P is poorly utilised by pigs because approximately two-thirds of P in feedstuffs of plant origin is present as phytate, which is largely unavailable for hydrolysis in the digestive tract( Reference Viveros, Centeno and Brenes 1 ). The phytate molecule is also capable of binding to other nutrients including starch and proteins, thus preventing their absorption( Reference Noureddini and Dang 2 ). The enzyme phytase (PHY) has been shown to improve P availability and subsequently improve growth, feed efficiency, nutrient utilisation and bone mineralisation to levels comparable to those induced by a diet supplemented with inorganic P, but to reduce nutrient excretion levels( Reference Varley, Callan and O'Doherty 3 ).
Studies have shown that the dietary inclusion of PHY has an effect on the availability of a number of nutrients( Reference Harper, Kornegay and Schell 4 ). The inclusion of PHY improves the digestibility of amino acids, starch, Ca, gross energy (GE), Fe, fat and Zn( Reference Selle, Cowieson and Cowieson 5 – Reference Lei, Ku and Miller 9 ). The action of PHY is likely to increase the availability of free nutrients including amino acids, peptides, fatty acids, glucose, galactose and fructose in the digesta as the phytate molecule undergoes hydrolysis and the bound starch, protein and fat are released. Although it is apparent that nutrient digestibility improves with the dietary inclusion of exogenous PHY, the physiological response of intestinal nutrient transporters to PHY is not well characterised.
Intestinal enterocytes are constantly exposed to fluctuations in dietary nutrients and therefore have to respond to fluctuations in luminal nutrients( Reference Dyer, Vayro and Shirazi-Beechey 10 ). Fluctuations in nutrient availability may affect the gene expression of nutrient transporters through a mechanism of nutrient sensing( Reference Dyer, Salmon and Zibrik 11 ). Nutrient transporters, expressed on the apical membrane of intestinal absorptive cells, are directly exposed to an environment that changes significantly with diet, and consequently their expression is adaptively regulated by dietary substrates( Reference Dyer, Al-Rammahi and Waterfall 12 ).
The hypothesis that improvements in growth performance, skeletal bone mineralisation and nutrient digestibility following supplementation of a low-P (LP) diet with PHY are accompanied by changes in the gene expression of intestinal nutrient transporters involved in peptide, mineral, carbohydrate and fatty acid transport was tested in the present study.
Materials and methods
All procedures used in the present experiment were conducted under experimental licence from the Irish Department of Health in accordance with the Cruelty to Animals Act 1876 and the European Communities (Amendment of the Cruelty to Animals Act 1876) Regulations, 1994.
Experimental design and diets
An experiment with a completely randomised design was carried out to investigate the effects of three dietary treatments on growth performance, coefficient of apparent ileal digestibility (CAID), coefficient of apparent total tract digestibility (CATTD), bone mineral accretion and intestinal nutrient transporter gene expression in growing pigs (43 kg body weight). The experimental period was divided into two components delineated by a diet change to match the requirements of pigs as they progressed from a phase when fed a nutritionally rich diet after weaning (days 0–23) to a phase when fed a lower-specification diet (days 23–44)( Reference Varley, Sweeney and Ryan 13 ). The composition and chemical analysis of the experimental diets are summarised in Table 1. Dietary treatments during period 1 (days 0–23) were as follows: (1) a high-P (HP) diet containing 5·9 g/kg total P, 3·4 g/kg available P and 7 g/kg Ca; (2) a LP diet containing 4·9 g/kg total P, 1·9 g/kg available P and 5·8 g/kg Ca; (3) a PHY diet containing LP diet ingredients+1020 phytase units (FTU)/kg of PHY (Ronozyme®, DSM Nutritional Products Limited). Dietary treatments during period 2 (days 23–44) were as follows: (1) a HP diet containing 5·9 g/kg total P, 3 g/kg available P and 6·7 g/kg Ca; (2) a LP diet containing 4·1 g/kg total P, 1·7 g/kg available P and 4·7 g/kg Ca; (3) a PHY diet containing LP diet ingredients+1030 FTU/kg of PHY. The HP diet was designed to match the standards set out by the NRC( 14 ) for Ca and P levels, while the LP diet was designed to contain both Ca and P levels 16 % lower than those outlined by the NRC during period 1 and 30 % lower during period 2( 14 ). All diets were designed to contain similar levels of standard ileal digestible lysine (13·3 and 11·1 g/kg during periods 1 and 2, respectively) and digestible energy (14·7 and 14·3 MJ/kg during periods 1 and 2, respectively). All diets were offered in meal form.
