Effect of phytase on nutrient digestibility and expression of intestinal tight junction and nutrient transporter genes in pigs

Abstract

The study was conducted to determine the effects of high levels of phytase on growth performance, nutrient digestibility, phytate breakdown, and expression of mucosal tight junction and nutrient transporter genes in weanling pigs. A total of 128 barrows were penned in groups of four and used in a randomized completely block design and assigned to four treatments for a 28-d study. A two-phase feeding was implemented (phase 1: day 1 to 14; phase 2: day 15 to 28). The diets differed in dietary calcium (Ca) and phosphorus (P) levels (positive control [PC]: 8.1 to 7.1 g/kg Ca and 6.5 to 6.8 g/kg P; negative control [NC]: 6.6 to 5.5 g/kg Ca and 5.6 to 5.3 g/kg P) from phase 1 to phase 2, respectively. NC diets were supplemented with phytase at 0 (NC), 1,500 (NC + 1,500), or 3,000 (NC + 3,000) phytase units (FTU)/kg. Blood was collected after fasting (day 27) or feeding (day 28) for the measurement of plasma inositol concentrations. On day 28, two pigs per pen were euthanized. Duodenal–jejunal and ileal digesta samples and feces were collected to determine inositol phosphates (InsP_3-6) concentrations. Phytase supplementation increased the body weight on days 14 and 28 (P < 0.05). Average daily gain and feed efficiency compared with NC were increased by phytase with the majority of its effect in phase 1 (P < 0.05). The apparent ileal digestibility and apparent total tract digestibility of P were increased in piglets fed phytase-supplemented diets (P < 0.01) compared with NC piglets. Disappearance of InsP₆ and total InsP_3-6 up to the duodenum–jejunum, ileum, and in feces was increased by both phytase application rates (P < 0.01). Plasma concentrations of myo-inositol were higher (P < 0.001) in the phytase-supplemented diets than PC and NC diets, irrespective of whether pigs were fed or fasted. Expression of claudin 3 was higher in pigs fed both phytase-supplemented diets in the duodenum and jejunum compared with PC and NC. Mucin 2 expression was lower in the ileum of NC + 3,000 fed piglets compared with PC (P < 0.05), whereas expression of GLUT2 (solute carrier family 2-facilitated glucose transporter member 2) was increased (P < 0.05) by the NC + 3,000 treatment in all sections. In summary, high phytase supplementation increased the growth performance of nursery pigs. The increased expression of GLUT2 by phytase may indicate an upregulation of glucose absorption from the intestine by phytase.

Introduction

Phosphorus is an essential nutrient for development due to its involvement in many metabolic processes (National Research Council, 2012). It is essential for the growth of bones, cellular integrity, and multiple enzymatic processes. Meeting the phosphorus requirements of pigs for growth is difficult, largely because approximately 60% to 70% of phosphorus in plant-based feed ingredients occurs as phytate phosphorus. Phytate (myo-inositol hexakisphosphate) is a major antinutrient in pigs, which cannot efficiently degrade it due to their low phytase activity in the gastrointestinal tract (GIT). Phytate complexes with minerals, reducing the digestibility of cations, such as calcium (Ca), zinc (Woyengo et al., 2009; Woyengo and Nyachoti, 2013), and iron (Laird et al., 2018). Phytate may also reduce protein and carbohydrate digestibility by complexing with proteins such as digestive enzymes that break down proteins and carbohydrates (e.g., trypsin and α-amylase) leading to inhibition of their activities (Singh and Krikorian, 1982; Deshpande and Cheryan, 1984). Therefore, supplementation of diets with phytase from fungal or bacterial sources is associated with many benefits, such as increases in phytate phosphorus utilization, enhancement of growth performance, and energy and amino acid digestibility (Dersjant-Li and Dusel, 2019).

A portion of the growth performance responses to phytase is associated with its extra-phosphoric effects (Gehring et al., 2013; Walk et al., 2013; Lu et al., 2019a). Specifically, some of the growth-enhancing effects of phytase have been attributed to the release of myo-inositol from phytate (Zyła et al., 2004; Cowieson et al., 2013, 2014; Walk et al., 2014). Myo-inositol may enter peripheral circulation, where it is involved in a plethora of metabolic functions and signaling pathways (Gonzalez-Uarquin et al., 2020), and may lead to increased growth performance (Lee and Bedford, 2016). For example, the work by Józefiak et al. (2010) demonstrated a potential relationship between phytase supplementation and the levels of insulin-like growth factor 1 gene expression in the liver, suggesting that phytase could regulate the expression of hormones that significantly regulate growth in animals. Shaw et al. (2011) also showed an increase of IL-17 gene expression in the cecum and duodenum of cocci-vaccinated chickens offered a phytase. Vigors et al. (2014) showed in pigs that phytase supplementation induced an upregulation of the expression of the oligopeptide transporter PEPT1 (solute carrier family 15 member 1, peptide transporter), an indication that phytase may potentially increase peptide absorption. Martínez-Montemayor et al. (2008) provided evidence that phytase might regulate oxidative stress response by upregulating expression of GSTM4 (glutathione S-transferase mu 4) in the liver, an effect that was dependent on the concentration of zinc in the diet. However, several gaps still remain on the molecular targets of phytase in the GIT of pigs. There is a lack of information on the effects of phytase on several tight junction genes that play a role in nutrient absorption and the maintenance of intestinal epithelial integrity and nutrient transporters in the GIT of growing pigs. Although phytase is typically supplemented in pig diets at 500 FTU/kg (Wilcock and Walk, 2016), it has been shown that 2,000 to 2,500 FTU phytase/kg feed could lead to an additional increase in growth performance of pigs beyond the expected growth response from the release of extra phosphorus (Santos et al., 2014; Laird et al., 2018). Therefore, the objective of this work was to determine the effects of high levels of phytase supplementation on growth performance and nutrient digestibility and to determine the effects of phytase on the expression of intestinal tight junction and nutrient transporter genes in weanling pigs.

Materials and Methods

Animals

All animal procedures were approved by the Purdue Animal Care and Use Committee. Pigs were kept in an environmentally controlled room with an average temperature of 26 °C and 10 h of lighting per day. Experiment involved a total of 128 barrows (Duroc × Yorkshire × Landrace) which were weaned at 21 d old and then fed ad libitum a common corn–soy nursery diet (calculated on an as-fed basis to contain 3,544.5 kcal/kg ME, 23.97 % crude protein (CP), 1.55% SID Lysine, 0.85% total Ca, 0.75% total P, 0.6 % non-phytate P, and 600 FTU phytase) for 10 d before being assigned to four treatments on day 31 (initial body weight [BW] 7.4 ± 0.5 kg) in a randomized complete block design. Experiment was for 28 d (32 to 59 d of age). Pigs were fed according to a two-phase feeding program: phase 1 (14 d total, 32 to 45 d of age) and phase 2 (14 d total, 46 to 59 d of age). There were 8 replicates of 4 pigs each per diet (32 pigs per treatment). At the end of the experiment, 2 pigs per pen (64 pigs total) were killed for the collection of duodenal–jejunal and ileal mucosa and digesta samples. Digesta samples were used for the analysis of Ca, P, inositol phosphates (InsP₃, InsP₄, InsP₅, InsP₆), myo-inositol, and titanium.

