100-[\left(\mathrm{T}{\mathrm{i}}_{\mathrm{I}}/\mathrm{T}{\mathrm{i}}_{\mathrm{O}}\right)\times \left({\mathrm{N}}_{\mathrm{O}}/{\mathrm{N}}_{\mathrm{I}}\right)\times 100]}\end{array}}$$

where Ti_I and Ti_O are the concentrations of titanium (g/kg DM) in diets and feces, respectively; N_I and N_O are the concentration of nutrients (g/kg DM) in diets and feces, respectively. In experiments 1 and 2, the estimated total P excreted (g/period) in growing pigs fed the PC was calculated as follows:

$${\displaystyle \begin{array}{ll}{\displaystyle \mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{P}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{d},\mathrm{g}/\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{o}\mathrm{d}=}& \mathrm{A}\mathrm{D}\mathrm{F}{\mathrm{I}}_{\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}}\times D_{\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}}\\ & \times \mathrm{P}\mathrm{I}\times [1-\mathrm{P}\mathrm{R}\mathrm{e}{\mathrm{t}}_{\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}}]\end{array}}$$

where ADFI_time is the average daily feed intake for each time point, D_time is the number of days during each time point, PI is the analyzed P of the intake in g/kg at each phase of the current study, and PRet_time is the proportion of P retained at each time point, determined by multiplying the coefficient of the ATTD of P with a factor of 99.9%. This factor is the average retention of digested P from a P balance trial carried out in grower and finisher pigs fed experimental diets similar to the current study (Jendza and Adeola, 2009).

The estimated total P excretion (g/period) for growing pigs fed either the NC or NC + 1,000 diets was calculated using the following formula:

$${\displaystyle \begin{array}{ll}{\displaystyle \mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{P}\mathrm{e}\mathrm{x}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{d},\mathrm{g}/\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{o}\mathrm{d}=}& [(\mathrm{A}\mathrm{D}\mathrm{F}{\mathrm{I}}_{\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}}\times {\mathit{D}}_{\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}})\\ & +(\mathrm{D}\mathrm{i}\mathrm{f}{\mathrm{f}}_{\mathrm{B}\mathrm{W}}/\mathrm{G}:{\mathrm{F}}_{\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}})]\\ & \times \mathrm{P}\mathrm{I}\times [1-\mathrm{P}\mathrm{R}\mathrm{e}{\mathrm{t}}_{\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}}]\end{array}}$$

where Diff_BW is the BW of pigs fed the PC minus the BW of pigs fed either the NC or NC +1,000 diets at each time point while G:F_time is the G:F at each time point; and ADFI_time, D_time, PI, and PRet_time are as defined above. The extra complexity in the calculation of the estimated total P excreted is because the BW of pigs fed the NC, or the NC + 1,000 diets were slightly different from that of the pigs fed the PC at each time point. The modifications to the equation account for the extra days and FI required for the pigs fed the NC or phytase supplemented NC to attain parity with the BW of pigs fed the PC at each time point. Thus, it accounts for BW and FI differences among treatments. Estimates for WSP excreted by growing pigs at each time point in both experiments was determined by multiplying the total P excretion estimates described above by the percentage of total P in the WSP form.

All data were analyzed using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) as a 3 × 4 factorial with 3 diets, 4 sampling time points, and their interaction as a fixed variables except for the plasma mineral concentrations in experiment 2 which was analyzed as a 3 × 2 factorial with 3 diets and 2 sampling time points. Blocks by BW and sex served as random variables while pen was the experimental unit for all analyses. Contrasts were used to examine the effects of diets (PC vs. NC; PC vs. NC + 1,000; and NC vs. NC + 1,000) and the linear and quadratic effects of time on pig responses. Statistical significance was declared at P ≤ 0.05 and a trend was set at 0.05 < P ≤ 0.1.

Results

All pigs were healthy and readily consumed diets throughout the duration of both experiments.