Table 1 Composition and chemical analysis of the experimental diets (as-fed basis; g/kg unless otherwise stated)
HP, high P; LP, low P; PHY, phytase; FTU, phytase units.
* The premix provided vitamins and minerals (per kg diet) as follows: Cu 25; Zn 100; Se 0·3; Mn 25; I 0·2; retinol 0·3; cholecalciferol 0·05; α-tocopherol 40.
† Calculated for the tabulated nutritional composition( Reference Sauvant, Perez and Tran 48 ).
Experimental protocol
A total of forty-eight pigs (Meatline boars × (Large White × Landrace) sows) with an average initial body weight of 11·76 (sd 0·75) kg were used in a 44 d weaner performance study. Pigs were penned in mixed-sex groups of two (eight replicates per treatment) on fully slatted floors (1·68 m × 1·22 m). Feed and water were available ad libitum with precaution taken to avoid feed wastage. Feed was kept in the feeders until the time the pigs were weighed, and then the feed was weighed again to calculate the feed conversion ratio. Pigs were weighed weekly. The ambient environmental temperature within the houses was thermostatically controlled. The temperature was set at 28°C during the 1st week and was reduced by 2°C per week to 22°C. Multiple fresh faecal samples were collected daily from all pens on days 10–15 during period 1 and on days 25–30 during period 2 and stored in sterile containers (Sarstedt). During the experiment, feed samples were collected at the time of feeding and stored until chemical analysis. Celite (300 mg/kg) was added to the feed during manufacture to measure the CAID and CATTD using the acid-insoluble ash technique( Reference McCarthy, Bowland and Aherne 15 ). On day 44, the male pig from each pen was slaughtered. Pigs were killed by lethal injection of Euthatal (pentobarbitone sodium BP; Merial Animal Limited) at a rate of 1 ml/1·4 kg body weight. After slaughter, the right front foot of twenty-four pigs was cleaned of all skin, muscle and connective tissue to remove the third and fourth metacarpals. Following removal, the metacarpals were again cleaned of any remaining flesh. These bones were subsequently used for the assessment of bone ash, P, and Ca levels and bone density. All metacarpals collected were individually stored at − 20°C to prevent desiccation until analysis.
Following a 3 h fast and slaughter, the entire digestive tract was removed by blunt dissection. Following tract removal, digesta samples were recovered aseptically from the ileum as sections of approximately 30 cm in length from the ileocaecal valve to measure the CAID of nutrients (N, DM and GE). Tissue samples from the jejunum (60 cm from the stomach) and ileum (8 cm from the ileocaecal valve) were collected and emptied by dissecting along the mesentery and rinsing using sterile PBS (Oxoid) as described previously( Reference Heim, Walsh and Sweeney 16 , Reference Sweeney, Collins and Reilly 17 ). Sections measuring 1 cm2, which had been stripped of the overlying smooth muscle, were cut from the tissue samples and stored in RNAlater solution (Applied Biosystems) overnight at 4°C. RNAlater was removed later, and tissue samples were stored at − 70°C until RNA extraction.