Dietary treatments

Dietary treatments were 1) positive control (PC) treatment which had dietary phosphorus concentration that met the nutrient requirement of weanling pigs (NRC, 2012); 2) negative control (NC) diet compared with PC diets with the reduction in standardized total tract digestible (STTD) level of Ca (−1.5 g/kg) and P (−0.7 g/kg) during phase 1 and Ca (−1.6 g/kg) and P (−1.4 g/kg) in phase 2; (3) NC + 1,500 in which phytase (Quantum Blue, AB Vista, Marlborough, UK) was added to the NC diet at 1,500 FTU/kg; and (4) NC + 3,000 in which phytase was added to the NC diet at 3,000 FTU/kg. Titanium dioxide (TiO₂) was added at 0.5% to phase 2 diets to determine the nutrient digestibility. The diet formulations are presented in Tables 1 and 2.

Table 1.

Ingredient composition of phase 1 diets used in the experiment (g/kg), as-fed¹

Ingredients	PC	NC	NC + 1,500	NC + 3,000
Corn	390.4	396.4	381.4	366.4
Soybean meal, 47.5% CP	250	250	250	250
Soy protein concentrate	88	88	88	88
Whey, dried	180	180	180	180
Lactose	30	30	30	30
Fish meal	30	30	30	30
Soybean oil	12.00	12.00	12.00	12.00
Limestone	10.00	8.00	8.00	8.00
Monocalcium phosphate	4.00	0.00	0.00	0.00
dl-Methionine	0.60	0.60	0.60	0.60
Phytase premix2	0	0	15	30
Salt	1.00	1.00	1.00	1.00
Vitamin premix3	2.50	2.50	2.50	2.50
Mineral premix4	1.50	1.50	1.50	1.50
Total	1,000	1,000	1,000	1,000
Calculated composition
DE, kcal/kg	3,585	3,606	3,606	3,606
ME, kcal/kg	3,414	3,434	3,434	3,434
CP	249	249	249	249
Ca	8.1	6.6	6.6	6.6
Total P	6.5	5.6	5.6	5.6
STTD P	4.1	3.4	3.4	3.4
SID Lys	13.3	13.3	13.3	13.3
Analyzed phytase activity, FTU	<50.0	<50.0	1,640.0	2,900.0

¹PC, positive control diet with adequate Ca and P; NC, negative control diet with inadequate Ca and P; NC⁺1,500, NC containing 1,500 FTU/kg phytase; NC⁺3,000, NC containing 3,000 FTU/kg phytase.

²Phytase premix made to 0.01 g enzyme/kg to be added at 10 g/kg to provide 0.1 g/kg diet or 100 mg/kg.

³Vitamin premix supplied per kilogram of diet: 3,635 IU vitamin A, 363 IU vitamin D3, 26.4 IU vitamin E, 3.6 mg vitamin K, 1,206 µg menadione, 21.2 µg vitamin B12, 4.2 mg riboflavin, 13.5 mg d-pantothenic acid, and 19.5 mg niacin.

⁴Mineral premix supplied per kilogram diet: 9 mg Cu (as copper sulfate),0.34 mg I (as Ca iodate), 97 mg Fe (as ferrous sulfate), and 97 mg Zn (as zinc oxide).

Table 2.

Ingredient composition of phase 2 diets used in the experiment (g/kg as-fed)¹

Ingredients	PC	NC	NC + 1,500	NC + 3,000
Corn	429.9	443.8	428.8	413.8
Soybean meal, 47.5% CP	300	300	300	300
Soy protein concentrate	58.5	58.5	58.5	58.5
Whey, dried	100	100	100	100
Lactose	51.00	51.00	51.00	51.00
Soybean oil	9.00	3.50	3.50	3.50
Limestone	8.75	7.60	7.60	7.60
Monocalcium phosphate	11.25	4.00	4.00	4.00
l-Lysine-HCl	1.00	1.00	1.00	1.00
dl-Methionine	0.60	0.60	0.60	0.60
Phytase premix2	0	0	15	30
Titanium dioxide premix3	25	25	25	25
Salt (NaCl)	1.00	1.00	1.00	1.00
Vitamin premix4	2.50	2.50	2.50	2.50
Mineral premix5	1.50	1.50	1.50	1.50
Total	1,000	1,000	1,000	1,000
Calculated composition
DE, kcal/kg	3,426	3,426	3,426	3,426
ME, kcal/kg	3,270	3,270	3,270	3,270
CP	229	230	230	230
Ca	7.1	5.5	5.5	5.5
Total P	6.8	5.3	5.3	5.3
STTD P	4.4	3.0	3.0	3.0
SID Lys	12.5	12.5	12.5	12.5
Analyzed nutrient composition
GE, kcal/kg	4,303	4,296	4,315	4,259
CP, g/kg	217	218	218	214
Ca, g/kg	8.0	6.8	6.3	6.3
P, g/kg	6.0	5.4	5.3	5.3
Myo-inositol, mmol/kg	1.1	1.1	1.1	1.1
InsP6, mmol/kg	22.0	22.9	22.9	22.4
Ins(1,2,3,4,5)P5, mmol/kg	0.5	0.6	0.6	0.5
Ins(1,2,4,5,6)P5, mmol/kg6	1.0	1.0	1.0	1.0
Analyzed phytase activity, FTU	<50.0	<50.0	1,200.0	2,570.0

¹PC, positive control diet with adequate Ca and P; NC, negative control diet with inadequate Ca and P; NC⁺1,500, NC containing 1,500 FTU/kg phytase; NC⁺3,000, NC containing 3,000 FTU/kg phytase.

²Phytase premix made to 0.01 g enzyme/kg to be added at 10 g/kg to provide 0.1 g/kg diet or 100 mg/kg. Data are means of 8 observations pigs per treatment.

³TiO₂ premix resulting in 5 g TiO₂/kg feed.

⁴Vitamin premix supplied per kilogram of diet: 3,635 IU vitamin A, 363 IU vitamin D3, 26.4 IU vitamin E, 3.6 mg vitamin K, 1,206 µg menadione, 21.2 µg vitamin B12, 4.2 mg riboflavin, 13.5 mg d-pantothenic acid, and 19.5 mg niacin.

