Extra-phosphoric effects of super dosing phytase on growth performance of pigs is not solely due to release of myo-inositol

Abstract

Two experiments were conducted to determine the effects of myo-inositol and phytase on growth performance, plasma metabolites, and nutrient digestibility of growing pigs. In experiment 1, 96 growing pigs with average initial body weight (BW) of 26.2 kg were used in a 25-d growth performance study. Pigs were assigned to four dietary treatments with three pigs per pen and eight replicate pens per treatment in a randomized complete block design. The four treatments were control diet (CD); CD + 2 g/kg inositol; CD + 1,000 FYT/kg phytase and CD + 3,000 FYT/kg phytase. Pigs were weighed individually every week. On day 25, blood sample was collected from one pig per pen to measure plasma metabolites concentrations. In experiment 2, 16 barrows (initial BW 34.8 ± 8.2 kg) were surgically fitted with T-cannulas. Pigs were allotted to four blocks based on BW and assigned to a quadruplicate 4 × 2 incomplete Latin square design with same four dietary treatments and two periods. Ileal digesta samples were collected from each pig on days 6 and 7 of each period to determine apparent ileal digestibility (AID) of nutrients. Phytase supplementation increased final BW and average daily gain (ADG) compared with CD (P < 0.05) with no effects on average daily feed intake (ADFI) and gain to feed (G:F) was higher in 3,000 FYT/kg phytase (P < 0.05). Inositol supplementation had no effects on growth performance. Plasma myo-inositol concentration was increased by inositol supplementation, and 3,000 FYT/kg phytase increased myo-inositol in the plasma by 97.2% (P < 0.05). Plasma P concentration was increased by 1,000 or 3,000 FYT/kg phytase with no effects on alkaline phosphatase (ALP), glucose, triglycerides (TAG), calcium (Ca), and urea concentrations. Phytase supplementation reduced (P < 0.05) the phytate-P concentration in the ileal digesta and increased the digestibility of phytate-P and total P with no effects on the AID of dry matter (DM), gross energy (GE), nitrogen (N), and Ca. In conclusion, the beneficial effects of 3,000 FYT/kg phytase on feed efficiency may due to the increased release of both myo-inositol and phosphorus (P), and may not be solely due to myo-inositol release by this level of phytase.

INTRODUCTION

Phosphorus (P) is the second most abundant mineral in animal body, and it is important in animal physiology and cellular functions, including as a component of adenosine triphosphate (ATP), bone structure, in cell replication and pH and ionic buffering. The P present in the cereal grains are mainly bound to phytic acid (Lott et al., 2000). In addition, phytic acid binds to other cations (Ca²⁺, Zn²⁺) to form the phytate complex. Low phytase activity and poor solubility of phytate contribute to limited utilization of P and other bound minerals in the gastrointestinal tracts (GIT) of monogastric animals, thus necessitating the use of exogenous phytases.

The effects of exogenous phytase addition to swine diets to increase nutrient digestibility and improve the growth performance has been fairly well studied (Braña et al., 2006; Adedokun et al., 2015). Most recent focus has been on the use of high doses of phytase (>1,500 FYT/kg) to achieve increased growth performance beyond that which may be solely due to the performance benefits from the extra P released by phytase. Guggenbuhl et al. (2016) reported a greater average daily gain (ADG) with the addition of 3,000 FYT/ kg phytase to a P deficient diet compared with a P-adequate diet positive control diet (CD). This potentially indicated extra-phosphoric effects and benefits of high dietary level of phytase supplementation.

It has been speculated that the extra-phosphoric effects of phytase might be related to release of myo-inositol and to complete (and rapid) destruction of anti-nutritive inositol phosphate esters (Walk et al., 2013). Myo-inositol plays an important role in cellular processes and cell function as phospholipids or inositol phosphates (Croze and Soulage, 2013). Several studies have reported that myo-inositol supplementation could improve the growth performance of chickens (Zyła et al., 2004; Cowieson et al., 2013; Sommerfeld et al., 2018) and phytase supplementation increased plasma myo-inositol concentration in pigs (Cowieson et al., 2017). The assumption that myo-inositol is a major mediator of the extra-phosphoric effect of phytase in pigs needs to be verified. In addition, the effects of myo-inositol on nutrient digestibility in pigs is currently unknown. Therefore, the objectives of current study were to determine the effects of myo-inositol on growth performance and nutrient digestibility of pigs, and to determine whether the extra-phosphoric effects of phytase in pigs is related to the release of myo-inositol.