Experiment 1

The average initial BW of pigs was 19.8 ± 1.16 kg. Growing pigs fed the PC had a greater (P < 0.01) BW, ADG, average daily feed intake (ADFI), and G:F when compared with pigs fed the NC (Table 2). As pigs grew from day 0 until day 28, the difference in BW of pigs fed the NC when compared with pigs fed PC or NC + 1,000 diets increased with time thus, resulting in a time × diet interaction (P < 0.01). A 17% or 22% difference was observed between the ADG of pigs fed the NC and PC, or the NC and phytase supplemented NC at day 7, respectively. In addition, a 37% or 38% difference in ADG was observed for both groups, respectively, at day 28 resulting in an interaction (P < 0.01) between time and diet. A linear increase (P < 0.01) in ADFI was observed over time with pigs consuming 0.85 kg/d at day 7 and up to 1.64 kg/d at day 28. Similarly, pigs fed the NC + phytase diets had increased (P < 0.01) ADFI when compared with pigs fed the NC diets (1.17 vs. 1.32 kg). There was a quadratic decrease (P < 0.01) in feed efficiency as pigs grew from days 0 to 28 while an overall improvement in G:F (P < 0.01) was observed when comparing pigs fed the NC and the phytase supplemented NC. There were no differences in the growth performance of pigs fed the PC and the phytase-supplemented NC diets.

Table 2.

Performance of growing pigs in response to experimental diets over time, experiment 1

Time (d)	Diet1	Final BW2, kg	ADG, kg/d	ADFI, kg/d	G:F, kg/kg	Replicates
7	PC	23.0	0.47	0.90	0.51	8
	NC	22.6	0.39	0.78	0.48	8
	NC + 1,000	23.2	0.48	0.86	0.55	8
14	PC	28.8	0.82	1.34	0.61	8
	NC	27.3	0.64	1.18	0.54	8
	NC + 1,000	29.2	0.86	1.31	0.66	8
21	PC	34.7	0.77	1.52	0.50	8
	NC	31.9	0.55	1.29	0.42	8
	NC + 1,000	35.0	0.77	1.43	0.54	8
28	PC	41.3	1.04	1.78	0.58	8
	NC	37.1	0.66	1.44	0.46	8
	NC + 1,000	41.3	0.90	1.71	0.54	8
7		22.9	0.44	0.85	0.52	24
14		28.4	0.78	1.28	0.61	24
21		33.9	0.69	1.41	0.49	24
28		39.9	0.87	1.64	0.53	24
	PC	32.0	0.77	1.39	0.55	32
	NC	29.7	0.56	1.18	0.48	32
	NC + 1,000	32.2	0.75	1.32	0.57	32
SEM3		0.46	0.03	0.04	0.02
P-values
PC vs. NC		<0.01	<0.01	<0.01	<0.01
Time × diet		<0.01	<0.01	0.26	0.39
Time linear		<0.01	<0.01	<0.01	<0.01
Time quadratic		0.36	<0.01	0.13	<0.01
PC vs. 1,000		0.83	0.69	0.10	0.57
NC vs 1,000		<0.01	<0.01	<0.01	<0.01

¹PC, positive control; NC, negative control; NC + 1,000, NC + 1,000 phytase units/kg.

²Initial BW were similar for all treatments with an average of 19.8 ± 1.16 kg.

³Standard error of mean is for the simple effects.

There was no difference in the ATTD of DM among diets fed to pigs (Table 3). There was no interaction between time and diets on the ATTD of DM; however, there was a linear increase (P < 0.01) in the ATTD of DM over time. Pigs fed the NC had lower (P < 0.01) ATTD of P and Ca as compared to pigs fed the PC; however, the supplementation of phytase improved (P < 0.01) the ATTD of P and Ca. There was no effect of time on P and Ca digestibility. The effects of diets on total P excretion increased with time thus, resulting in a time × diet interaction (P < 0.01). A similar interaction between time and diet occurred with the WSP excreted. Pigs fed phytase supplemented NC diets had a lower (P < 0.01) amount of WSP excreted per day when compared with pigs fed the PC or NC diets. Pigs fed the PC had an increased (P < 0.01) plasma concentration of P but not Ca when compared with pigs fed the NC (Figure 1). There was no interaction between time and diet on the plasma concentrations of P and Ca in pigs, but the inclusion of phytase improved (P < 0.01) the plasma concentration of P in pigs when compared with those fed the NC diets (Figure 1A). Meanwhile, a quadratic decrease (P < 0.01) was observed on the plasma concentrations of Ca over time (Figure 1B).

Table 3.