⁵Mineral premix supplied per kilogram diet: 9 mg Cu (as copper sulfate), 0.34 mg I (as Ca iodate), 97 mg Fe (as ferrous sulfate), and 97 mg Zn (as zinc oxide).

⁶InsP₄ and InsP₃ isomers were below the limit of detection.

Experimental procedures and sample collection

Pigs were weighed every 2 wk. Pigs were fed experimental diets ad libitum from day 0 to 26. Feed intakes were calculated every 2 wk after deducting leftovers from the amount supplied, and feed efficiency (G:F) was calculated based on the ratio of average daily gain and average daily feed intake. On day 26 of the experiment, pigs were fasted overnight for 12 h and two pigs per pen, representing the average pen weight, were bled the next day (day 27 of the experiment) through jugular venipuncture to obtain fasting jugular blood (approximately 12 h after fasting) concentrations of analytes. A vacutainer tube containing lithium heparin (Franklin Lakes, NJ) was used to collect the blood samples. Pigs were returned to ad libitum feeding afterward, and on day 28 of the experiment, the same two pigs per pen were similarly bled approximately 45 min after feeding (after lights were switched back on to encourage feeding) to obtain fed concentrations of analytes. Feces were collected by rectal palpation. The same pigs that were bled earlier were then euthanized with an intramuscular injection of a mixture of Telazol (Zoetis Inc., MI) and Xylazine (RXV Inc., CA) that provided 0.2 mg tiletamine base, 0.2 mg zolazepam base, and 0.2 mg mannitol per kg BW of pig to induce sedation followed by CO₂ asphyxiation and exsanguination. Jugular and portal blood were then collected. Digesta from the duodenum and proximal jejunum (duodenum–jejunum), about 50 cm length from the duodenal bulb, and distal ileum (about 50 cm length up from ileal–cecal junction) were flushed with cold water. Collected small intestinal digesta and feces were pooled from the two pigs per pen and frozen at −20 °C.

Gross energy, dry matter, phosphorus, calcium, nitrogen, and InsP determination

Freeze-dried duodenal–jejunal, ileal digesta, and fecal samples were ground to pass through a 0.5-mm screen. Diets, digesta, and fecal samples were ground and dried at 105 °C in a drying oven (Precision Scientific Co., Chicago, Illinois) for 24 h to determine the dry matter (DM) content (method 934.01; AOAC International, 2006). Gross energy (GE) was determined on a bomb calorimeter (Parr 1261 bomb calorimeter, Parr Instrument Co., Moline, IL). Titanium concentration in the digesta was determined according to the procedures of Myers et al. (2004). Briefly, samples were digested in H₂SO_4. Then, 30% H₂O₂ was added and the absorbance of samples was measured at 406 nm wavelength. The concentration of P was analyzed by first digesting samples in concentrated nitric acid and 70% perchloric acid. Absorbance of samples was measured on SpectraCount spectrophotometer (model AS1000; Packard, Meriden, CT; AOAC International, 2006) at 620 nm as described by Zhai and Adeola (2013). The concentration of Ca was determined using an atomic absorption spectrometer (AAnalyst 300; PerkinElmer, Norwalk, CT) according to the method 968.08 (AOAC International, 2000). Nitrogen content was determined with the combustion method on a model FP-2000 nitrogen analyzer (Leco Corp., St. Joseph, MI). Phytase activity in feeds was determined at a commercial laboratory (AB Vista Laboratories, Hengoed, UK).

Digestibility of GE, P, Ca, Nitrogen, and disappearance InsP₆ and cumulated InsP_3-6 were calculated using the following equation:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{A}\mathrm{p}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y},\mathrm{\%}=[1-\left(\mathrm{T}{\mathrm{i}}_{\mathrm{i}}/\mathrm{T}{\mathrm{i}}_{\mathrm{o}}\right)\times \left({\mathrm{Y}}_{\mathrm{o}}/{\mathrm{Y}}_{\mathrm{i}}\right)]\times 100;}\end{array}}$$

The disappearance coefficients of InsP₆ and cumulated InsP_3-6 were calculated using the following equation:

$${\displaystyle \begin{array}{l}{\displaystyle \mathrm{D}\mathrm{i}\mathrm{s}\mathrm{a}\mathrm{p}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}=1-\left(\mathrm{T}{\mathrm{i}}_{\mathrm{i}}/\mathrm{T}{\mathrm{i}}_{\mathrm{o}}\right)\times \left({\mathrm{X}}_{\mathrm{o}}/{\mathrm{X}}_{\mathrm{i}}\right)}\end{array}}$$

Where Ti_i and Ti_o are the titanium concentrations of the diet and output (duodenal–jejunal digesta, ileal digesta, or feces), respectively (mg/kg of DM); and Y_o and Y_i are the concentrations of GE, P, Ca, N, InsP₆, or total InsP_3-6 in the output (duodenal–jejunal digesta, ileal digesta, or feces) and diet, respectively (mg/kg DM or nmol/g DM).

The extraction and measurement of InsP_3-6 isomers in feed, digesta, and feces were conducted according to the method of Zeller et al. (2015) with slight modifications as described by Sommerfeld et al. (2018b) and measured by high-performance ion chromatography (ICS-3000 system, Dionex, Idstein, Germany). It was not possible to separate enantiomers with this procedure to distinguish between the d- and l-forms. In addition, some InsP₃ isomers could not be identified because standards were unavailable. Due to coelution, a clear separation between the isomers Ins(1,2,6)P₃, Ins(1,4,5)P₃, and Ins(2,4,5)P₃ was not possible. Feed, digesta, and plasma myo-inositol concentration were determined according to Sommerfeld et al. (2018a) on a gas-chromatograph/mass spectrometer after samples had been derivatized.

Analysis of insulin and glucose

Blood was centrifuged at 2,000 × g in heparinized tubes for 15 min at 4 °C. The resulting supernatant was transferred to a new tube and stored at −80 °C prior to analysis. Plasma glucose concentrations were determined using the Autokit glucose kit (Wako Pure Chemical Industries Ltd., Chuo-Ku Osaka, Japan) according to the manufacturer’s protocol. Plasma insulin concentration was determined using the porcine insulin ELISA kit following the manufacturer’s protocol (Inter and intra-assay coefficient of variation: 2.7% and 3.5%, respectively; Mercodia, Uppsala, Sweden).