Laboratory analysis of samples
Feed and faecal samples were analysed for N, DM, organic matter, ash, GE and neutral-detergent fibre. Following collection, faecal samples were dried at 100°C for 48 h. Feed and dried faecal samples were milled through a 1 mm screen (Christy and Norris Hammer Mill). Diet, faecal and digesta samples were analysed for DM (method 934.01) and crude ash (method 942.05) according to the AOAC( 18 ). The DM content was determined after drying for 24 h at 100°C. The crude ash content was determined after ignition of a weighed sample in a muffle furnace (Nabertherm) at 550°C for 6 h. The ash was then digested in aqua regia (HCl–HNO3 mixture). In the first round of digestion, ash was digested using 20 % aqua regia (4:1 HCl and nitric acid (v/v)) and subsequently digested using 25 % aqua regia. This solution was used for the determination of P and Ca concentrations. The concentration of Ca in feed, digesta and faecal samples was determined using an atomic absorption spectrophotometer (Varian 50; Varian, Inc.) using the method of Ramakrishna & Robinson( Reference Ramakrishna and Robinson 19 ). The concentration of P was determined spectrophotometrically (PU 8600 UV/visible spectrophotometer, Pye Unicam, Philips) using the method of Cavell( Reference Cavell 20 ). The GE was determined using an adiabatic bomb calorimeter (Parr Instruments) as described by O'Shea et al. ( Reference O'Shea, McAlpine and Sweeney 21 ). The neutral-detergent fibre fraction was analysed using the Fibertec Extraction Unit (Fibertec, Tecator) as described by Van Soest et al. ( Reference Van Soest, Robertson and Lewis 22 ). The concentration of N in diet, faecal and digesta samples was determined using a LECO FP 528 (Leco Instruments (U.K.) Limited) as described by O'Shea et al. ( Reference O'Shea, McAlpine and Sweeney 21 ). The concentration of acid-insoluble ash was determined according to the method of McCarthy et al. ( Reference McCarthy, Aherne and Okai 23 ). Feed samples were analysed for PHY activity according to the method reported by Brady et al. ( Reference Brady, Callan and Cowan 24 ). PHY activity is expressed as FTU per unit of feed and is defined as the quantity of enzyme that liberates 1 μmol of inorganic P per min from a 1·5 mmol/1 solution of sodium phytate at pH 5·5 and 37°C( Reference Engelen, van der Heeft and Randsdorp 25 ).
Bone analysis
Bone samples were analysed for DM, density, ash, Ca and P. Bone density was calculated using a balance (Scout Pro balance 200 g × 0·01 g, Ohaus Limited). The samples were first weighed in air and then weighed by submerging in distilled water using the integral weigh-below hook facility. Bone volume was calculated by subtracting the wet weight from the dry weight, and bone density was determined by dividing the dry weight by the volume( Reference Giancoli, Corey and Mullaney 26 ). The samples were placed in an oven at 100°C for 16 h to determine DM weight. The samples were then ashed at 650°C in a muffle furnace, and the ash was digested in aqua regia (HCl–HNO3 mixture) and analysed for Ca and P as described above for faecal and digesta samples.
RNA extraction and real-time RT-PCR
Total RNA was extracted from ileal and jejunal samples (25 mg) using TRIzol Reagent (Sigma-Aldrich) according to the manufacturer's instructions. The crude RNA extract was further purified using the GenElute Mammalian Total RNA Miniprep Kit (RTN70, Sigma-Aldrich) according to the manufacturer's instructions. A DNase removal step was included (DNASE7-E70, Sigma-Aldrich). The total RNA was quantified using a NanoDrop-ND1000 spectrophotometer (Thermo Fisher Scientific, Inc.). The purity of RNA was assessed by determining the ratio of the absorbance at 260 nm to that at 280 nm. All total RNA samples had 260:280 nm ratios above 1·8. In addition, the integrity of RNA was verified using the Agilent RNA 6000 NanoChip Bioanalyzer Kit (Agilent Technologies). All samples had a RNA integrity number above 8 (average 8·3 (se 0·59)). Total RNA (1 μg) was reverse-transcribed using a commercially available complementary DNA synthesis kit (First Strand cDNA Synthesis Kit, Fermentas) using oligo-deoxy-thymine (dT) primers in a final reaction volume of 20 μl according to the manufacturer's instructions, and minus-RT and no-template controls were