MATERIALS AND METHODS

All animal procedures were approved by the Purdue University Animal Care and Use Committee. All the pigs used in this study were obtained from Purdue University Swine Research Unit.

Experiment 1: Growth Performance

A total of 96 pigs (48 barrows and 48 gilts) with an average initial body weight (BW) of 26 kg were used in a 25-d growth performance study. Pigs were randomly assigned to four treatments with three pigs per pen and four replicate pens of barrows and four replicate pens of gilts in a randomized complete block design. Pigs were housed in floor pens (2.4 m × 1.8 m) with a feeder and nipper drinker. The pens were located in an environmentally regulated building maintained at 23 ± 2 °C with a 12-h light (0700 to 1900) cycle. The four treatments used in this study were: a control diet (CD); CD + 2 g/kg myo-inositol; CD + 1,000 FYT/kg phytase, and CD + 3,000 FYT/kg phytase. The CD (Table 1) was formulated to meet the total P and other nutrient requirements set by the NRC (NRC, 2012), where more than 80% of total P was from organic soucres. Pigs had ad libitum access to water and feed during the whole experimental period and were weighed individually weekly. Feed intake was recorded every week. On day 25, one pig close to the average pen weight was selected and nonfasting blood samples were drawn from the jugular vein into heparinized tubes. After collection, blood tubes were immediately put on ice. Blood samples were then centrifuged at 2,000 × g at 4 °C for 10 min to separate the plasma and stored at −80 °C until further analysis.

Table 1.

Ingredient composition of experimental diets for experiment 1 (as-fed basis)

	Dietary treatment
Ingredient, g/kg	CD	CD + inositol	CD + 1,000 phytase	CD + 3,000 phytase
Corn	440.45	420.45	430.45	410.45
Canola meal	200	200	200	200
Rice bran*	200	200	200	200
Soybean meal	100	100	100	100
Limestone	14.2	14.2	14.2	14.2
Monocalcium phosphate	5	5	5	5
Salt	3.5	3.5	3.5	3.5
Soybean oil	26.5	26.5	26.5	26.5
Lysine—HCl	5.2	5.2	5.2	5.2
DL-methionine	0.5	0.5	0.5	0.5
l-threonine	1.3	1.3	1.3	1.3
l-tryptophan	0.45	0.45	0.45	0.45
Vitamin premix†	1.5	1.5	1.5	1.5
Mineral premix‡	0.9	0.9	0.9	0.9
Selenium premix||	0.5	0.5	0.5	0.5
Inositol premix$	0	20	0	0
Phytase premix¶	0	0	10	30
Total	1,000	1,000	1,000	1,000
Analyzed nutrient composition
GE, kcal/kg	4,308	4,305	4,312	4,310
CP, g/kg	188	190	192	192
P, g/kg	8.6	8.5	8.7	8.7
Ca, g/kg	12.1	11.8	12.5	12.3
Inositol, g/kg	0.3	2.3	0.3	0.3
Total phytate-P (g/kg DM)&	6.4	6.5	6.2	5.8
Phytase, FYT/kg	94	91	1,243	2,968
Calculated standardized ileal digestibile AA composition, g/kg
Arg	9.8	9.8	9.8	9.8
His	4.0	4.0	4.0	4.0
Ile	6.2	6.2	6.2	6.2
Leu	11.4	11.4	11.4	11.4
Lys	10.8	10.8	10.8	10.8
Met	3.2	3.2	3.2	3.2
Cys	2.9	2.9	2.9	2.9
Phe	7.0	7.0	7.0	7.0
Tyr	4.1	4.1	4.1	4.1
Thr	6.3	6.3	6.3	6.3
Trp	2.0	2.0	2.0	2.0
Valine	6.8	6.8	6.8	6.8

*Purchased from Provimi, OH. Analyzed nutrient composition: DM (88.6%), CP (172.5 g/kg), Ca (2.8 g/kg), and P (20.6 g/kg).

^†Supplied the following per kg of diet: vitamin A, 1,089 μg; vitamin D3, 9·1 μg; vitamin E, 23·7 mg; menadione,1·32 mg; riboflavin, 5·28 mg; d-pantothenic acid, 13·1 mg; niacin, 19·8 mg; choline chloride, 771 mg; vitamin B12, 23·1 μg.

