Transport of valine across the small intestinal epithelium in pigs fed different valine levels and Bacillus subtilis.

Mutants of Bacillus subtilis overproducing valine (B. subtilis VAL) could be an approach to supply pigs dietary valine (Val). In the study, 18 gilts were fed: (i) negative diet with a standardized ileal digestible (SID) Val:Lys of 0.63:1 (Neg); (ii) Neg added B. subtilis VAL (1.28 × 1011  cfu/kg as-fed) or; (iii) Neg added L-Val to a Val:Lys of 0.69:1. Using the Ussing chamber method, the study aimed to investigate whether (i) the diets affect intestinal transport of additions of 0, 5, 10 or 20 mmol Val/L from the mucosal to the serosal side and (ii) the B. subtilis VAL contributes to a net transport of Val produced in situ. The results showed that the Isc (ΔIscVal ) and release of Val to the serosal side solution (Srel ; µmol cm-2  min-1 ) increased with Val addition (linear and quadratic, p < .0001) but was similar for 5, 10 or 20 mmol Val/L and not affected by diet. No net transport of in situ produced Val by B. subtilis VAL was detected. In conclusion, feeding a Val-deficient diet with or without B. subtilis VAL or a Val sufficient diet did not affect the Val transport across intestinal epithelia. No in situ Val production by B. subtilis VAL was observed in the Ussing chambers.

Evaluation of in situ valine production by Bacillus subtilis in young pigs.

Mutants of Bacillus subtilis can be developed to overproduce Val in vitro. It was hypothesized that addition of Bacillus subtilis mutants to pig diets can be a strategy to supply the animal with Val. The objective was to investigate the effect of Bacillus subtilis mutants on growth performance and blood amino acid (AA) concentrations when fed to piglets. Experiment 1 included 18 pigs (15.0±1.1 kg) fed one of three diets containing either 0.63 or 0.69 standardized ileal digestible (SID) Val : Lys, or 0.63 SID Val : Lys supplemented with a Bacillus subtilis mutant (mutant 1). Blood samples were obtained 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding and analyzed for AAs. In Experiment 2, 80 piglets (9.1±1.1 kg) were fed one of four diets containing 0.63 or 0.67 SID Val : Lys, or 0.63 SID Val : Lys supplemented with another Bacillus subtilis mutant (mutant 2) or its parent wild type. Average daily feed intake, daily weight gain and feed conversion ratio were measured on days 7, 14 and 21. On day 17, blood samples were taken and analyzed for AAs. On days 24 to 26, six pigs from each dietary treatment were fitted with a permanent jugular vein catheter, and blood samples were taken for AA analysis 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding. In experiment 1, Bacillus subtilis mutant 1 tended (P<0.10) to increase the plasma levels of Val at 2 and 3 h post-feeding, but this was not confirmed in Experiment 2. In Experiment 2, Bacillus subtilis mutant 2 and the wild type did not result in a growth performance different from the negative and positive controls. In conclusion, results obtained with the mutant strains of Bacillus subtilis were not better than results obtained with the wild-type strain, and for both strains, the results were not different than the negative control.

Microbial phytase addition resulted in a greater increase in phosphorus digestibility in dry-fed compared with liquid-fed non-heat-treated wheat-barley-maize diets for pigs.

The objective was to evaluate the effect of microbial phytase (1250 FTU/kg diet with 88% dry matter (DM)) on apparent total tract digestibility (ATTD) of phosphorus (P) in pigs fed a dry or soaked diet. Twenty-four pigs (65±3 kg) from six litters were used. Pigs were housed in metabolism crates and fed one of four diets for 12 days; 5 days for adaptation and 7 days for total, but separate collection of feces and urine. The basal diet was composed of wheat, barley, maize, soybean meal and no mineral phosphate. Dietary treatments were: basal dry-fed diet (BDD), BDD with microbial phytase (BDD+phy), BDD soaked for 24 h at 20°C before feeding (BDS) and BDS with microbial phytase (BDS+phy). Supplementation of microbial phytase increased ATTD of DM and crude protein (N×6.25) by 2 and 3 percentage units (P<0.0001; P<0.001), respectively. The ATTD of P was affected by the interaction between microbial phytase and soaking (P=0.02). This was due to a greater increase in ATTD of P by soaking of the diet containing solely plant phytase compared with the diet supplemented with microbial phytase: 35%, 65%, 44% and 68% for BDD, BDD+phy, BSD and BSD+phy, respectively. As such, supplementation of microbial phytase increased ATTD of P in the dry-fed diet, but not in the soaked diet. The higher ATTD of P for BDS compared with BDD resulted from the degradation of 54% of the phytate in BDS by wheat and barley phytases during soaking. On the other hand, soaking of BDS+phy did not increase ATTD of P significantly compared with BDD+phy despite that 76% of the phytate in BDS+phy was degraded before feeding. In conclusion, soaking of BDS containing solely plant phytase provided a great potential for increasing ATTD of P. However, this potential was not present when microbial phytase (1250 FTU/kg diet) was supplemented, most likely because soaking of BDS+phy for 24 h at 20°C did not result in a complete degradation of phytate before feeding.