included. The final reverse transcription product was adjusted to a volume of 120 μl using nuclease-free water. The mRNA expression profiles of selected candidate genes were analysed by quantitative real-time PCR using the ABI Prism 7500 FAST Sequence Detection System (Applied Biosystems). PCR amplification was performed in a total volume of 20 μl containing 10 μl of master mix (SYBR PCR Master Mix, Applied Biosystems), 1·0 μl of forward and reverse primers (300 pm final), 6·5 μl of RNAse-free water, and 2·5 μl of template complementary DNA (5·0 ng of RNA equivalents). The two-step PCR programme was as follows: 95°C for 10 min for one cycle, followed by 95°C for 15 s and 60°C for 1 min for forty cycles. All reactions were performed in duplicate. Primers were designed for each gene of interest (Primer Express Software version 2.0, Applied Biosystems), and the specificity of all primers was confirmed by melting curve analysis. Primer efficiency was determined using a serial dilution of Sus scrofa-derived complementary DNA (1:4 dilution series over seven points). Primers for all the selected nutrient transporters (peptide transporter 1 (PEPT1/SLC15A1); sodium–glucose-linked transporter 1 (SGLT1/SLC5A1); GLUT2/SLC2A2, GLUT5/SLC2A5, GLUT7/SLC2A7, and GLUT8/SLC2A8; fatty acid-binding protein 2 (FABP2); Fe-regulated transporter (SLC40A1); cluster of differentiation 36/fatty acid translocase (CD36/FAT); membrane Ca channel (TRPV6); Ca-binding protein (calbindin); plasma membrane Ca2+ ATPase (PMCA1) and vitamin D receptor (VDR)) are given in Table 2. The optimal number of reference targets for this sample set was identified using the geNorm application within the qbasePLUS software package( Reference Hellemans, Mortier and De Paepe 27 ) (Biogazelle) and confirmed for the present study (geNorm V< 0·15). The normalisation factor was calculated as the geometric mean of the reference targets glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hydroxymethylbilane synthase (HMBS). Calibrated normalised relative quantities of gene expression for each analysed sample were generated using the qbasePLUS package (Biogazelle) and incorporated efficiency correction.
Table 2 Porcine specific primers used for real-time PCR
GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HMBS, hydroxymethylbilane synthase; PEPT, peptide transporter; SGLT, sodium–glucose-linked transporter; FABP, fatty acid-binding protein; CD, cluster of differentiation; VDR, vitamin D receptor; TRPV6, membrane Ca channel; PMCA1, plasma membrane Ca2+ ATPase; SLC, solute carrier.
Statistical analysis
Data were analysed as a completely randomised block design using the general linear model procedure of SAS (SAS Institute, Inc.)( 28 ). The pen served as the experimental unit for performance and CATTD. For all the other parameters, the individual pig served as the experimental unit. Data were checked for normality using the PROC UNIVARIATE function of SAS (SAS Institute, Inc.). All data presented in the tables are expressed as least-squares means with their standard errors. Means were separated using the Tukey–Kramer method. P values < 0·05 were considered to be statistically significant.
Results
Growth performance
The effect of PHY and P levels on the growth performance of pigs is summarised in Table 3. At the end of the experiment, pigs fed the LP diet had lower overall average daily gain (P< 0·05) and final body weight (P< 0·01) and an increased feed conversion ratio (P< 0·05) compared with those fed the HP and PHY diets.
Table 3 Effect of phosphorus and phytase (PHY) levels on the growth performance of pigs during period 1 (days 0–23) and period 2 (days 23–44) (Least-squares means with their standard errors)
HP, high P; LP, low P; BW, body weight; ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio.
a,bLeast-squares mean values within a row with unlike superscript letters were significantly different (P< 0·05).
* A total of eight replicates were used per treatment.
Apparent ileal digestibility
The effect of PHY and P levels on the CAID of pigs is summarised in Table 4. Pigs fed the PHY diet had higher CAID of GE (P< 0·001) and N (P< 0·05) compared with those fed the HP diet. Pigs fed the PHY diet had higher CAID of P (P< 0·01), ash (P< 0·05) and GE (P< 0·001) compared with those fed the LP diet.