^‡Supplied the following per kg of diet: iodine, 0·46 mg; Mn, 15 mg; Cu, 11·3 mg; Fe, 121·3 mg; Zn, 121·2 mg.

^||Selenium premix provided 0·3 mg Se/kg diet.

^$Inositol premix made to 0·1 g/kg which when added at 20 g/kg provided 2 g inositol/kg diet.

^¶Phytase premix (RONOZYME HiPhos; DSM Nutritional Products) made to 0·01 g/kg which when added at 10 or 30 g/kg provided 0·1 g/kg or 0·3 g/kg diet (1,000 or 3,000 FYT/kg, respectively).

^&Phytate bound P.

Plasma Metabolites Measurement

Plasma samples were sent to a DSM laboratory (Center for Animal Nutrition and Health, Village Neuf, Saint Louis, France) to determine the concentrations of alkaline phosphatase (ALP), glucose, triglycerides (TAG), calcium (Ca), P, urea, and myo-inositol. Plasma concentrations of P, Ca, ALP, glucose, TAG, and urea were determined in duplicates with an automated analyzer (Cobas 6000, Roche Diagnostics). To determine plasma myo-inositol concentrations, samples were prepared in duplicates as described by Frieler et al. (2009). Plasma samples were extracted in chloroform/methanol and the upper phase was collected with after two centrifugations. Then, then the extracts were analyzed by HPLC/MS according to the method of Leung et al. (2011).

Experiment 2: Nutrient Digestibility

Sixteen barrows (initial BW 34.8 ± 8.2 kg), surgically fitted with T-cannulas at the distal ileum as described by Dilger et al. (2004) were used. Pigs were allotted to four blocks based on BW and assigned to a quadruplicate 4 × 2 incomplete Latin square design with four dietary treatments and two periods. The four diets were similar to those used in experiment 1 (Table 2). No inorganic source of P was added to the diets. Therefore, the digestible P content was low in this experiment and amount of limestone was also reduced to maintain the Ca:tP. Acid insoluble ash (AIA, Celite) was added at 2% as indigestible marker. Pigs were housed individually in metabolism crates equipped with a feeder and a nipple drinker with ad libitum access to water. During the experimental periods, pigs received their assigned diet at 4% of BW of the lightest pig in each block daily in two equal meals at 0700 and 1700 h to ensure complete ingestion of feed in all the pigs. Each experimental period consisted of 5 d of adaptation and 2 d of ileal digesta collection. On days 6 and 7 of the experimental period, plastic sample bags (Whirl-Pak bag; NASCO, Fort Atkinson, WI) containing 10 mL of 10% formic acid were attached to the T-cannulas from 0730 to 1700 h. Attached bags were inspected every 30 min and changed whenever they were full. Collected ileal digesta samples were immediately stored at −20 °C. After each experimental period, ileal digesta samples were slightly thawed and pooled within pigs and subsampled for freeze-drying.

Table 2.

Ingredient composition of experimental diets of experiment 2 (as-fed basis)

	Dietary treatment
Ingredient, g/kg	CD	CD + inositol	CD + 1,000 phytase	CD + 3,000 phytase
Corn	431.05	411.05	421.05	401.05
Canola meal	200	200	200	200
Rice bran*	200	200	200	200
Soybean meal	100	100	100	100
Limestone	12.8	12.8	12.8	12.8
Salt	3	3	3	3
Soybean oil	23	23	23	23
Lysine—HCl	5.2	5.2	5.2	5.2
DL-methionine	0.4	0.4	0.4	0.4
l-threonine	1.2	1.2	1.2	1.2
l-tryptophan	0.45	0.45	0.45	0.45
Vitamin premix†	1.5	1.5	1.5	1.5
Mineral premix‡	0.9	0.9	0.9	0.9
Selenium premix||	0.5	0.5	0.5	0.5
Celite	20	20	20	20
Inositol premix$	0	20	0	0
Phytase premix¶	0	0	10	30
Total	1,000	1,000	1,000	1,000
Analyzed nutrient composition
GE, kcal/kg	4,205	4,212	4,125	4,212
CP, g/kg	181	181	185	183
P, g/kg	7.1	7.3	7.3	7.5
Ca, g/kg	12.0	11.4	12.2	12.9
Inositol, g/kg	0.3	2.2	0.3	0.3
Total phytate-P (g/kg DM)&	6.5	6.5	6.6	6.7
Phytase, FYT/kg	74	83	1094	3142
Calculated standardized ileal digestibile AA composition, g/kg
Arg	9.8	9.8	9.8	9.8
His	4.0	4.0	4.0	4.0
Ile	6.2	6.2	6.2	6.2
Leu	11.3	11.3	11.3	11.3
Lys	10.8	10.8	10.8	10.8
Met	3.1	3.1	3.1	3.1
Cys	2.8	2.8	2.8	2.8
Phe	6.9	6.9	6.9	6.9
Tyr	4.1	4.1	4.1	4.1
Thr	6.9	6.9	6.9	6.9
Trp	2.0	2.0	2.0	2.0
Valine	6.7	6.7	6.7	6.7