Effect of particle size and microbial phytase on phytate degradation in incubated maize and soybean meal.

The objective of the study was to evaluate the effect of screen size (1, 2 and 3 mm) and microbial phytase (0 and 1000 FTU/kg as-fed) on phytate degradation in maize (100% maize), soybean meal (100% SBM) and maize-SBM (75% maize and 25% SBM) incubated in water for 0, 2, 4, 8 and 24 h at 38°C. Samples were analysed for pH, dry matter and phytate phosphorus (P). Particle size distribution (PSD) and average particle size (APS) of samples were measured by the Laser Diffraction and Bygholm method. PSD differed between the two methods, whereas APS was similar. Decreasing screen size from 3 to 1 mm reduced APS by 48% in maize, 30% in SBM and 26% in maize-SBM. No interaction between screen size and microbial phytase on phytate degradation was observed, but the interaction between microbial phytase and incubation time was significant ( P<0.001). This was because microbial phytase reduced phytate P by 88% in maize, 84% in maize-SBM and 75% in SBM after 2 h of incubation ( P<0.05), whereas the reduction of phytate P was limited (<50%) in the feeds, even after 24 h when no microbial phytase was added. The exponential decay model was fitted to the feeds with microbial phytase to analyse the effect of screen size and feed on microbial phytase efficacy on phytate degradation. The interaction between screen size and feed affected the relative phytate degradation rate ( Rd) of microbial phytase as well as the time to decrease 50% of the phytate P ( t1 =2) ( P<0.001). Thus, changing from 3 to 1 mm screen size increased Rd by 22 and 10%/h and shortened t1 =2 by 0.4 and 0.2 h in maize and maize-SBM, respectively ( P<0.05), but not in SBM. Moreover, the screen size effect was more pronounced in maize and maize-SBM compared with SBM as a higher phytate degradation rate constant (Kd) and Rd, and a shorter t1 =2 was observed in maize compared with SBM in all screen sizes ( P<0.05). However, a higher amount of degraded phytate was achieved in SBM than in maize because of the higher initial phytate P content in SBM. In conclusion, reducing screen size from 3 to 1 mm increased Kd and Rd and decreased t1 =2 in maize and maize-SBM with microbial phytase. The positive effect of grinding on improving microbial phytase efficacy, which was expressed as Kd, Rd and t1/2, was greater in maize than in SBM.

Isoleucine requirement of pigs weighing 8 to 18 kg fed blood cell-free diets.

The aim of the present study was to determine the minimum requirement of Ile in young pigs, enabling feeding of balanced low-CP diets. Most previous studies have used experimental diets that included blood cells, which are particularly high in Leu and known to antagonize the use of Ile. One week after weaning at d 28, 100 crossbred female pigs weighing 7.9 ± 0.7 kg were allocated to 1 of 5 dietary treatments. Diets were formulated to contain 1.15 g standardized ileal digestible (SID) Lys/MJ NE and were free of blood cells. The SID Ile was 0.42, 0.47, 0.53, 0.58, and 0.62 relative to Lys. The other indispensable AA were supplied according to requirements. Representative samples from the 5 diets were analyzed in 4 replicates at 3 different laboratories. The pigs were fed ad libitum and individually housed in 7 identical rooms during a 21-d period. At d 0, 7, 14, and 21, the pigs were weighed, and feed intake was determined. At d 15, blood samples were collected from the jugular vein to determine the plasma urea and free AA content. There were differences among the 3 laboratories in the analyzed content of several AA, and also Ile and Lys showed a large variation within the diets, which may cause variation in published requirement estimates. The concentration of Ile in plasma increased linearly (P < 0.01), and Lys in plasma decreased linearly (P = 0.02) with increasing SID Ile:Lys. A tendency for a linear decrease in plasma concentration was found for Thr (P = 0.10). Both ADFI and ADG were reduced when Ile was supplied above the Ile requirement estimate. Quadratic regression curves on ADFI, ADG, and G:F all showed the maximum at 0.52 SID Ile:Lys. Modeling with 2-sloped quadratic broken-line curves showed the maximum at 0.50, 0.53, and 0.54 SID Ile:Lys for ADFI, ADG, and G:F, respectively. In conclusion, the average estimation of requirement in this dose-response study using blood cell-free diets was 0.52 SID Ile:Lys during a 21-d experimental period from 8 kg BW.