Table 4 Effect of phosphorus and phytase (PHY) levels on the coefficient of apparent total tract digestibility (CATTD) (periods 1 and 2) and the coefficient of apparent ileal digestibility (CAID) of pigs (after slaughter (day 43)) (Least-squares means with their standard errors)
HP, high P; LP, low P.
a,b,cLeast-squares mean values within a row with unlike superscript letters were significantly different (P< 0·05).
* CATTD was calculated based on data obtained from two pigs in each of the eight pens, while ileal digestibility was calculated based on data obtained from eight individual pigs after slaughter.
Total tract digestibility
The effect of PHY and P levels on the CATTD of pigs is summarised in Table 4. Pigs fed the PHY diet had higher CATTD of Ca, P and ash compared with those fed the HP and LP diets (P< 0·001) during periods 1 and 2. Pigs fed the LP diet had a lower CATTD of P compared with those fed the HP diet (P< 0·001) during periods 1 and 2.
Bone parameters
The effect of PHY and P levels on the bone parameters of pigs is summarised in Table 5. Pigs fed the LP diet had decreased bone ash content (P< 0·001), bone P content (P< 0·01) and bone density (P< 0·01) compared with those fed the HP and PHY diets.
Table 5 Effect of phosphorus and phytase (PHY) levels on bone parameters in the fourth metacarpal of the right front leg of pigs during slaughter (day 43) (Least-squares means with their standard errors)
HP, high P; LP, low P.
a,bLeast-squares mean values within a row with unlike superscript letters were significantly different (P< 0·05).
* A total of eight replicates were used per treatment.
† Density was calculated according to the method of Giancoli et al.( Reference Giancoli, Corey and Mullaney 26 ).
Jejunal nutrient transporter gene expression
The effect of PHY and P levels on the jejunal nutrient transporter gene expression of pigs is summarised in Table 6. Pigs fed the PHY diet exhibited a numerical increase in the gene expression of the Ca transporter TRPV6 (P< 0·10) and the P transporter SLC34A2 (P< 0·10) compared with those fed the HP diet. Pigs fed the HP diet exhibited an increased gene expression of the amino acid transporter SLC7A11 compared with those fed the PHY diet (P< 0·05). Pigs fed the HP diet exhibited a numerical increase in the gene expression of the amino acid transporter SLC7A1 compared with those fed the LP diet (P< 0·10).
Table 6 Effect of phosphorus and phytase (PHY) levels on the normalised relative abundance of nutrient transporter mRNA in the jejunal tissue of weaned pigs (Least-squares means with their standard errors)
HP, high P; LP, low P; SGLT1, sodium–glucose-linked transporter 1; PEPT1, peptide transporter 1; CD36, cluster of differentiation 36; FABP2, fatty acid-binding protein 2; VDR, vitamin D receptor; TRPV6, membrane Ca channel; PMCA1, plasma membrane Ca2+ ATPase.
a,bLeast-squares mean values within a row with unlike superscripts were significantly different (P< 0·05).
* A total of eight replicates were used per treatment.
Ileal nutrient transporter gene expression
The effect of PHY and P levels on the ileal nutrient transporter gene expression of pigs is summarised in Table 7. Pigs fed the PHY diet exhibited an increased gene expression of PEPT1 compared with those fed the LP diet (P< 0·05). Pigs fed the PHY diet exhibited a numerical increase in the gene expression of FABP2 (P< 0·10) compared with those fed the LP diet. Pigs fed the LP diet exhibited a lower gene expression of GLUT2 and SGLT1 compared with those fed the HP diet (P< 0·05). Pigs fed the LP diet exhibited an increase in the gene expression of the Ca transporters TRPV6, calbindin and PMCA1 compared with those fed the HP and PHY diets (P< 0·001).
Table 7 Effect of phosphorus and phytase (PHY) levels on the normalised relative abundance of nutrient transporter mRNA in the ileal tissue of weaned pigs (Least-squares means with their standard errors)
HP, high P; LP, low P; SGLT1, sodium–glucose-linked transporter 1; PEPT1, peptide transporter 1; CD36, cluster of differentiation 36; FABP2, fatty acid-binding protein 2; VDR, vitamin D receptor; TRPV6, membrane Ca channel; PMCA1, plasma membrane Ca2+ ATPase.
a,bLeast-squares mean values within a row with unlike superscript letters were significantly different (P< 0·05).