Real-time PCR analysis

The abundance of the following tight junction and nutrient transporter genes was analyzed with RT-PCR. Genes analyzed were CLDN1, CLDN3, CLDN4 (tight junction proteins), ASCT2 (SLC1A5, solute carrier family 1 [neutral amino acid transporter] member 5, Na⁺-dependent neutral amino acid transporter), GLUT2 (solute carrier family 2-facilitated glucose transporter member 2), GLUT5 (solute carrier family 2-facilitated glucose/fructose transporter, member 5), MUC2 (mucin 2, oligomeric mucus/gel-forming), PEPT1, SGLT1 (solute carrier family 5-sodium/glucose cotransporter member 1), SLC5A3 (solute carrier family 5, inositol transporters, member 3), SLC2A13 (H⁽⁺⁾-myo-inositol symporter), and ALPI (alkaline phosphatase, intestinal). Expression of genes was normalized using GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Primers were designed with the Primer Blast software (NCBI-NIH, Bethesda, MD). Sequences of real-time polymerase chain reaction (PCR) primers used are provided in Supplementary Table S1. The QIAzol lysis reagent (Qiagen, Valencia, CA) was used to extract total ribonucleic acid (RNA) from intestinal samples. Concentrations of RNA were measured on a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE). RNA integrity was determined by agarose gel electrophoresis. Reverse transcription of RNA was done with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) using oligo dT_12–18 primers. PCR was conducted on a Bio-Rad CFX thermocycler (Bio-Rad, Temecula, CA) with the SYBR real-time PCR mix (Biotool, Houston, TX) in a total reaction volume of 20 μL. A melt curve analysis was performed for each gene at the end of the PCR run. The expression level for each gene was calculated after its cycle threshold (Ct) was normalized to the Ct for GAPDH using the ΔΔCt method.

Statistical analysis

Data were analyzed using the Proc GLM procedure of SAS (SAS Inst. Inc., Cary NC) for a randomized complete block design with diet as the main effect. Pen was the experimental unit. The model included diet and replicate (block). Results are reported as least square means and standard errors of the means. Means were different at P ≤ 0.05. When the diet effect was significant at P < 0.05, differences between means were compared using the Tukey’s test. Superscript designations were used to indicate significant mean differences.

Results

Growth performance and nutrient digestibility

Phosphorus concentrations in the PC, NC, NC + 1,500, and NC + 3,000 diets were 6.5, 5.6, 5.6, 5.6 and 6.8, 5.3, 5.3, 5.3g/kg feed for phases 1 and 2, respectively (Tables 1 and 2). Analyzed Ca was about 1.0 g higher than estimated values in phase 2 diets. BW of NC pigs was significantly lower (P < 0.05) than the PC, NC + 1,500, and NC + 3,000 pigs on days 14 and 28 (Table 3). Feed intake just differed in phase 1 with lower intake in NC pigs compared with those of the other treatments (P < 0.05). Gain during phase 1 was also lower in NC compared with other treatments (P < 0.05) but not different across treatments during phase 2. The overall gain in the NC diet was lower than NC + 1,500 (P < 0.05) but not different from the other two treatments. G:F was lower in the NC during phase 1 than in other treatments (P < 0.05), but not different across treatments during phase 2 and overall. Duodenal–jejunal digestibility and the amount of absorbed P were negative in the PC and NC diets but significantly increased by the NC + 1,500 and NC + 3,000 diets (P < 0.01) (Table 4). The apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of P followed a similar pattern with phytase significantly increasing (P < 0.01) P digestibility and P absorption compared with NC. However, the ATTD of GE and DM were lower in the NC+1,500 diet than NC (P < 0.01). Irrespective of location, Ca digestibility was not significantly affected by treatment.

Table 3.

Growth performance of weanling pigs fed different levels of phytase^1,2

Period	Item	PC	NC	NC + 1,500	NC + 3,000	SD	P-value
	Day 0 wt, kg	7.4	7.4	7.5	7.4	0.1	0.65
	Day 14 wt, kg	12.3a	11.7b	12.5a	12.3a	0.5	<0.01
	Day 28 wt, kg	19.2a	17.9b	19.5a	19.1a	1.2	0.04
Phase 1	Gain, g/d	348a	303b	366a	348a	36.2	<0.01
	FI, g/d	569a	507b	533ab	530ab	28.7	<0.01
	G:F, g/kg	612b	600c	690a	650ab	49.6	<0.01
Phase 2	Gain, g/d	491	443	496	490	68.2	0.38
	FI, g/d	957	885	957	972	116.2	0.43
	G:F, g/kg	515	501	521	504	47.2	0.81
Overall	Gain, g/d	419ab	373b	431a	419ab	41.9	0.04
	FI, g/d	763	696	745	751	61.8	0.14
	G:F, g/kg	551	536	580	557	37.3	0.15

¹PC, positive control diet with adequate Ca and P; NC, negative control diet with inadequate Ca and P; NC⁺1,500, NC containing 1,500 FTU/kg phytase; NC₊3,000, NC containing 3,000 FTU/kg phytase.

²Data are means of eight observations per treatment.

^a–cWithin a row, means without a common superscript differ (P < 0.05).

Table 4.

Nutrient digestibility in pigs fed different levels of phytase^1,2

Section	PC	NC	NC + 1,500	NC + 3,000	SEM	P-value
Duodenum–jejunal digestibility, %
GE	24.9	29.7	27.0	27.9	4.90	0.92
DM	30.3	33.5	31.7	34.5	4.07	0.89
P	−10.7c	−5.5bc	31.1ab	34.4a	9.76	<0.01
Ca	25.2	18.5	24.4	37.3	6.08	0.20
N	14.6	14.3	19.1	15.9	8.93	0.98
Ca absorbed, g/d	1.9	1.1	1.5	2.2	0.34	0.15
P absorbed, g/d	−0.7b	−0.3ab	1.6a	1.7a	0.51	<0.01
AID, %
GE	55.5	61.2	52.5	58.0	3.73	0.42
DM	53.6	60.1	50.8	57.0	3.66	0.33
P	40.8bc	30.0c	50.6ab	57.5a	3.99	<0.01
Ca	62.3	57.3	66.4	66.1	3.62	0.28
N	59.5	68.1	62.6	64.1	2.70	0.18
Ca absorbed, g/d	4.8	3.5	4.0	4.0	0.35	0.08
P absorbed, g/d	2.4ab	1.4b	2.6a	2.9a	0.25	<0.01
ATTD, %
GE	76.2a	77.1a	72.7b	75.9ab	0.82	<0.01
DM	76.6ab	77.9a	74.3b	78.2a	0.76	<0.01
P	48.9ab	42.3b	53.3ab	61.4a	3.48	<0.01
Ca	79.3	77.6	76.6	81.9	2.50	0.48
N	68.0ab	69.9a	64.8b	68.5ab	1.26	0.05
Ca absorbed, g/d	6.1a	4.7b	4.6b	5.0ab	0.29	<0.01
P absorbed, g/d	2.8ab	2.0b	2.7ab	3.2a	0.22	0.01

¹PC, positive control diet with adequate Ca and P; NC, negative control diet with inadequate Ca and P; NC⁺1,500, NC containing 1,500 FTU/kg phytase; NC⁺3,000, NC containing 3,000 FTU/kg phytase.