*Purchased from Provimi, OH. Analyzed nutrient composition: DM (88.6%), CP (172.5 g/kg), Ca (2.8 g/kg), and P (20.6 g/kg).

^†Supplied the following per kg of diet: vitamin A, 1,089 μg; vitamin D3, 9·1 μg; vitamin E, 23·7 mg; menadione,1·32 mg; riboflavin, 5·28 mg; d-pantothenic acid, 13·1 mg; niacin, 19·8 mg; choline chloride, 771 mg; vitamin B12, 23·1 μg.

^‡Supplied the following per kg of diet: iodine, 0·46 mg; Mn, 15 mg; Cu, 11·3 mg; Fe, 121·3 mg; Zn, 121·2 mg.

^||Selenium premix provided 0·3 mg Se/kg diet.

^$Inositol premix made to 0·1 g/kg which when added at 20 g/kg provided 2 g inositol/ kg diet.

^¶Phytase premix (RONOZYME HiPhos; DSM Nutritional Products) made to 0·01 g/kg which when added at 10 or 30 g/kg provided 0·1 g/kg or 0·3 g/kg diet (1,000 or 3,000 FYT/kg, respectively).

^&Phytate bound P.

Chemical Analyses

Diets and ileal digesta samples were ground to pass through a 0.5-mm screen before analysis. Ground samples were dried at 105 °C in a drying oven (Precision Scientific Co., Chicago, IL) for 24 h to determine the dry matter (DM) content (method 934.01; AOAC, 2006). Gross energy (GE) was determined in a bomb calorimeter (Parr 1261 bomb calorimeter, Parr Instruments Co., Moline, IL). To determine P and Ca concentration, diets and ileal digesta samples were ashed in a muffle furnace at 600°C for 16 h. The ashed samples were digested in 20 mL 4N HCl and 5 drops of concentrated nitric acid for about 7 min on a hot plate, and the digested samples were moved into 250 mL volumetric flasks. P concentrations were measured by spectrophotometric reading of absorbance at 620 nm using the method described by Zhai and Adeola (2013). Concentration of Ca in the supernatant was determined using flame atomic absorption spectrometry (Varian FS240 AA Varian, Inc., Palo Alto, CA). Feed samples were determined for nitrogen (N) content by the combustion method (TruMac N analyzer; Leco Corp., St. Joseph, MI), Acid-insoluble ash content was determined using AOCS Official Method Ba 5b-68 (AOCS, 2000). Freeze dried diets and ileal digesta samples were also sent to DSM laboratory to determine the phytate-P concentration according to the method of Skoglund et al. (1997) with some modifications. Briefly 0.5 g of ground diet and ileal digesta samples were extracted with 10 mL 0.5 M HCl for 2 h at 40 °C under agitation. The extracts were then frozen overnight, thawed, and extracted again under agitation for 2 h at 40 °C and centrifuged. Inositol phosphates were separated from the crude extract by filtration through Amicon Ultra 30 K filter (Millipore, Molsheim, France) after centrifugation for 1 h at 10 °C. Samples (150 μL) were transferred to chromatographic vials for phytate-P determination using HPLC (Aureli et al., 2017).