Effect of microbial phytase on phosphorus digestibility in non-heat-treated and heat-treated wheat-barley pig diets.

The objective was to evaluate effects of microbial phytase on apparent total tract digestibility (ATTD) of P in a non-heat-treated and a heat-treated wheat (Triticum aestivum)-barley (Hordeum vulgare) diet fed without inorganic P in a 2 × 3 factorial arrangement. The basal diet was ground and half of the batch was steam pelleted at 81°C and crumbled. Phytase was added at 0, 250, and 500 phytase units (FTU)/kg as-fed (Aspergillus niger). The study comprised 36 pigs from 6 litters. Pigs were housed in metabolism crates and fed 1 of 6 diets for 12 d: 5 d for adaptation and 7 d for total collection of feces. The ATTD of P was highest (P < 0.01) for the non-heat-treated diets and highest (P < 0.01) for the phytase-supplemented diets. Heat treatment reduced plant phytase activity by 25% whereby the ATTD of P decreased (P < 0.01) from 57 to 49%. Microbial phytase increased the ATTD of P to a maximum of 64 and 61% in the non-heat-treated and heat-treated diets corresponding to an increase of 7 and 12%-units. Responses for ATTD of P did not differ between 250 vs. 500 FTU/kg as-fed. In conclusion, processing of feed (meal or pellets) containing plant phytase should be considered to avoid over- or underestimation of effects of microbial phytase.

High-moisture air-tight storage of barley and wheat improves nutrient digestibility.

Barley (Hordeum vulgare) and wheat (Triticum aestivum) are often stored dry with 14% or less moisture, which during rainy periods may require that grains are dried after harvest. The hypothesis is that air-tight storage of high-moisture barley and wheat will increase nutrient digestibility due to chemical conversions prior to feeding. The objective was to evaluate the effect of high moisture compared to dry storage of barley and wheat on digestibility of P and CP. The crops were grown on 1 field keeping other factors constant. Half of the grains was harvested in the morning after a rainy day and stored in air-tight silos (DM, %: barley, 85.2; wheat, 82.8) and the other half was harvested later the same day (windy and sunny) and stored dry (DM, %: barley, 89.8; wheat, 88.3). After 6 mo of storage, 1 low- and 1 high-moisture diet were prepared with a barley:wheat ratio of 1:1 mixed with soybean (Glycine max) meal and rapeseed cake to produce a compound diet without inorganic P and microbial phytase. Sixteen 45-kg pigs housed in metabolism crates were fed either the low- or the high-moisture diet for 5 d for adaptation and 7 d for total collection of feces. Digestibility of P was 12% higher (P < 0.01) and of CP was 4% higher (P = 0.08) in the high-moisture diet. Phytase activity of dry-stored grain was lower (P < 0.01) and phytate P was 4% higher in the high-moisture stored grain vs. the grains stored dry. Overall, high-moisture storage increased digestibility of P and CP when the grain was fed to finishing pigs. Therefore, high-moisture air-tight storage saved energy (without drying) and at the same time enhanced P digestibility and increased the nutritional value of grain probably through enzymatic activity during storage.

The presence of inositol phosphates in gastric pig digesta is affected by time after feeding a nonfermented or fermented liquid wheat- and barley-based diet.

The objective was to quantify the retention of digesta and evaluate the degradation of phytate or inositol hexakisphosphate (InsP(6)) and lower inositol phosphates (InsP5, InsP4, InsP3, and InsP2) in the stomach at different times after feeding pigs a fermented liquid diet with microbial phytase or a nonfermented diet with or without microbial phytase. Six barrows fitted with gastric cannulas were used. The experiment was a 3 × 3 Latin square with 3 pigs fed 3 diets during 3 wk in 2 replicates. Each experimental period lasted for 7 d, comprising 3 d of adaptation and 4 d of total collection of gastric digesta. For each pig, the digesta was collected once daily at 1, 2, 3, or 5 h after feeding the morning meal. A basal wheat- and barley-based diet was steam-pelleted at 90°C. The dietary treatments were a nonfermented basal diet (NF-BD), the NF-BD with microbial phytase (750 phytase units of phytase/kg, as-fed basis; NF-BD + phytase), and the NF-BD + phytase fermented for 17.5 h (F-BD + phytase). Gastric InsP6-P was not detected at all in pigs fed F-BD + phytase because of complete InsP6 degradation during fermentation of the feed before feeding. Gastric InsP6-P decreased over time (P < 0.05) in pigs fed NF-BD and NF-BD + phytase. The decreases were 45, 54, 56, and 61 percentage points greater at 1, 2, 3, and 5 h, respectively, in pigs fed NF-BD + phytase compared with NF-BD. However, substantial amounts of InsP6 still passed into the small intestine in pigs fed NF-BD + phytase, especially within the first hour (estimated to 17% of InsP6-P intake). The accumulation of lower inositol phosphates in gastric digesta was very small for all treatments and at all times because of a rapid and almost complete degradation. In conclusion, phytase addition to the nonfermented diet increased the degradation of gastric InsP6. However, considerable amounts of intact InsP6 still passed into the small intestine because of a shortage of time for InsP6 degradation in the stomach. Therefore, to increase the apparent digestibility of plant P in dry wheat- and barley-based diets, the development of phytases that can degrade InsP6 effectively immediately after ingestion of the feed at an initial gastric pH from 6.5 to 5.0 is needed. Feeding F-BD + phytase compensated for the shortage of time because the InsP6 degradation was completed during fermentation before feeding. The degradation of InsP6 to InsP5 is the bottleneck for plant P utilization in pigs because the degradation of the lower inositol phosphates is rapid and almost complete.