Apparent total tract digestibility (ATTD) of nutrients and relative P excretion of growing pigs fed experimental diets over time, experiment 1

Time (d)	Diet1	ATTD DM, %	ATTD P, %	ATTD Ca, %	Total P excreted2, g/period	WSP excreted3, g/period	WSP, % of total fecal P	Replicates
7	PC	84.3	54.5	60.5	16.1	12.9	80.4	8
	NC	83.8	40.5	51.3	15.0	13.2	88.5	8
	NC + 1,000	83.8	62.2	61.3	8.7	7.6	87.0	8
14	PC	84.3	55.9	67.6	23.2	18.3	78.9	8
	NC	83.5	41.6	52.7	24.4	20.6	84.4	8
	NC + 1,000	84.5	66.1	69.3	11.0	9.5	86.1	8
21	PC	84.7	58.5	66.4	24.6	19.0	76.9	8
	NC	84.0	43.2	53.2	33.2	27.3	82.4	8
	NC + 1,000	84.8	66.5	69.4	12.1	10.0	83.1	8
28	PC	85.7	54.8	66.4	32.1	23.9	74.5	8
	NC	85.6	43.5	53.3	43.6	35.1	80.5	8
	NC + 1,000	85.7	62.8	67.3	17.1	13.9	81.3	8
7		84.0	52.4	57.7	13.3	11.2	85.3	24
14		84.1	54.5	63.2	19.6	16.1	83.1	24
21		84.5	56.0	63.0	23.3	18.8	80.8	24
28		85.7	53.7	62.3	30.9	24.3	78.8	24
	PC	84.8	55.9	65.2	24.0	18.5	77.7	32
	NC	84.3	42.2	52.6	29.1	24.1	84.0	32
	NC + 1,000	84.7	64.4	66.8	12.2	10.3	84.4	32
SEM4		0.51	2.62	3.37	2.16	1.74	0.72
P-values
PC vs. NC		0.28	<0.01	<0.01	0.03	<0.01	<0.01
Time × diet		0.94	0.96	0.95	<0.01	<0.01	0.33
Time linear		<0.01	0.69	0.74	<0.01	<0.01	<0.01
Time quadratic		0.31	0.27	0.92	0.18	0.21	0.81
PC vs. 1,000		0.97	<0.01	0.85	<0.01	<0.01	<0.01
NC vs 1,000		0.39	<0.01	<0.01	<0.01	<0.01	0.68

¹PC, positive control; NC, negative control; NC + 1,000, NC + 1,000 phytase units/kg.

²Estimated total P excreted (g/period) in growing pigs fed the PC was calculated using the formula: [ADFI_time × D_time × PI × (1 − PRet_time)]; where ADFI_time is the average daily feed intake for each time point; D_time is the number of days between each time point; PI is the analyzed P (g/kg) of the intake in experiment 1; PRet_time is the proportion of P retained at each time point, determined by multiplying the coefficient of the ATTD of P with a factor of 99.9%. This factor is the average retention of digested P retention of digested P from a P balance trial conducted in grower and finisher pigs fed experimental diets similar to the current study (Jendza and Adeola, 2009). The estimated total P excretion (g/period) for growing pigs fed either the NC or NC + 1,000 diets was calculated using the formula: [(ADFI_time × D_time) + (Diff_BW/G:F_time)] × PI × [1 − PRet_time]; where Diff_BW is the BW of pigs fed the PC minus that of pigs fed either the NC or NC +1,000 diets at each time point; G:F_time is the gain-to-feed ratio at each time point, and ADFI_time, D_time, PI, and PRet_time are as defined above.

³Estimates for WSP excreted by growing pigs at each time point was determined by multiplying the total P excretion estimates by the percentage of total P in the WSP form.

⁴Standard error of mean is for the simple effects.

Figure 1.

Plasma concentration (mg/L) of minerals in growing pigs (20 kg) fed experimental diets over time (experiment 1). Panel A represents the time × diet effect on plasma P concentrations in pigs fed experimental diets for 28 d; average initial plasma concentration of P was 77.3 mg/L; Each data point represents 8 replicate pens. Panel B represents the main effect of time on plasma Ca concentrations in pigs fed experimental diets for 28 d. Average initial plasma concentration of Ca was 102.8 mg/L; Each data point represents 24 replicate pens. PC = positive control; NC = negative control; NC + 1,000 = NC + 1,000 phytase units/kg; time L and time Q = linear and quadratic effects of time; SEM is for the simple effects.

Experiment 2

The average initial BW of pigs in this trial was 49.8 ± 3.21 kg. A significant difference (P < 0.05) was observed for all growth performance parameters between pigs fed the PC and NC in experiment 2 (Table 4). A trend (P < 0.1) was observed in the interaction between time and diet on the BW and ADG of pigs, as the magnitude of differences within the BW and ADG of pigs fed the PC and NC, and the NC and NC + phytase, respectively, increased over time. However, a quadratic increase (P < 0.01) in the BW and ADG of pigs was observed over time while phytase improved (P < 0.05) both parameters in pigs fed phytase supplemented NC diets when compared with pigs fed the NC diets. There was a linear increase (P < 0.01) in the ADFI of pigs over time however, phytase inclusion did not affect the ADFI. As pigs grew from days 0 to 55, the feed efficiency was reduced quadratically (P < 0.01) by ~ 15%; however, the inclusion of phytase improved the feed efficiency of pigs by 11% when compared with pigs fed the NC.