Calculations and Statistical Analysis

The apparent ileal digestibility (AID) of GE and nutrient were calculated using the following equations described by Adeola (2001):

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{A}\mathrm{I}\mathrm{D},\mathrm{\%}=100\times [1-\left(\frac{\mathrm{A}\mathrm{I}{\mathrm{A}}_i}{\mathrm{A}\mathrm{I}{\mathrm{A}}_o}\right)\times \left(\frac{Y_o}{Y_i}\right)],}\end{array}}$$

in which AIA_i and AIA_o represent the concentration of AIA (g/kg DM) in diets and ileal digesta, respectively; and Y_i and Y_o represent the concentration of GE or nutrients (g/kg DM) in diets and ileal digesta, respectively.

All the data were analyzed using GLM procedure of SAS (SAS, 2006, SAS Institute, Inc., Cary, NC). For experiment 1, pen was the experimental unit, the model included replicate, sex, diet, sex × diet interaction, and replicate within sex as independent variable. Because sex had no effect on growth performance and plasma metabolite concentrations, the data presented in Tables 3 and 4 reflect pooled data for barrows and gilts. For experiment 2, individual pig was the experimental unit, and the model included period, diet, period × diet interaction, and block within period as independent variable. Tukey’s multiple comparison was used to separate the means if diet effect was significant, and statistical significance was declared at P < 0.05.

Table 3.

Effects of myo-inositol and phytase on growth performance of pigs in experiment 1

	Dietary treatment1
Item	CD	CD + inositol	CD + 1,000 phytase	CD + 3,000 phytase	SEM	P-value
Initial BW, kg	26.3	26.2	26.2	26.2	0.2	0.99
Final BW, kg	44.3b	44.6ab	46.5a	46.5a	0.5	0.01
ADG, g	723b	736ab	812a	814a	19.8	0.004
ADFI, g	1,704	1,762	1,797	1,757	42.9	0.51
G:F, g/kg	424bc	418c	452ab	464a	7.9	0.001

¹Data are means of eight replicate pens per treatment, means with different superscript in same row are different (P < 0.05). CD, control diet.

Table 4.

Effects of myo-inositol and phytase on plasma metabolites of pigs in experiment 1

	Dietary treatment1
Item	CD	CD + inositol	CD + 1,000 phytase	CD + 3,000 phytase	SEM	P-value
ALP, U/L	157	165	168	148	17.0	0.83
Glucose, mg/dL	106	113	108	104	3.5	0.34
TAG, mg/dL	35	39	42	49	8.2	0.67
Ca, mg/dL	11.0	11.1	10.5	10.6	0.2	0.08
P, mg/dL	7.1c	7.2c	8.5a	8.1ab	0.3	0.002
Urea, mg/dL	21.9	16.2	18.4	21.0	1.5	0.06
myo-inositol, µmol/L	35.6c	86.3a	48.7bc	70.2a	5.7	<0.001

¹Data are means of eight pigs per treatment, means with different superscript in same row are different (P < 0.05). CD, control diet; ALP, alkaline phosphates; TAG, triacylglycerol.

RESULTS

Phytase Activity

The analyzed phytase activity in the feed for both experiment were similar to the expected values. In experiment 1, the analyzed phytase activities in the CD and myo-inositol supplemented diets were 94 and 91 FYT/kg, respectively. Analyzed activities were 1,243 and 2,968 FYT/kg for the 1,000 and 3,000 FYT diets, respectively. In experiment 2, phytase activities were 74, 83, 1,094, and 3,142 FYT/kg for the CD, myo-inositol, 1,000 and 3,000 FYT diets, respectively.

Experiment 1: Growth Performance

The growth performance and plasma metabolites response of myo-inositol and phytase are presented in Tables 3 and 4. Phytase supplementation increased final BW (P < 0.05) compared with CD and the myo-inositol supplemented diet. However, myo-inositol had no effect on final BW. Overall ADG was increased by phytase supplementation compared with control (812 and 814 g/d for 1,000 and 3,000 FYT phytase, respectively vs. 723 g/d P < 0.05). Diet had no effect on average daily feed intake (ADFI), whereas gain to feed (G:F) was increased by 3,000 FYT/kg phytase compared with CD. The 1,000 and 3,000 FYT/kg phytase diets increased G:F compared with the inositol diet (P < 0.05). However, myo-inositol had no effect on ADG, ADFI and G:F. Phytase supplementation increased plasma P concentration on d 25 (P < 0.05). As expected, myo-inositol supplementation increased plasma myo-inositol concentration. In addition, 3,000 FYT/kg phytase increased plasma myo-inositol concentration compared with CD, but with no difference compared with the inositol supplemented diet. Diet had no effect on plasma concentrations of ALP, glucose, TAG, Ca, and urea.