²Data are means of eight observations per treatment.

^a–cWithin a row, means without a common superscript differ (P < 0.05).

Intestinal phytate breakdown

Concentrations of InsP₆, Ins(1,2,3,4,5)P₅, and Ins(1,2,4,5,6)P₅ were significantly reduced in the duodenal–jejunal digesta of phytase-fed pigs, indicating that these InsPs were significantly degraded (Table 5). Ins(1,2,3,4,6)P₅ was undetectable in piglets fed phytase-supplemented diets. This corresponds to the observed elevation of Ins(1,2,5,6)P₄ and of myo-inositol concentrations in the duodenal–jejunal NC + 1,500, and NC + 3,000 digesta (P < 0.05). In the ileal digesta, Ins(1,2,3,4,6)P₅ was also undetectable in phytase-fed piglets. Ileal concentration of Ins(1,2,3,4,5)P₅ tended to be lower (P < 0.08) in the phytase-supplemented diets and Ins(1,2,4,5,6)P₅ was undetectable in the NC + 3,000 diets and lower in the NC + 1,500 than in PC and NC digesta (P < 0.01). Ileal concentration of myo-inositol was elevated (P < 0.001) in phytase-fed piglets. A relevant amount of InsP₆ was still detectable in the feces of PC piglets (Table 5). The amount of fecal InsP₆ in the NC was reduced to 36% of the PC treatment and further reduced to 6% and 9% of the PC in the two phytase treatments (P < 0.001), respectively. Lower levels of Ins(1,2,4,5,6)P₅ and Ins(1,2,3,4,5)P₅ were found in the feces of NC pigs compared with PC, and their concentrations were negligible in phytase treatments. The InsP₃ isomers were not detectable in the feces. Fecal myo-inositol levels were low and not different among treatments. Disappearances of InsP₆ and InsP_3-6 up to the duodenum–jejunum, ileum, and feces were increased by phytase at both application levels (P < 0.001) (Table 6).

Table 5.

Concentrations of inositol phosphates or myo-inositol (µmol/g DM) in duodenum–jejunal, ileal, and fecal samples of piglets fed with experimental diets^1,2

Item	PC	NC	NC + 1,500	NC + 3,000	SEM	P-value
Duodenum–Jejunum
Ins(1,2,6;1,4,5;2,4,5)P3	0.58	0.69	2.65	2.12	1.76	0.88
Ins(1,2,3,4)P4	0.42	0.35	0.58	n.d.3	—	—
Ins(1,2,5,6)P4	0.97b	0.97b	6.90a	4.93a	3.66	0.02
Ins(1,2,3,4,6)P5	0.57	0.52	n.d.	n.d.	—	—
Ins(1,2,3,4,5)P5	1.45a	1.38a	0.69b	0.35b	0.4	0.02
Ins(1,2,4,5,6)P5	2.08a	2.05a	0.46b	n.d.	0.4	0.003
InsP6	22.10a	22.23a	2.69b	1.84b	4.3	<0.001
myo-inositol	1.02bc	0.82c	1.80ab	2.47a	0.65	<0.001
Ileum
Ins(1,2,6;1,4,5;2,4,5)P3	0.61	0.63	3.69	1.93	1.66	0.29
Ins(1,2,3,4)P4	0.54	0.45	n.d.	n.d.	0.12	0.19
Ins(1,2,5,6)P4	1.76	1.52	7.20	4.74	4.89	0.23
Ins(1,2,3,4,6)P5	0.54	0.61	n.d.	n.d.	0.07	0.10
Ins(1,2,3,4,5)P5	2.37	2.02	0.87	0.34	0.72	0.08
Ins(1,2,4,5,6)P5	3.13a	3.11a	0.33b	n.d.	0.70	0.01
InsP6	34.28a	31.38a	2.31b	1.61b	4.55	<0.001
myo-inositol	0.50b	0.52b	2.15a	2.63a	0.99	<0.001
Feces
Ins(1,2,6;1,4,5;2,4,5)P3	n.d.	n.d.	n.d.	n.d.	—	—
Ins(1,2,3,4)P4	n.d.	n.d.	n.d.	n.d.	—	—
Ins(1,2,5,6)P4	0.43	n.d.	0.43	n.d.	—	—
Ins(1,2,3,4,6)P5	0.49	n.d.	n.d.	n.d.	—	—
Ins(1,2,3,4,5)P5	1.04a	0.37b	0.32b	n.d.	0.15	0.01
Ins(1,2,4,5,6)P5	1.79a	0.76b	n.d.	n.d.	0.38	0.01
InsP6	24.55a	8.95b	2.23c	1.38c	3.7	<0.001
myo-inositol	0.22	0.23	0.11	0.11	0.1	0.16

¹PC, positive control diet with adequate Ca and P; NC, negative control diet with inadequate Ca and P; NC⁺1,500, NC containing 1,500 FTU/kg phytase; NC⁺3,000, NC containing 3,000 FTU/kg phytase.

²Data are means of eight observations per treatment.

³n.d., not detectable in the majority of samples.

^a–cWithin a row, means without a common superscript differ (P < 0.05).

Table 6.

Effect of different phytase application rates on the disappearance coefficients of InsP₆ and the sum of InsP_3-6 analyzed in piglets up to the duodenal–jejunal, ileal, and fecal sampling points^1,2

Item	PC	NC	NC + 1,500	NC + 3,000	SD	P-value
Duodenum–Jejunum
InsP6	0.12b	0.08b	0.89a	0.93a	0.10	<0.001
InsP3-6	0.05b	0.05b	0.61a	0.73a	0.08	<0.001
Ileum
InsP6	0.09b	0.26b	0.93a	0.95a	0.12	<0.001
InsP3-6	0.06b	0.17b	0.78a	0.84a	0.20	<0.001
Feces
InsP6	0.62c	0.87b	0.97a	0.98a	0.06	<0.001
InsP3-6	0.61c	0.87b	0.97a	0.98a	0.06	<0.001

¹PC, positive control diet with adequate Ca and P; NC, negative control diet with inadequate Ca and P; NC⁺1,500, NC containing 1,500 FTU/kg phytase; NC⁺3,000, NC containing 3,000 FTU/kg phytase.

²Data are means of eight observations per treatment.

^a–cWithin a row, means without a common superscript differ (P < 0.05).

Plasma parameters

Plasma concentrations of myo-inositol in blood collected from the portal vein were higher than those collected from the jugular vein with no differences found for PC compared with NC piglets (Table 7). Phytase application increased the plasma myo-inositol concentration (P < 0.001), with differences in the application rates found only in the jugular blood from fed piglets. There were no treatment differences in the plasma concentrations of insulin and glucose (Table 8).

Table 7.