High-performance ion chromatography method for separation and quantification of inositol phosphates in diets and digesta.

A gradient high-performance ion chromatographic method for separation and quantification of inositol phosphates (InsP(2)-InsP(6)) in feedstuffs, diets, gastric and ileal digesta from pigs was developed and validated. The InsP(2)-InsP(6) were separated on a Dionex CarboPac PA1 column using a gradient with 1.5 mol L(-1) methanesulfonic acid and water. The exchange of the commonly used HCl with methanesulfonic acid has two advantages: (i) the obtained baseline during the separation is almost horizontal and (ii) it is not necessary to use an inert HPIC equipment as the methanesulfonic acid is not as aggressive as HCl. Twenty-three of the 27 separated inositol phosphate isomers were isolated. ICP-MS was used for quantification of phosphorus in the isolated isomers and used for calculation of correction factors for each isomer allowing InsP(6) to be used as calibration standard. The detection limits for InsP(2)-InsP(6) were in the range of 0.9-4.4 mg phosphorus L(-1). The recovery of the major part of the inositol phosphates was 80-100%, and the CV for repeatability and reproducibility were 1-17% and 1-14%, respectively.

Heat-treatment, phytase and fermented liquid feeding affect the presence of inositol phosphates in ileal digesta and phosphorus digestibility in pigs fed a wheat and barley diet.

The aim was to evaluate the effect of heat-treatment, microbial phytase addition and feeding strategy (dry feeding v. fermented liquid feeding) on degradation of phytate (myo-inositol hexakisphosphate, InsP6) and formation and further degradation of lower inositol phosphates (myo-inositol pentakisphosphate-myo-inositol bisphosphate, InsP5-InsP2) at the distal ileum of pigs. Furthermore, the apparent ileal digestibility/degradability (AID) of phosphorus (P), InsP6-P and calcium (Ca) and the apparent total tract digestibility (ATTD) of P and Ca were studied. Pigs were fitted with a T-shaped ileal cannula for total collection of digesta at 2 h intervals during an 8 h sampling period after feeding the morning meal. Each period lasted for 2 weeks: 8 days of adaptation followed by 3 days of total collection of faeces and 3 days of total collection of ileal digesta. The experiment was designed as a 4 × 4 Latin square with four pigs fed four diets. A basal wheat/barley-based diet was fed either as non-heat-treated or heat-treated (steam-pelleted at 90°C). The heat-treatment resulted in an inactivation of plant phytase below detectable level. Diet 1 (non-heat-treated basal diet fed dry); diet 2 (heat-treated basal diet fed dry); diet 3 (as diet 2 but with microbial phytase (750 FTU/kg as fed) fed dry); diet 4 (as diet 3 fed liquid (fermented for 17.5 h nighttime and 6.5 h daytime at 20°C with 50% residue in the tank)). Chromic oxide (Cr2O3) was included as marker and ATTD was determined both by total collection of faeces (ATTDTotal) and Cr2O3 (ATTDCr). InsP6 was completely degraded in diet 4 before feeding resulting in no InsP6-P being present in ileal digesta. InsP6-P concentration in ileal digesta decreased with increasing dietary levels of plant or microbial phytase in pigs fed the dry diets. Consequently, AID and ATTD of P and Ca were greatest for pigs fed diet 4 followed by diets 3, 1 and 2. The ATTD of P depended on the used method as ATTDTotal of P was 72%, 61%, 44% and 34%, whereas ATTDCr of P was 65%, 52%, 38% and 23% for diets 4, 3, 1 and 2, respectively. In all pigs the ileal concentration of InsP5-InsP2-P was extremely small, and thus unimportant for maximisation of ATTD of plant P. In conclusion, fermented liquid feeding with microbial phytase seems to be an efficient approach to improve ATTD of plant P compared with dry feeding. This opens up for further reductions in P excretion.