* A total of eight replicates were used per treatment.
Discussion
PHY is used extensively to improve P digestibility in pigs, by increasing the availability of phytate-bound P and improving growth performance and bone mineralisation. PHY supplementation has also been shown to improve the digestibility of a number of nutrients( Reference Harper, Kornegay and Schell 4 ). We hypothesised that improvements in growth performance, skeletal bone mineralisation and nutrient digestibility following supplementation of a LP diet with PHY are accompanied by changes in the gene expression of intestinal nutrient transporters involved in peptide, mineral, carbohydrate and fatty acid transport.
The primary aim of the present study was to investigate the effects of PHY on nutrient and mineral digestibility, growth performance and bone mineralisation and the secondary aim was to investigate its effect on intestinal nutrient transporter gene expression in pigs. The experimental period was divided into two components delineated by a diet change to match the requirements of pigs as they progressed from a phase when fed a nutritionally rich diet after weaning (days 0–23) to a phase when fed a lower-specification diet (days 23–44)( Reference Varley, Sweeney and Ryan 13 ). In the present study, the PHY diet was found to improve the CATTD of Ca and P, the CAID of GE and P, growth performance and bone mineralisation and to increase the CAID of N when compared with the HP diet. The findings of the present study are in agreement with the findings that supplementation of a LP diet with PHY can improve nutrient digestibility, growth performance and bone mineralisation( Reference Harper, Kornegay and Schell 4 , Reference Lei and Stahl 29 , Reference Vats, Bhattacharyya and Banerjee 30 ). These improvements have been attributed to the capacity of PHY to release nutrients chelated by the phytate molecule. The PHY diet increased the CAID of GE compared with the LP diet and increased the CAID of N compared with the HP diet. However, there was no difference in the CATTD of N and GE among the dietary treatment groups, indicating that activity in the large intestine affects the CATTD of N and GE. Similarly, Woyengo et al. ( Reference Woyengo, Sands and Guenter 31 ) suggested that hindgut fermentation masks the effects of PHY. This is important, as nutrients absorbed in the small intestine are used with greater efficiency than those that undergo hindgut fermentation. Nutrients such as amino acids that are not absorbed in the small intestine are of no nutritional value to pigs. Although the effects of PHY on growth performance, nutrient digestibility and bone mineralisation( Reference Varley, Flynn and Callan 32 – Reference Selle, Ravindran and Ravindran 34 ) are well established, the underlying biological mechanisms involved in the uptake of nutrients following the degradation of the phytate molecule are not well understood. Intestinal enterocytes respond to fluctuations in intestinal nutrients by modifying the gene expression of intestinal nutrient transporters( Reference Dyer, Vayro and Shirazi-Beechey 10 , Reference Dyer, Salmon and Zibrik 11 ). This indicates that intestinal enterocytes can up-regulate the gene expression of nutrient transporters in response to increased nutrient availability. In previous studies, improvements in nutrient digestibility have been found to be accompanied by improvements in intestinal nutrient transporter gene expression( Reference Heim, Walsh and Sweeney 16 ). Heim et al. ( Reference Heim, Walsh and Sweeney 16 ) showed that changes in the expression of intestinal GLUT are directly related to GE digestibility. Similarly, changes in crude protein digestibility have been shown to be accompanied by changes in the gene expression of amino acid and peptide transporters( Reference Wang, Zeng and Feng 35 ). Therefore, in the present study, we aimed to investigate whether dietary PHY inclusion increases the expression of glucose, galactose, fructose, amino acid, peptide, fatty acid, vitamin and mineral transporters. Although not significant, the PHY diet was found to numerically increase ileal N digestibility compared with the LP diet in the present study. The ileal mRNA expression of PEPT1 was increased in pigs fed the PHY diet compared with that in pigs fed the HP diet. PEPT1 is a major transporter involved in the absorption of the products of protein digestion across the intestinal apical membrane. This indicates that the gene expression of this transporter was up-regulated in response to the increase in N availability as the phytate molecule was hydrolysed in situ ( Reference Fei 36 ). Although PHY supplementation increased the gene expression of PEPT1, no effect was observed on the gene expression of the apical membrane amino acid transporters (SLC1A4, SLC6A19 and SLC7A1) or the basolateral transporters (SLC7A11 and SLC1A2). This indicates that the increased N availability observed in the present study could be partially influenced by the increased gene expression of PEPT1 ( Reference Gilbert, Wong and Webb 37 ). This is in agreement with the findings of previous studies where increases in crude protein digestibility were found to be accompanied by changes in PEPT1 expression( Reference Wang, Zeng and Feng 35 ). The HP diet increased the jejunal gene expression of the apical membrane transporter SLC7A1 compared with the LP diet and increased the jejunal gene expression of SLC7A11 compared with the PHY diet.