Effects of different phytase application rates on myo-inositol concentrations (µmol/L) in fasting and fed jugular and portal vein plasma¹

Plasma source	PC	NC	NC + 1,500	NC + 3,000	SEM	P-value
Portal vein	123.7b	132.4b	178.1a	183.0a	8.23	<0.001
Fasting jugular	31.5b	37.2b	50.0a	53.8a	2.29	<0.001
Fed jugular	65.6c*	71.7c*	126.7b*	164.3a*	7.17	<0.001

¹Data are means of eight observations per treatment.

^a–cWithin a row, means without a common letter superscript differ (P < 0.015).

*Significant difference compared with corresponding fasting jugular concentration (P < 0.0001).

Table 8.

Plasma concentrations of insulin and glucose in pigs treated with two levels of phytase^1,2

Item	PC	NC	NC + 1,500	NC + 3,000	SEM	P-Value
Jugular blood Insulin3, µg/L	0.042	0.038	0.047	0.049	0.005	0.41
Fasting jugular blood glucose3, mg/dL	97.4	95.2	94.5	95.3	3.28	0.93
Fed jugular blood glucose, mg/dL	175.4	251.0	215.3	207.2	18.74	0.07
Portal vein blood glucose, mg/dL	488.5	477.2	491.9	473.8	7.49	0.27

¹PC, positive control diet with adequate Ca and P; NC, negative control diet with inadequate Ca and P; NC⁺1,500, NC containing 1,500 FTU/kg phytase; NC⁺3,000, NC containing 3,000 FTU/kg phytase.

²Data are means of eight observations per treatment.

³Insulin values were based on one pig per pen, and glucose data represent the average of two pigs per pen.

Gene expression

Expression of CLDN1 was not affected by treatment in the duodenum (Table 9) (P > 0.05). However, in the jejunum, CLDN1 was downregulated at the higher level of phytase supplementation (NC + 3,000) compared with the PC diet (Table 10) and compared with PC and NC pigs in the ileum (Table 11) (P < 0.05). Expression of CLDN3 was higher in the duodenum and jejunum of phytase fed compared with PC and NC pigs. However, there was no treatment effect on the expression of CLDN4 (P > 0.05). There was no change in the duodenal expression of ASCT2 relative to the PC; however, ASCT2 expression in the duodenum was higher (P < 0.05) in the NC compared with PC and NC + 3,000 treatments (Table 9). Expression of ileal MUC2 was lower in the NC + 3,000 compared with the PC treatment (P < 0.05). Expression of GLUT2 was increased (P < 0.05) by the NC + 3,000 treatment in all sections compared with PC. Expression of SLC2A13 in the duodenum was higher in the NC vs. NC + 1,500 and NC + 3,000 (P < 0.01), but not affected by treatment in the jejunum and ileum. Treatment did not affect the level of GLUT5 and PEPT1 in all sections. Expression of SGLT1 was higher in the NC + 3,000 treatment in the jejunum relative to NC (P < 0.01) (Table 10). Expression of ALPI was decreased (P < 0.05) by the NC + 3,000 in the duodenum (Table 9) and by the NC + 1,500 and NC + 3,000 treatments in the jejunum (Table 10) and ileum relative to PC (Table 11).

Table 9.

Effect of different dietary phytase application rates on relative gene expression in the duodenal tissue of pigs^1,2

Gene	PC	NC	NC + 1,500	NC + 3,000	SEM	P-value
CLDN1	1.00	0.42	0.50	0.60	0.33	0.86
CLDN3	1.00b	0.82b	1.41a	1.64a	0.18	0.01
CLDN4	1.00	0.98	0.93	0.94	0.15	0.93
ASCT2	1.00b	2.24a	1.50ab	1.29b	0.24	0.01
MUC2	1.00	0.99	0.85	0.84	0.11	0.32
GLUT2	1.00b	1.41b	1.70ab	3.15a	0.38	0.01
GLUT5	1.00	0.80	0.69	1.58	0.44	0.69
PEPT1	1.00	1.47	1.20	1.35	0.42	0.96
SGLT1	1.00	0.80	1.13	1.26	0.19	0.17
SLC5A3	1.00	1.35	0.69	0.93	0.21	0.09
SLC2A13	1.00ab	1.44a	0.70b	0.57b	0.16	0.01
ALPI	1.00ab	1.17a	0.63ab	0.58b	0.15	0.04

¹PC, positive control diet with adequate Ca and P; NC, negative control diet with inadequate Ca and P; NC⁺1,500, NC containing 1,500 FTU/kg phytase; NC⁺3,000, NC containing 3,000 FTU/kg phytase.

²Data are means of eight observations per treatment.

^a,bWithin a row, means without a common superscript differ (P < 0.05).

Table 10.

Effect of different dietary phytase application rates on relative gene expression in jejunal tissue of pigs^1,2

Gene	PC	NC	NC + 1,500	NC + 3,000	SEM	P-value
CLDN1	1.00a	0.62ab	1.24a	0.32b	0.40	0.02
CLDN3	1.00b	0.98b	1.17ab	1.65a	0.14	0.01
CLDN4	1.00	0.85	0.90	1.12	0.13	0.78
ASCT2	1.00ab	1.14ab	0.90b	1.82a	0.18	0.03
MUC2	1.00	1.39	1.46	1.77	0.2	0.20
GLUT2	1.00b	0.69b	0.78b	2.27a	0.25	0.01
GLUT5	1.00	0.98	1.05	0.87	0.21	0.69
PEPT1	1.00	1.52	1.23	1.19	0.31	0.96
SGLT1	1.00ab	0.92b	1.04ab	1.58a	0.15	0.02
SLC5A3	1.00	1.63	1.43	2.83	0.27	0.23
SLC2A13	1.00	1.20	1.08	0.74	0.22	0.08
ALPI	1.00a	0.83ab	0.30c	0.43bc	0.14	0.01

¹PC, positive control diet with adequate Ca and P; NC, negative control diet with inadequate Ca and P; NC⁺1,500, NC containing 1,500 FTU/kg phytase; NC⁺3,000, NC containing 3,000 FTU/kg phytase.

²Data are means of eight observations per treatment.

^a–cWithin a row, means without a common superscript differ (P < 0.05).

Table 11.

Effect of different dietary phytase application rates on relative gene expression in ileal tissue of pigs¹

Gene	PC	NC	NC + 1,500	NC + 3,000	SEM	P-value
CLDN1	1.00a	1.60a	0.93ab	0.78b	0.53	0.01
CLDN3	1.00	0.85	0.9	1.59	0.17	0.06
CLDN4	1.00	0.84	1.09	1.99	0.46	0.29
ASCT2	1.00	0.85	0.98	1.04	0.18	0.76
MUC2	1.00a	0.58ab	0.69ab	0.51b	0.17	0.03
GLUT2	1.00b	0.90b	1.12ab	2.60a	0.26	0.01
GLUT5	1.00	1.53	1.46	1.38	0.52	0.59
PEPT1	1.00	1.51	1.25	1.28	0.30	0.6
SGLT1	1.00	1.00	0.90	1.74	0.18	0.31
SLC5A3	1.00	0.89	0.96	1.19	0.26	0.81
SLC2A13	1.00	0.87	0.62	0.72	0.12	0.12
ALPI	1.00a	1.05a	0.40b	0.55b	0.17	0.04

¹Data are means of eight observations per treatment.