Table 4.

Performance of finishing pigs in response to experimental diets over time, experiment 2

Time (d)	Diet1	Final BW2, kg	ADG, kg/d	ADFI, kg/d	G:F, kg/kg	Replicates
14	PC	62.9	0.93	2.40	0.39	8
	NC	60.8	0.79	2.31	0.34	8
	NC + 1,000	62.2	0.89	2.33	0.38	8
26	PC	74.7	0.98	2.65	0.37	8
	NC	71.7	0.91	2.56	0.36	8
	NC + 1,000	73.9	0.98	2.62	0.37	8
42	PC	91.3	0.87	2.91	0.30	8
	NC	86.9	0.80	2.78	0.29	8
	NC + 1,000	90.9	0.87	2.85	0.31	8
55	PC	105.5	1.13	3.35	0.34	8
	NC	97.3	0.84	3.13	0.27	8
	NC + 1,000	103.6	1.08	3.20	0.34	8
14		62.0	0.87	2.35	0.37	24
26		73.4	0.96	2.61	0.37	24
42		89.7	0.84	2.85	0.30	24
55		102.2	1.02	3.23	0.32	24
	PC	83.6	0.98	2.83	0.35	32
	NC	79.2	0.84	2.70	0.31	32
	NC + 1,000	82.6	0.95	2.75	0.35	32
SEM3		1.12	0.04	0.07	0.01
P-values
PC vs. NC		<0.01	<0.01	0.03	<0.01
Time × diet		0.06	0.07	0.97	0.13
Time linear		<0.01	0.05	<0.01	<0.01
Time quadratic		<0.01	<0.01	0.18	<0.01
PC vs. 1,000		0.66	0.62	0.25	0.97
NC vs. 1,000		0.02	<0.01	0.47	<0.01

¹PC, positive control; NC, negative control; NC + 1,000, NC + 1,000 phytase units/kg.

²Initial bodyweight were similar for all treatments with an average of 49.8 ± 3.21 kg.

³Standard error of mean is for the simple effects.

As observed in experiment 1, there was no interaction or effect of diet on the ATTD of DM however, a linear increase (P < 0.01) was observed over time. Pigs fed the NC had lower (P < 0.01) ATTD of P and Ca when compared with pigs fed the PC diets (Table 5). Although there was no interaction between time and diets on the ATTD of P and Ca, there was a linear increase (P < 0.05) in the digestibility of both minerals over time. Phytase supplementation in the NC diet also improved (P < 0.01) the ATTD of P and Ca by 135% and 46%, respectively, when compared with pigs fed the NC diets without phytase. There were interactions (P < 0.01) between time and diets on the total P and WSP excretion with changes to the effects of diets on P excreted at each time point. The ratio of water soluble to total P in the feces differed among pigs fed experimental diets with pigs fed the PC having the lowest values while pigs fed the phytase diets having the highest values; however, this ratio reduced over time resulting in an interaction effect (P < 0.01). Phytase supplementation of the NC reduced total P excretion by 49%, 41%, or 42% over the growing, finishing, and entire grow-finish period, respectively (Figure 2A). Similarly, a 45%, 32%, and 35% reduction in WSP excretion was observed between pigs fed the PC and NC + 1,000 diets at the growing, finishing, and entire grow-finish period, respectively (Figure 2B). Pigs fed the PC had a higher (P < 0.01) plasma P when compared with pigs fed the NC (Figure 3A). Although the interaction between time and diet was not significant, a trend was observed (P < 0.1) as the concentration of P in the plasma of pigs fed the PC and NC + phytase diets increased slightly over time but reduced in pigs fed the NC diets during the same period. However, there was an increase (P < 0.05) in plasma P from days 28 to 55 and phytase inclusion improved (P = 0.04) the concentration of plasma P in pigs. There was no difference in the concentrations of Ca in the plasma of pigs fed the PC and NC, nor was there an interaction between diet and time (Figure 3B). However, phytase inclusion increased (P < 0.03) the plasma concentrations of Ca over the pigs fed the PC.

Table 5.