The HP diet up-regulated the gene expression of SGLT1 compared with the LP diet. The intestinal GLUT SGLT1 is the major transporter involved in the absorption of glucose and other sugars across the luminal membrane of porcine enterocytes( Reference Moran, Al-Rammahi and Arora 38 ). The high levels of Ca and P present in the inorganic P supplement may inhibit the ability of the phytate molecule to bind to free glucose, as observed when feeding the LP diet, causing an increased gene expression of SGLT1 ( Reference Yoon, Thompson and Jenkins 39 ). In a previous experiment, pigs supplemented with phytic acid have been found to exhibit a reduced expression of SGLT1 ( Reference Woyengo, Rodriguez-Lecompte and Adeola 40 ). This indicates that phytate chelates free glucose and makes it unavailable for digestion, which in turn causes the low expression observed when feeding the LP diet. Following the increased gene expression of the apical membrane transporter SGLT1, an increase in the gene expression of GLUT2, which is expressed on the basolateral membrane of intestinal enterocytes, is to be expected( Reference Moran, Al-Rammahi and Arora 38 ). The gene expression of GLUT2 was up-regulated in pigs fed the HP diet when compared with that in pigs fed the LP diet. SGLT1 and GLUT2 together are effectively responsible for glucose absorption( Reference Kellett and Brot-Laroche 41 ). The increased gene expression of GLUT2 indicates increased glucose availability from the HP diet, possibly due to the inhibition of the ability of the phytate molecule to bind to free glucose by the inorganic P supplement.
Similar to that observed for N digestibility, PHY supplementation was found to increase the CAID of GE in the present study. However, PHY supplementation had no effect on the gene expression of the studied GLUT. The increase in the gene expression of FABP2 indicates that PHY was effective at reducing the ability of the phytate molecule to bind to free fat and make it unavailable and could in part explain the increase in the CAID of GE. The fact that PHY has the ability to increase the availability of fat( Reference Selle, Ravindran and Ravindran 8 ) and up-regulate the expression of the fatty acid transporter FABP2, as observed in the present study, indicates a role for it in increasing the availability of fat. FABP found in the brush border membranes of enterocytes may play a role in fatty acid uptake( Reference Zaefarian, Romero and Ravindran 42 ). In a recent study in broilers, PHY supplementation has been found to improve the ileal digestibility of fat along with that of the fat constituents, SCFA and unsaturated fatty acids( Reference Zaefarian, Romero and Ravindran 42 ). The formation of mineral–phytate complexes has been reported to prevent the utilisation of lipids. PHY supplementation may increase fat digestibility by reducing the formation of soaps in the gut( Reference Ravindran, Selle and Ravindran 43 ).