^a,bWithin a row, means without a common superscript differ (P < 0.05).

Discussion

There is interest in supplementing phytase in swine diets at levels beyond 500 FTU/kg, a level which was found to release 0.3 to 1.5 g/kg available phosphorus (Augspurger et al., 2003; Wilcock and Walk, 2016). The higher levels of phytase are considered to have extra-phosphoric effects because the growth performance improvements seen at these levels are beyond what could be accounted for by the extra digestible P released by the enzyme (Walk et al., 2013; Guggenbuhl et al., 2016; Lu et al., 2019b). Diets that are deficient in non-phytate phosphorus are known to result in reduced performance especially gain and feed intake.

Effects on growth performance and nutrient digestibility

Current results during phase 1 of this study show that phytase supplementation increased animal performance with increased gain, FI, and G:F when added to a low P diet. This finding agrees with the results from our recent study (Lu et al., 2019) and those of others (Kornegay and Qian, 1996; Laird et al., 2018). Although a phytase effect was not observed in the second phase of the trial, at the end of the study, phytase-supplemented pigs were significantly heavier (approx. 8.9%) than pigs on the NC diet but not heavier than PC pigs. Phytase is known to increase the nutrient digestibility (Nyannor et al., 2007; Adedokun et al., 2015; Lu et al., 2019a). Although P digestibility and the amount of absorbed P were both negative in the duodenal–jejunal section of control animals, perhaps due to endogenous secretions into these regions, phytase significantly increased these parameters, consistent with a rapid effect of the current phytase to increase phytate P breakdown even in the anterior sections of the GIT. The AID of P and the daily amount of absorbed P followed a similar trend such that AID were 20.6% and 27.5% points and the daily amount of absorbed P was 91.67% and 107.1% higher in the NC + 1,500 and NC + 3,000 treatments, respectively than in the NC. Similarly, ATTD of P in the NC + 1,500 and NC + 3,000 compared with NC increased by 11.0% and 19.1% and daily amount of absorbed P increased by 0.7 and 1.2g/d, respectively. Nevertheless, the ATTD of P hardly exceeded a value of 60% in NC + 3,000, which is consistent with the results from a recent meta-analysis that indicated a maximum in ATTD of P of 65% in phytase-supplemented diets in pigs (Rosenfelder-Kuon et al., 2020b). The AID of GE, DM, and N were not affected by treatment. The ATTD of GE, DM, and N were unexpectedly lower in the NC + 1,500 compared with NC, but these were not different between NC and NC + 3,000. However, the intake of digestible GE and N was numerically 7% to 8% higher on the NC + 1,500 than the NC and this would compensate for the lower digestibility. This similar increase in final BW in both the NC + 1,500 and NC + 3,000 compared with the NC treatment could also be an indication that nutrients other than GE and N were responsible for the increased BW in phytase-supplemented pigs.

Inositol phosphates

Phytase is known to degrade InsP₆ following a step-wise dephosphorylation. However, because in vivo breakdown of phytate is never complete, a mixture of inositol phosphate esters is formed during phytate hydrolysis (Zeller et al., 2015; Kühn et al., 2016; Laird et al., 2018). In this trial, the low level of InsP₆ in the duodenal–jejunal and ileal digesta and feces and the resulting increase in InsP₆ disappearance with phytase supplementation within the small intestine demonstrate a near-complete hydrolysis of InsP₆ at the two levels used. There was no significant increase of InsP₅ isomers, whereas Ins(1,2,5,6)P₄ or its analytically indistinguishable E.coli phytase-specific enantiomers were increased in the duodenum–jejunum with phytase application. This finding agrees with others who showed an increase of InsP₄ in digesta of broilers (Zeller et al., 2015; Sommerfeld et al., 2018a, 2018b) and pigs (Kühn et al., 2016; Laird et al., 2016; Rosenfelder-Kuon et al., 2020a) when fed diets with added phytases. Despite the buildup of InsP₄ with phytase application, the overall InsP_3-6 disappearance up to the ileum, compared with the InsP₆ disappearance, was reduced by only 15% and 11% points in phytase-supplemented diets. However, the AID of P attained its maximum at about 58% with high phytase inclusion. This indicates that some P remained bound as InsP₂ and InsP₁ and hence undigestible to pigs. In fact, concentrations of InsP₂ in ileal digesta of pigs were increased upon phytase supplementation (Rosenfelder-Kuon et al., 2020a). Nevertheless, complete dephosphorylation of some InsP₆ by phytase supplementation is demonstrated by the increase in digesta myo-inositol concentrations in the duodenal–jejunal as well as the ileal digesta. Similarly, Laird et al. (2018) found a dose-dependent increase both in InsP degradation and in myo-inositol concentration in the gastrointestinal digesta when feeding either 500 or 2,500 FTU/kg feed of phytase. These two phytase levels increased InsP₆ disappearance up to the ileum by 39% and 50%, which was lower than the increase of 67% and 69% observed in NC + 1,500 and NC + 3,000 treatments, respectively, in this study. An increased prececal InsP₆ disappearance from 76.3% to 85% with increasing the application rate from 750 to 3,000 FTU/kg feed was found also by Rosenfelder-Kuon et al. (2020a). Thus, a more robust increase in InsP₆ disappearance is obtained by feeding higher levels of phytase.

A remarkable InsP₆ concentration was found in fecal material of NC and even more in PC. This is not consistent with other pig studies that have reported almost complete InsP₆ degradation in feces of pigs (Sandberg et al., 1993; Schlemmer et al., 2001; Rosenfelder-Kuon et al., 2020a). Rosenfelder-Kuon et al. (2020a) have suggested that that post-ileal InsP₆ disappearance may be the result of bacterial activity. Perhaps, fermentation processes were less developed in piglets of the current study than in heavier pigs used in the other aforementioned pig studies. It may also be possible that bacterial phytase production in the large intestine was impaired by non-digested mineral P entering the large intestine because phytate degradation by ruminal microorganism was found to be delayed by added phosphate in vitro (Haese et al., 2017). This can explain why InsP₆ concentrations were markedly higher in PC than NC pigs of the current study. InsP₆ degradation in the colon may also be reduced by Ca carbonate supplementation to the feed (Sandberg et al., 1993).

