Apparent digestibility (ileal and overall) of nutrients and endogenous nitrogen losses in growing pigs fed wheat (var. Soissons) or its by-products without or with xylanase supplementation

Livestock Production Science 62 (2000) 119–132 www.elsevier.com / locate / livprodsci

Apparent digestibility (ileal and overall) of nutrients and endogenous nitrogen losses in growing pigs fed wheat (var. Soissons) or its by-products without or with xylanase supplementation Y.-L. Yin a,f , J.D.G. McEvoy c , H. Schulze d , U. Hennig e , W.-B. Souffrant e , a,b , K.J. McCracken * a

Department of Agricultural and Environmental Science, The Queen’ s University of Belfast, Newforge Lane, Belfast BT9 5 PX, Northern Ireland, UK b Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5 PX, Northern Ireland, UK c Veterinary Sciences, Department of Agriculture for N. Ireland, Stoney Road, Belfast BT4 3 SD, Northern Ireland, UK d Finnfeeds International Ltd., Marlborough, Wiltshire SN8 1 AA, UK e ¨ ¨ die Biologie Landwirtschaftlicher Nutztiere, Forschungsbereich Ernahrungsphysiologie ‘‘ Oskar Kellner’’, Forschungsinstitut f ur Rostock, Germany f Present address: Department of Animal Science, University of Manitoba, Winnipeg, Canada R3 T 2 N2 Received 28 July 1998; received in revised form 3 February 1999; accepted 15 June 1999

Abstract The main aims of this experiment were: 1) to study the sites and extent of digestion of the nutrients in wheat (var. Soissons) and its by-products (bran, middlings) and in recombined wheat (flour 1 bran 1 middlings); 2) the contribution of microbial action (VFA apparent production) and endogenous secretion of N in the small intestine; and 3) interactions with an exogenous non-starch polysaccharide (NSP) degrading enzyme (xylanase). The post valve ‘‘T’’ caecal cannulae (PVTC, 12 pigs) and end-to-end ileo-rectal anastomosis (IRA, 30 pigs) ileal digesta collection methods with TiO 2 and Cr 2 O 3 as the indigestible markers, and the 15 N-isotope dilution technique were used in this study. Four diets, based on wheat (W), recombined wheat (WR), wheat plus bran (WB) or wheat plus middlings (WM) were used without or with xylanase addition. NSP content was highest in the diet based on wheat bran and lowest in the W and WR diets. Recombination of the milled fractions had no effect on digestibility values relative to the ground wheat. Negative correlations (P , 0.001) occurred for apparent overall and ileal digestibility of dry matter (DM), energy, crude protein (CP) and amino acids with NSP content. As shown in some previous studies, NSP contributed to a higher ileal flow of endogenous nitrogen (P , 0.001) and volatile fatty acids (VFA) (P 5 0.021) and the proportion of DM fermented in the large intestine was increased (P , 0.001). Although statistically significant (P , 0.05), the improvements associated with enzyme addition in overall apparent digestibility of DM, CP and gross energy were only of the order of 1% and those at the ileal level were less than 2%

*Corresponding author. Tel.: 144-1232-255-368; fax: 144-1232-662-007. E-mail address: [email protected] (K.J. McCracken) 0301-6226 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 99 )00129-3

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(P . 0.05). However, with the WB diet the improvements for all three parameters were approximately 2% for overall apparent digestibility and 4% for ileal apparent digestibility.  2000 Elsevier Science B.V. All rights reserved. Keywords: Wheat; Wheat by-products; Overall and ileal apparent digestibility; Xylanase

1. Introduction There is considerable interest in the use of fibrous feeds for pigs. However, pigs do not produce the endogenous enzymes required to break down cell wall non-starch polysaccharides (NSP). The negative relationship between dietary fibre and digestibility of nutrients is well established (Low, 1985; McClean, 1993; Yin, 1994; Jørgensen et al., 1996). The reduced apparent digestibility of energy from highfibre feeds can be attributed to differences in the physical or chemical structure of feeds and the shorter transit time of digesta (Graham et al., 1986). The decreased apparent digestibility of crude protein and amino acids, however, might be due to increased endogenous nitrogen (N) secretion (de Lange et al., 1990) or reduced digestibility of feed N due to the physical and chemical structure of the feed affecting accessibility of protein-degrading enzymes or a combination of both. Addition of exogenous enzymes to diets based on barley and wheat improves both nutrient digestibility and performance of pigs (Taverner and Campbell, ¨ 1988; Bohm, 1990; Li et al., 1994; Dierick and Decuypere, 1995; Baidoo et al., 1997). Therefore, the enzymes are capable of reducing some of the anti-nutritive properties of cell wall NSP. In broiler chickens, reduced digesta viscosity, following enzyme supplementation of barley- and wheat-based diets, is correlated with improved performance, particularly in relation to feed efficiency (Bedford and Classen, 1992; Choct et al., 1994; Preston, 1997). Similar relationships have not been established in pigs (Bedford et al., 1992). The aims of this experiment were to determine the extent of ileal and overall digestion of the nutrients in pig diets containing wheat and its by-products, the contribution of microbial action (VFA apparent production) and endogenous secretion of N in the small intestine to digestion and interactions with an