Apparent total tract digestibility (ATTD) of nutrients and P excretion in finishing pigs fed experimental diets over time, experiment 2

Time (d)	Diet1	ATTD DM, %	ATTD P, %	ATTD Ca, %	Total P excreted2, g/period	WSP excreted3, g/period	WSP, % of total fecal P	Replicates
14	PC	84.9	36.7	50.4	94.3	63.9	67.8	8
	NC	84.5	17.8	40.1	96.1	72.6	75.5	8
	NC + 1,000	85.1	45.3	60.7	57.0	44.9	78.7	8
26	PC	85.1	37.5	53.0	87.4	57.0	65.2	8
	NC	85.1	20.2	43.6	93.1	68.1	73.2	8
	NC + 1,000	85.5	48.2	64.6	53.5	41.1	76.9	8
42	PC	86.0	38.5	55.0	112.7	72.8	64.6	8
	NC	85.6	22.5	46.8	129.1	92.8	71.8	8
	NC + 1,000	85.9	52.5	66.8	63.0	46.7	74.0	8
55	PC	86.3	41.1	57.7	101.6	64.9	63.9	8
	NC	86.0	24.3	48.6	149.8	106.1	70.9	8
	NC + 1,000	86.7	54.1	69.7	61.4	44.7	73.0	8
14		84.8	33.3	50.4	82.5	60.4	74.0	24
26		85.3	35.3	53.7	78.0	55.4	71.8	24
42		85.8	37.8	56.2	101.6	70.7	70.1	24
55		86.3	39.8	58.6	104.2	71.9	69.3	24
	PC	85.6	38.5	54.0	99.0	64.7	65.4	32
	NC	85.3	21.2	44.8	117.0	84.9	72.9	32
	NC + 1,000	85.8	50.0	65.4	58.7	44.3	75.6	32
SEM4		0.44	2.58	2.74	6.27	4.46	0.21
P-values
PC vs. NC		0.77	<0.01	<0.01	0.02	<0.01	<0.01
Time × diet		0.99	0.97	1.00	<0.01	<0.01	<0.01
Time linear		<0.01	0.04	0.03	<0.01	<0.01	<0.01
Time quadratic		1.00	0.87	0.99	<0.01	<0.01	0.01
PC vs. 1,000		0.79	<0.01	<0.01	<0.01	<0.01	<0.01
NC vs. 1,000		0.39	<0.01	<0.01	<0.01	<0.01	<0.01

¹PC, positive control; NC, negative control; NC + 1,000, NC + 1,000 phytase units/kg.

²Estimated total P excreted (g/period) in growing pigs fed the PC was calculated using the formula: [ADFI_time × D_time × PI × (1 − PRet_time)]; where ADFI_time is the average daily feed intake for each time point; D_time is the number of days between each time point; PI is the analyzed P (g/kg) of the intake in experiment 1; PRet_time is the proportion of P retained at each time point, determined by multiplying the coefficient of the ATTD of P with a factor of 99.9%. This factor is the average retention of digested P retention of digested P from a P balance trial conducted in grower and finisher pigs fed experimental diets similar to the current study (Jendza and Adeola, 2009). The estimated total P excretion (g/period) for growing pigs fed either the NC or NC + 1,000 diets was calculated using the formula: [(ADFI_time × D_time) + (Diff_BW/G:F_time)] × PI × [1 − PRet_time]; where Diff_BW is the BW of pigs fed the PC minus that of pigs fed either the NC or NC +1,000 diets at each time point; G:F_time is the gain-to-feed ratio at each time point, and ADFI_time, D_time, PI, and PRet_time are as defined above.

³Estimates for WSP excreted by growing pigs at each time point was determined by multiplying the total P excretion estimates by the percentage of total P in the WSP form.

⁴Standard error of mean is for the simple effects

Figure 2.