Regulation of myo-inositol concentration

Complete hydrolysis of phytate produces myo-inositol that is absorbed and can be detected in portal and peripheral blood of pigs (Guggenbuhl et al., 2016; Laird et al., 2016; Cowieson et al., 2017; Lu et al., 2019). Phytase increased myo-inositol concentration of portal and jugular blood (fasting and fed) compared with the NC, with the highest levels seen in portal blood. This agrees with the findings of Laird et al. (2016), who also found higher myo-inositol concentrations in portal compared with peripheral plasma of phytase-fed pigs. However, Laird et al. (2018) reported lower portal plasma myo-inositol concentrations than recorded in this study. The differences in plasma myo-inositol concentrations in fed vs. fasted states, with higher concentrations seen in the fed state, suggest that feeding and sampling points affect plasma myo-inositol concentrations. This could explain why the increase in myo-inositol concentration by phytase varied between 25% and 228% compared with the plasma concentrations in the NC treatment. Although an insulin-like effect of myo-inositol is assumed, no differences in blood insulin and glucose concentrations were detected. This was probably due to the complexity of metabolic regulation between glucose and insulin. However, the lack of phytase effect on blood glucose and insulin concentrations agrees with the results of others in pigs (Cowieson et al., 2017), where blood glucose concentration was measured over a 6-h period after feeding, and in chickens (Cowieson et al., 2017).

Regulation of intestinal tight junction and nutrient transporters gene expression

The intestinal mucin layer is part of the brush border protection against invasion by pathogenic organism and its abundance reflects brush border integrity (Jiang et al., 2013). Increased mRNA of MUC2, the gene that codes for mucin, has been linked to feeding of a high-fiber diet in pigs (Ferrandis Vila et al., 2018), which is typically associated with increased endogenous nutrient loss from the sloughing of the mucosa. Such losses may be responsible for the increased MUC2 expression noted here as it might be a compensatory mechanism to replace lost mucin. Therefore, it is possible that the lower MUC2 expression in the ileum in the NC + 3,000 could be an indirect indication of stabilization of the mucin layer by this high phytase application. This may suggest additional mechanisms by which phytase is contributing to intestinal health and animal performance through stabilization of the mucin layer. However, because the mucin content of the digesta or brush border was not measured in this study, and MUC2 gene expression was only affected in the ileum, additional work is warranted to fully determine the effects of phytase on mucin turnover.

Glucose absorption into the enterocyte is primarily through SGLT1 (Pappenheimer, 2001) and potentially through basolateral to apical cycling of GLUT2 (Kellett, 2001). The expression of both transporters is increased in the gut in response to greater substrate availability (Miyamoto et al., 1993). Phytase supplementation, especially at 3,000 FTU/kg feed, increased the expression of GLUT2 in all sections of the GIT and significantly increased SGLT1 expression in the jejunum. Although starch digestibility is high in the pig (Knudsen et al., 1993), it is unknown if the expression of SGLT1 and GLUT2 could be limiting maximal glucose absorption in nursery pigs, and our results indicate a possibility that phytase supplementation could be enhancing the glucose uptake through increased expression of these transporters. However, the timing of measurement of blood glucose concentration in this experiment did not allow an accurate assessment of potential phytase effect on glucose absorption. The claudins are important tight junction proteins that regulate paracellular permeability (Garcia-Hernandez et al., 2017) of intestinal epithelial cells. Reduction in expression of these proteins is associated with compromised intestinal tight junctions and reduced animal performance (Pinton et al., 2010; Xia et al., 2019). Phytase appears to alter the composition of the tight junction claudins by increasing the expression of claudin 3 (CLDN3) in the duodenum and jejunum and the tendency to increase it in the ileum (especially at the higher level of phytase), and by decreasing the expression of claudin 1 in the jejunum and ileum at NC + 3,000. The lack of effect of phytase on claudin 4 may indicate that the effects of phytase on claudin genes are very specific to each claudin. Phytase protects the intestinal barrier by reducing phytate-induced mucin loss (Onyango et al., 2009), and this may represent a potential mechanism by which phytase helps to maintain intestinal integrity. Changes in epithelial integrity may also be coupled to changes in tight junction gene expression, and we speculate that the regulation of expression of tight junction genes may represent another mechanism by which phytase protects the intestinal epithelium. Additionally, phytase leads to the release of myo-inositol, a precursor for membrane phosphatidylinositol, a component of membrane phospholipids (Huber, 2016). The relative proportion of the membrane phospholipids (phosphatidylserine, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine) affects membrane properties, such as fluidity and receptor expression (Fajardo et al., 2011). Alteration of membrane phospholipid composition may also affect the integrity of the membrane and expression of tight junction proteins. Additional studies are needed on the potential mechanism of phytase regulation of claudin genes and the implication on tight junction integrity. The downregulation of the myo-inositol transporter (SLC2A13) in the duodenum and that of ALPI in all three sections of the GIT could be a feedback mechanism to limit the excessive uptake of myo-inositol or P in the presence of elevated concentrations of these molecules in the GIT.

In conclusion, the use of elevated dietary phytase levels in low-P diets results in increased growth performance and P utilization in nursery pigs, which is accompanied by a near-complete breakdown of InsP₆ and elevation of circulating myo-inositol concentrations. Phytase may also be regulating glucose transport through the upregulation of GLUT2 expression. Finally, phytase may be altering intestinal brush border mucin and tight junction composition through changes in the MUC2, CLDN1, and CLDN3 gene expression. Future functional studies are needed to decipher how phytase may regulate epithelial tight junction integrity and permeability and nutrient transport across the intestinal brush border.

Glossary

Abbreviations

AID

apparent ileal digestibility

ALPI

alkaline phosphatase, intestinal

ASCT2

solute carrier family 1 (neutral amino acid transporter) member 5

ATTD

apparent total tract digestibility

BW

body weight

CLDN1

claudin 1

CLDN3

claudin 3

CLDN4

claudin 4

DM

dry matter

FI

feed intake

G:F

feed efficiency

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

GE

gross energy

GIT

gastronintestinal tract

GLUT2

solute carrier family 2-facilitated glucose transporter member 2

GLUT5

solute carrier family 2-facilitated glucose/fructose transporter, member 5

GSTM4

glutathione S-transferase mu 4

InPs

inositol phosphates

MUC2

mucin 2 oligomeric mucus/gel-forming

PEPT1

solute carrier family 15 member 1, peptide transporter

RNA

ribonucleic acid

SGLT1

solute carrier family 5-sodium/glucose cotransporter member 1

SLC2A13

H⁽⁺⁾-myo-inositol symporter

SLC5A3

solute carrier family 5, inositol transporters, member 3

STTD

standardized total tract digestibility

Conflict of interest statement

The authors disclose that there was no conflict of interest.