Experiment 2: Nutrient Digestibility

Table 5 shows the AID digestibility of nutrients in the cannulated pigs. The AID of DM, GE, N, and Ca were not affected by phytase or inositol supplementation. The phytate-P concentrations in the diets were similar (0.67–0.71%, Table 6). Phytase supplementation significantly reduced phytate-P concentration in the ileal digesta (0.97 and 0.75% for 1,000 and 3,000 FYT/kg phytase, respectively vs. 1.51% for CD; Table 6, P < 0.05). Similarly, phytase significantly increased phytate-P digestibility (Table 6, P < 0.05). The AID of total P was also increased by phytase supplementation (33.8% and 36.1% for 1,000 and 3,000 FYT/kg phytase, respectively, vs. 18.6% for CD P < 0..05), whereas inositol supplementation had no effect on AID of P.

Table 5.

Effects of myo-inositol and phytase on AID of nutrients of pigs in experiment 2

	Dietary treatment1
Item	CD	CD + inositol	CD + 1,000 phytase	CD + 3,000 phytase	SEM	P-value
DM, %	58.5	57.3	58.7	57.9	0.92	0.72
GE, %	64.7	64.0	63.2	62.0	0.88	0.19
N, %	62.8	63.7	65.2	65.1	1.02	0.32
Ca, %	22.4	23.5	25.9	31.2	2.84	0.16

¹Data are means of eight pigs per treatment. CD, control diet.

Table 6.

Effects of myo-inositol and phytase on ileal phytate-P concentration and digestibility of pigs in experiment 2

	Dietary treatment1
Item	CD	CD + inositol	CD + 1,000 phytase	CD + 3,000 phytase	SEM	P-value
Dietary phytate-P, %	0.67	0.69	0.69	0.71	—	—
Ileal phytate-P concentration, %	1.51a	1.45a	0.97b	0.75c	0.04	<0.001
AID of phytate-P, %	6.1c	8.0c	40.1b	55.7a	1.95	<0.001
AID of total P, %	18.6b	24.0b	33.8a	36.1a	2.29	<0.001

¹Data are means of eight pigs per treatment, means with different superscript in same row are different (P < 0.05). CD, control diet.

DISCUSSION

Phytase is typically added to swine diets at 500 to 1,000 FYT/kg feed to release 0.3–1.7 g/kg available P (Augspurger et al., 2003; Wilcock and Walk, 2016), and phytase concentrations of >1,500–2,000 FYT/kg are considered super dosing. The effects of super dosing phytase on growth performance in pigs have been reported elsewhere (Kies et al., 2006; Nyannor et al. 2007; Santos et al., 2014; Zeng et al., 2016; Laird et al., 2018). Studies done by Kies et al. (2006) and Zeng et al. (2014) showed that phytase at 20,000 FYT/kg diet increased ADG and feed efficiency of weanling pigs. In addition, a linear effect of phytase on ADG and feed efficiency was observed by Nyannor et al. (2007) in growing pigs receiving phytase at 16,500, 33,000, and 49,500 FYT/kg. Similar results were observed in the current study where phytase at 3,000 FYT/kg increased feed efficiency but not at 1,000 FYT/kg. Thus, extra-phosphoric effects of phytase could be obtained, especially at higher levels of phytase supplementation. One potential underlying reason for the extra-phosphoric effect of phytase is the release of myo-inositol through complete hydrolysis of the phosphate groups on the phytate. Myo-inositol may have insulin mimetic effect in stimulation of glucose uptake into tissues. Insulin stimulates glucose uptake by increasing the translocation of intracellular glucose transporter type 4 vesicles into the plasma membrane of myocytes (Kahh, 1996), and it has been reported that glucose transport is rate limiting for muscle glycogen synthesis (Richter and Hargreaves, 2013). However, supplementation of myo-inositol at 2 g/kg in the current study did not affect growth performance. In contrast, the work done by Sommerfeld et al. (2018) reported a positive effects of myo-inositol on growth performance in broiler chickens when supplemented to a nutrient sufficient diet. The reason for the different response in the current study, besides species differences, could be the source of P. More than 80% of the total P in the current study was from organic sources, with phytate-P content in the diets ranging from 66.7% to 76.5% to the total P content. Therefore, the digestible P content would be low, although the total P content is adequate and meets the nutrient requirement. In contrast, inorganic P contributed more than 50% of the total P in the work done by Sommerfeld et al. (2018). Therefore, it is possible that myo-inositol itself may have limited effect on growth performance when digestible P is insufficient in pigs. The inclusion of high phytate-P content in the current study is not typical of commercial swine diets. Therefore, it will be interesting to determine how pigs will respond in the presence of added myo-inositol and super dosed phytase in typical swine diets with low phytate (corn-DDGS, animal byproducts, etc.) and high digestibile P content.