Sum of estimated P excretion (g/period) in growing pigs fed the PC and phytase supplemented NC (NC + 1,000) diets over each time point in experiments 1 and 2. Panel A represents the sum of estimated total P excreted by pigs over the grower phase (28 d), finisher phase (55 d), and entire grow-finish phase (83 d). Panel B represents the sum of estimated water-soluble P (WSP) excreted by pigs over the grower phase (28 d), finisher phase (55 d), and entire grow-finish phase (83 d). Each bar represents a mean of 8 observations. Estimated total P excreted (g/period) in growing pigs fed the PC was calculated using the formula: [ADFI_time × D_time × PI × (1 − PRet_time)]; where ADFI_time is the average daily feed intake for each time point; D_time is the number of days between each time point; PI is the analyzed P (g/kg) of the intake in experiment 1; PRet_time is the proportion of P retained at each time point, determined by multiplying the coefficient of the ATTD of P with a factor of 99.9%. This factor is the average retention of digested P retention of digested P from a P balance trial conducted in grower and finisher pigs fed experimental diets similar to the current study (Jendza and Adeola, 2009). The estimated total P excretion (g/period) for growing pigs fed either the NC + 1,000 diets was calculated using the formula: [(ADFI_time × D_time) + (Diff_BW/G:F_time)] × PI × [1 − PRet_time]; where Diff_BW is the BW of pigs fed the PC minus that of pigs fed either the NC +1,000 diets at each time point; G:F_time is the gain-to-feed ratio at each time point, and ADFI_time, D_time, PI, and PRet_time are as defined above. Estimates for WSP excreted by growing pigs at each time point was determined by multiplying the total P excretion estimates by the percentage of total P in the WSP form.

Figure 3.

Plasma concentration (mg/L) of minerals in growing pigs (50 kg) fed experimental diets over time. Panel A represents the time × diet effect on plasma P concentrations in pigs fed experimental diets for 55 d; average initial plasma concentration of P was 73.8 mg/L; each data point represents 8 replicate pens. Panel B represents the main effect of diet on plasma Ca concentrations in pigs fed experimental diets for 55 d. Main effect means of diet with different superscripts differ (P < 0.05). Average initial plasma concentration of Ca was 100.1 mg/L; each data point represents 32 replicate pens. PC = positive control; NC = negative control; NC + 1,000 = NC + 1,000 phytase units (FYT)/kg; SEM is for the simple effects.

Discussion

The use of phytase in monogastric nutrition is widespread due to its proven benefits in improving productivity and reducing manure P. Phytase is known to hydrolyze the phytate complex present in most cereals and oilseeds thus releasing P and other nutrients in the upper section of the gastrointestinal tract (Adeola and Cowieson, 2011). Pigs are unable to effectively hydrolyze the phytate compounds in feed ingredients due to an inadequacy of endogenous enzymes capable of breaking the phytic bonds hence, the increased use of exogenous phytase in swine diets. Although there is a lot of information on the effects of phytase on the performance and nutrient utilization of pigs (Almeida et al., 2013; Humer et al., 2015), there is little information that reveals the effects of phytase on responses of pigs at different time points within the growing and finishing phases. Even though it is common practice to include phytase in the diets of pigs until market weight, it may be important to know if there are changes in the effects of phytase on P utilization at various time points during each phase as it could help farmers decide when or how much phytase to include at a particular time for optimum productivity. It could also be that there are slight changes in the P release capacity of phytase, or in the amount of P being lost into the environment by pigs at different time points. Thus, information from this study will contribute to literature, and may provide more insight on the action of phytase on P utilization.

Growth performance is often used as an indicator of phytase effects on P utilization particularly when P deficient diets are fed to pigs (Jones et al., 2010). Pigs fed the low-P diets in the grower or finisher phases had reduced performance as compared to pigs fed P-adequate diets which indicates the importance of P bioavailability to the productivity of pigs and as observed with previous studies (Jendza et al., 2005; Brana et al., 2006; Blavi et al., 2019). There were interactions between time and diets on BW and ADG in pigs at the grower phase and tendencies for interactions in pigs at the finisher phase. It was observed that the impact of P deficiency on growth performance seemed to increase at each time point in both trials. For instance, in experiments 1 and 2, there was approximately a 17% and 15% difference between the ADG of pigs fed the PC and NC diets at days 7 and 14, respectively, while at days 28 and 55, the difference had increased to 37% and 26%, respectively. Similarly, the impact of phytase in ameliorating the effects of the P deficiency on growth performance seemed to increase at each time point in both trials. When examining the growth curve of pigs, the growing and finishing phases are characterized by rapid growth and development which requires that adequate nutrients be supplied to meet these physiological needs. A disturbance in the supply of a nutrient such as P, either due to the presence of phytate or the absence of IP, could negatively impact the growth curve of pigs even when other nutrients are in adequate supply. Hereby indicating the important role P plays in several biochemical processes necessary to sustain life (Jendza et al., 2005; Brana et al., 2006; Blavi et al., 2019). This means that as pigs grow older, the impact of P deficiency only gets worse as pigs struggle to meet their physiological needs and catch up with other pigs fed P adequate diets.