Ca is a mineral that is known to be chelated by the phytate molecule( Reference Graf 44 ). During period 1, the PHY diet was found to improve the CATTD of Ca by 27 and 29 % compared with the LP and HP diets, respectively. Similarly during period 2, the PHY diet was found to improve the CATTD of Ca by 31 and 28 % compared with the LP and HP diets, respectively. Due to low levels of Ca and P in the LP diet used in the present study, pigs fed the LP diet exhibited reduced growth and bone mineralisation. PHY supplementation ameliorated the negative effects of the LP diet. The mineral transporters TRPV6, calbindin and PMCA1 were studied as these three Ca transporters are involved in transcellular Ca transport (uptake, intracellular movement, and extrusion)( Reference Song, Peng and Porta 45 ). A numerical increase was observed in the gene expression of the basolateral Ca transporter PMCA1 in the jejunum of pigs fed the PHY diet when compared with that in pigs fed the HP diet. These results indicate that the trend of an increased gene expression of TRPV6 could partially explain the increased digestibility of Ca observed in the present study. The gene expression of the Ca channel TRPV6 was increased in the ileum of pigs fed the LP diet when compared with that in pigs fed the other two diets. Similarly, an increased expression of calbindin was observed in pigs fed the LP diet, and a significant trend towards an increased gene expression of the basolateral Ca transporter PMCA1 was also observed when comparing pigs fed the LP diet with those fed the HP and PHY diets. These results are in agreement with the results of the study carried out by Li( Reference Li 46 ), where low levels of Ca were found to up-regulate the expression of calbindin in broilers. It has previously been established that low levels of dietary Ca can lead to an increased expression of PMCA1 and calbindin in the kidney of mice( Reference Song, Peng and Porta 45 ). These results indicate that animals deficient in Ca respond by up-regulating the expression of Ca transporters to maximise the utilisation of available Ca in their ileum. In previous experiments( Reference Yoon, Thompson and Jenkins 39 ), it has been shown that the degradation of the phytate molecule predominantly occurs in the stomach and proximal small intestine. This mechanism could explain why the studied Ca transporters were differentially expressed in the ileum and pigs fed the HP and PHY diets exhibited a lower expression. The majority of Ca absorption in pigs fed the HP and PHY diets will take place in the proximal region of the small intestine due to the easy absorption of Ca from the inorganic source and the increased availability of Ca following the degradation of the phytate molecule. This is supported by the trend towards an increased expression of TRPV6 in the jejunum.
Similar to that observed for Ca digestibility, the PHY diet was found to improve the CATTD of P by 33 and 22 % compared with the LP and HP diets during period 1, respectively. During period 2, the PHY diet was found to improve the CATTD of P by 33 and 17 % compared with the LP and HP diets, respectively. The PHY diet improved ileal P digestibility by 25 % compared with the LP diet. A trend towards an increased gene expression of the transporter SLC34A2 was observed in the jejunum of pigs fed the PHY diet compared with the expression in those fed the HP diet. This increased gene expression could partially explain the increased P digestibility observed in the present study. In the present study, these transporters were not found to be differentially expressed in the ileum. The majority of P absorption in pigs occurs through the transcellular route and some P is transported by intestinal transporters( Reference Fan, Shen and Yin 47 ). The majority of P present in both HP and PHY diets will be released and absorbed in the proximal region of the intestine.
In summary, the PHY diet improved growth performance and bone mineralisation when compared with the HP diet and improved the ileal digestibility of GE and total tract nutrient digestibility of Ca and P when compared with the LP diet. The PHY diet improved the gene expression of the peptide transporter PEPT1, the Ca transporter TRPV6, the P transporter SLC34A2 and the fatty acid transporter FABP2 compared with the LP diet. The increase in the gene expression of these nutrient transporters indicates that intestinal nutrient transporter gene expression is a mechanism involved in the uptake of nutrients following the degradation of the phytate molecule.
Acknowledgements
The authors thank Ms Bernie Flynn, Mr Paddy Reilly, Mr James Callan and Ms Denise Cunningham for their technical assistance.
The present study was funded under the Earth and Natural Sciences Doctoral Studies Programme, which is funded under the Programme for Research in Third-Level Institutions and co-funded under the European Regional Development Fund.
The authors' contributions are as follows: S. V., T. S., C. J. O., J. A. B. and J. V. O. contributed to the conception and design of the study and the analysis and interpretation of the data; S. V. and J. V. O. wrote and edited the article.
None of the authors has any conflicts of interest to declare.