As a metabolic precursor for PIP3, myo-inositol may have distal intracellular insulin-like effects. By enhancing the concentration of PIP3 in the cell, myo-inositol may increase insulin sensitivity. In addition, myo-inositol may also promote insulin secretion from pancreatic β cells. In mammalian cells, pyrophosphates (IP₇) are synthesized from myo-inositol through a series of chemical reactions involving many enzymes such as inositol hexakisphosphate (IP₆) kinases, diphosphoinositol pentakisphosphate (PP-IP₅) kinases (Wilson et al., 2013). It has been reported that overexpression of IP₆ kinases in pancreatic β cells increased production of IP₇, and this stimulated exocytosis of insulin in a dose-dependent manner (Illies et al., 2007). In agreement with this finding, Bhandari et al. (2008) found a significant reduction of plasma insulin in IP₆ kinase knock-out mice. A decrease in extracellular phosphate has been linked to a decrease in IP₇ (Lonetti et al., 2011) and restoring the phosphates concentration in the medium resulted in a rapid increase in IP₇ levels in Saccharomyces cerevisiae (Losito et al., 2009). As expected, in the current study, dietary myo-inositol supplementation increased plasma myo-inositol concentration, but with no effect on plasma P concentration. With the lack of effect of myo-inositol on growth performance, it is possible that the mostly plant-based (more than 80%) digestible P concentration in these diets was not high enough to support the synthesis of IP₇, therefore limiting the insulin-like effects of myo-inositol in this study. Furthermore, although phytase at 1,000 FYT/kg increased plasma P concentration, it did not increase plasma myo-inositol concentration, thus potentially limiting the insulin-like effects at the level of supplementation. However, phytase at 3,000 FYT/kg increased both plasma P and myo-inositol concentration. Taken together, the increase in feed efficiency by phytase at 3,000 FYT/kg may not only be due to the release of P or myo-inositol, but a combination effects of both P and myo-inositol release. However, more researches are needed to test this hypothesis by enough inorganic source of P and myo-inositol.

The effects of myo-inositol and phytase on nutrient digestibility were also tested in the current study. To our knowledge, there is no report of effects of myo-inositol on nutrient digestibility in pigs. Our results show that myo-inositol had no effects on AID of nutrients. Phytase is thought to improve nutrient digestibility by increasing the solubility of nutrients in the small intestine through mediating a reduction in the chelating capacity of IP esters. However, phytase had no effect on AID of DM, GE, N, and Ca in the current study. These results are in agreement with previous reports (Traylor et al., 2001; Zeng et al., 2016; She et al., 2018). However, others reported a positive effects of phytase on GE, and AA digestibility (Adedokun et al., 2015; Velayudhan et al., 2015; Kiarie et al., 2016). The different nutrient digestibility response to phytase may be due to the different origins of phytase, different dietary phytate (and other nutrient) concentrations and the different Ca:tP in the different experiments. However, in agreement with previous studies (Veum et al., 2006; Kerr et al., 2010; Adedokun et al., 2015; She et al., 2018), phytase supplied at either 1,000 or 3,000 FYT/kg significantly increased AID of total P. The P source in experiment 2 was all from organic materials, with phytate-P content ranging from 89% to 91.5% in the diets (Table 2). The AID of phytate-P was increased by phytase (40.1% and 55.7% for 1,000 and 3,000 FTY phytase compared with 6.1% in the CD) (Table 6). This indicates that the phytase is effective in hydrolyzing the phytate to release P.

In summary, myo-inositol supplementation by itself to a nutrient-adequate diet had no effect on growth performance and digestibility of nutrients was not affected by myo-inositol when added to a diet limiting in digestible P in pigs. In addition, the beneficial effect of super dosed phytase (3,000 FYT/kg) on feed efficiency may be related to the release of sufficient amounts of both P and myo-inositol.