It is logical that the impact of phytase on growth performance would follow similar trends as the absence of available P since the release of P and other nutrients by phytase would have met the increasing demand of pigs as they grew older. From the current trials, the impact of P deficiency and the efficacy of phytase on growth performance were more evident during the grower phase (experiment 1) than the finisher phase (experiment 2). This suggest that age may impact the efficacy of phytase on growth performance as have been observed with previous trials in pigs (Cambra-Lopez et al., 2020) and in broiler chickens (Babatunde et al., 2019a, b). It was also evident that supplementing pig diets with phytase while completely removing IP supported the growth performance of pigs when compared with pigs fed diets with IP in both trials.

Several studies have used the ATTD of nutrients particularly P and Ca as indicators of phytase efficiency in swine (Kerr et al., 2010; Almeida et al., 2013; Wensley et al., 2020). Although there were no interactions between time and diet on the ATTD of nutrients in both experiments, there were effects of either diet or time on the ATTD of nutrients. As observed by Tsai et al. (2020), there was no effect of P status in diets or phytase supplementation on the ATTD of DM in both trials; however, the ATTD of DM increased over time in both trials. This could be indicative of the increased demand of nutrients and the efficiency in utilizing the nutrients present in diets as pigs grew older. There was an effect of diets (P deficiency and phytase supplementation) on the ATTD of P and Ca in pigs at both phases and as observed in previous trials (Olukosi et al., 2007; Arredondo et al., 2019). The presence of phytate has been known to hinder the digestibility of P, Ca, and other nutrients in pigs as they bind tightly to the phytic hexose structures thus preventing them from being hydrolyzed and utilized by pigs (Selle and Ravindran, 2008). However, the presence of phytase in diets was able to break the phytate bonds thus, increasing the digestibility of both P and Ca in pigs at both growth phases. There was no effect of time on the ATTD of P and Ca in pigs at the growing phase (experiment 1); however, there was an increase in the ATTD of both nutrients in pigs at the finishing phase (experiment 2). We speculate that the demand for P and Ca in adolescent pigs at the growing phase was very similar and being that the pigs were fed the experimental diets for only 28 d, there may have been no changes in the digestion of P and Ca from week to week. However, in the older pigs which were on the experimental diets for 55 d, the efficiency, at which nutrients such as P and Ca were digested, may have improved as pigs grew older and as observed with the ATTD of DM as previously observed (Olukosi et al., 2007) and in the current study. Although there was no interaction between time and diets, it may be possible to tap into the increased digestive potential of pigs in the finisher phase by supplying an increased dose of phytase. This may further boost the digestion of nutrients, subsequently getting pigs to market weight faster, while reducing the amount of P lost to the environment. However, further studies are required to confirm this hypothesis.

Although we did not conduct a P balance trial in the current study, we considered the amount of P lost from pigs through feces particularly in the water-soluble form as an important tool in determining phytase effects on P utilization over time. Previous P balance studies in pigs have indicated that more than 99% of absorbed P is retained (Jendza et al., 2009; Sorensen et al., 2018). Thus, the amount of P lost in the urine is minimal when compared with the feces (Sorensen et al., 2018) and since all the P in the urine will be soluble, we assume that the contribution of urinary P to the WSP excreted would also be minimal. Water-soluble P is of more importance when the impact of waste from the livestock industry on the environment is evaluated (Powers et al., 2006). It has been established that WSP contribute to the run-off of nutrients from manure-applied soils into water courses hereby causing issues such as eutrophication (Smith et al., 2004). From both trials, phytase supplementation reduced the total P and WSP excreted per period by an average of 44% and 37%, respectively, when compared with pigs fed the PC diets.

To blunt the disparity in BW of pigs fed the PC and NC diets at each time point, we accounted for the extra days and FI required by pigs fed the NC or NC + 1,000 diets to attain similar BW with pigs fed the PC. This correction resulted in meaningful assessment of the environmental cost savings of phytase when compared with feeding IP to pigs because of the adjustment for differences in growth performance and P retention. This environmental savings is broadly defined in terms of P loss that may contribute to eutrophication, the resulting damage that may have an unquantifiable cost implication, as well as interventions that will be required to repair the damage to the environment. Although pigs fed the phytase-supplemented NC diet may have required some extra feed and days to catch up with the BW of pigs fed the PC, the excretion of WSP by pigs that received the PC was 80% (18.5 vs. 10.3 g) or 46% (64.7 vs. 44.3 g) more than pigs that received the phytase-supplemented NC diet during the grower or finisher phases, respectively. The ramifications of this observation becomes more vivid when one considers that the values of 74 vs. 41 g in the grower period and 259 vs. 177 g in the finisher period were the estimated amounts of WSP excreted by a group of 4 pigs/pen fed either the PC or NC + 1,000 diets, respectively. Therefore, a group of 4 pigs raised from 20 to 100 kg and fed either the PC or NC + 1,000 diets for 83 days would excrete an estimated 333 or 218 g of WSP, respectively. When phytase completely replaces the inclusion of IP in pig diets, the amount of savings, in terms of WSP lost to the environment, becomes more apparent in commercial herds with thousands of pigs. Since WSP is of greater importance to the environment due to its role in nutrient runoff and eutrophication (Powers et al., 2006), the implication is that phytase inclusion at 1,000 FYT/kg reduces the environmental impact of pig production by ~40%. If the environmental issues, arising from the loss of nutrients such as P, from commercial swine production are to be addressed, then the complete replacement of IP with phytase should be considered commercially. From the current data, it was clear that regardless of the age of pigs or the form of P, the amount of P lost increased over time, however this could be related to the increase in FI over time. In agreement with Powers et al. (2006), we also observed that the WSP as a percentage of total fecal P was higher in pigs fed the phytase supplemented diets as compared with those fed the PC. This may be due to the action of microbes in the feces or the presence of undigested IP and unhydrolyzed phytate in the feces of pigs fed the NC or PC (Angel et al., 2005; Jendza et al., 2009). However, this did not reduce the impact of phytase on WSP loss as the quantity of P in terms of mass (g) lost from the pigs fed the PC was significantly higher than pigs fed the NC + 1,000 diet. With information from these studies, it is possible to project how much P is lost from pigs per time during the grow-finish phase hereby supporting the use of time-sensitive interventions that could reduce nutrient losses and protect the environment.

The P and Ca status of animals can usually be evaluated by measuring their concentrations in the blood (Oster et al., 2016). Regardless of the absence of interactions on plasma P concentrations in the younger pigs (experiment 1), the P status of the diets influenced the plasma concentration of P with pigs fed the NC having lower levels of P in the plasma when compared with pigs fed either the PC or the NC + phytase. Similar observations have been made with previous studies where P deficiency led to low concentrations of P in the blood; however, the inclusion of phytase mitigates these effects (Adeola et al., 2004; Jendza et al., 2005; Madrid et al., 2013). Given that plasma P concentrations were similar in pigs fed the PC and phytase supplemented NC, the almost 40% difference in WSP between both groups of pigs suggests that pigs fed the phytase supplemented NC diets had greater tissue retention of P or increased deposition of P in the bones. There was an effect of time on plasma Ca concentration in experiment 1 but not in experiment 2, which could be due to age or physiological state of pigs. Concentrations of circulating Ca decreased over time in the younger pigs and may have been due to the rapid deposition of minerals on the bones as pigs grew exponentially during the growing phase or the reduced requirements of minerals as pigs grow older (Xu et al., 2002). However, in the finisher phase, pigs are at a mature stage, and the mechanism for regulating Ca should be well developed such that it is not necessarily impacted by time. Furthermore, blood was sampled at 5 time points in experiment 1, but at 3 time points in experiment 2. In agreement with Madrid et al. (2013), there was no effect of diets on the plasma Ca levels in the pigs for experiment 1. Pigs fed the phytase-supplemented diets had the highest concentrations of Ca in the blood when compared with pigs fed the PC in experiment 2. Calcium is more tightly regulated than P in the body (Votterl et al., 2021) and may explain why there was no effects of diets on plasma Ca levels in younger pigs (experiment 1) despite the time effects observed. However, pigs fed the NC + phytase in experiment 2 absorbed ~21% more Ca than pigs fed the PC, which may explain the difference in circulating Ca levels regardless of the sampling time.

In conclusion, the positive effects of phytase on the responses of growing and finishing pigs fed diets without IP are irrefutable. However, the magnitude of these phytase effects on responses vary over time. Therefore, time-sensitive application of phytase may be carried out with pigs depending on the aim of the farmer or researcher. Similarly, the cost saving effects of phytase to the environment are established when the amount of WSP lost from commercial swine production is considered.

Glossary

Abbreviations

ADG

average daily gain

ADFI

average daily feed intake

ADFI_time

average daily feed intake at each time point

ATTD

apparent total tract digestibility

BW

body weight

DM

dry matter

FI

feed intake

FYT

phytase units

G:F

gain-to-feed ratio

G:F_time

gain-to-feed ratio at each time point

IP

inorganic phosphorus

Conflicts of Interest Statement

The authors declare no real or perceived conflicts of interest.