15

N-isotope technique; Endogenous N flow; VFA production;

NSP degrading enzyme (xylanase). One source of wheat (var. Soissons) and its by-products was used to eliminate differences due to the nature and composition of dietary fibre. Ground wheat was compared with its recombined fractions (flour 1 bran 1 middlings; designated recombined wheat) to determine the extent to which physical separation of the components might affect digestion. This experiment was conducted in collaboration with the Research Institute for the Biology of Farm Animals, Department of Nutritional Physiology ‘‘Oskar Kellner’’, Rostock, Germany. The same diets were used at both institutions, except that the WR diet was only used in Belfast. Whereas PVTC cannulae were used in Belfast, the end-to-end ileo-rectal anastomosis (IRA) method was used in Rostock.

2. Materials and methods

2.1. Experiment 1 2.1.1. Animals and experimental design Twelve male Large White 3 Landrace pigs, approximately 26 kg, were fitted with post valve ‘‘T’’ caecal cannulae (PVTC), according to the procedures of van Leeuwen et al. (1991). The pigs were randomly allocated, in a four period crossover design, to eight diets based on wheat (W), wheat 1 middlings (WM), wheat 1 bran (WB) or recombined wheat (WR), given without or with xylanase addition (Finnfeeds International Ltd) (Table 1). Each of the four main diets was mixed as a single batch and then divided for addition of the enzyme. The bran, flour and middlings were the mill fractions from the same whole grain wheat and were produced in a commercial flour mill (Andrews Milling) to normal specifications. The wheat was the French variety, Soissons, which had been grown in England. The diets, which contained both TiO 2 and Cr 2 O 3 as indigest-

Y.-L. Yin et al. / Livestock Production Science 62 (2000) 119 – 132 Table 1 Composition of experimental diets a (g / kg) Diet

W

Wheat Wheat flour Wheat bran Wheat middlings Soybean meal Fish meal Soybean oil Dicalcium phosphate Limestone Lysine Binder b Mineral / vitamins c Markers d

760

170 30 10 5 10.1 0.4 9 2.5 3

WR 570 60 130 170 30 10 5 10.1 0.4 9 2.5 3

121

followed by two 12 h collections of ileal digesta (08.00 to 20.00 h on consecutive days). WM

WB

380

380

2.2. Experiment 2

380

2.2.1. Animals and experimental design Thirty German Landrace barrows were obtained from the farm of the Research Institute for the Biology of Farm Animals (Dummerstorf, Rostock, Germany). When the pigs weighed approximately 15 kg an ileo-rectal anastomosis was performed according to the procedure described by Hennig et al. (1986). In each series, six of the diets used in Belfast (W, WM and WB, each without or with xylanase addition) were allocated at random to five pigs per diet. On day 10 after surgery, the pigs were individually housed in metabolism cages and given the appropriate experimental diet. On day 15 following the IRA surgery two indwelling silicone (Silastic, Dow Corning, Michigan) catheters (internal diameter 1.5 mm) were surgically implanted into the pig, one into the external jugular vein (for the infusion of 15 Nleucine) and the other into the carotid artery (for blood sampling), according to the procedures described by Weirich et al. (1970). The animals were allowed 48 h to recover from the second surgical manipulation before starting a ten-day experimental period.

380 170 30 10 5 10.1 0.4 9 2.5 3

170 30 10 5 10.1 0.4 9 2.5 3

a

Enzyme (xylanase, Trichoderma longibrachiatum, 0.321 g / kg diet, guaranteed minimum enzyme activity 5000 units / kg diet, added to one half of each diet. Enzyme activity was determined by the manufacturer, Finnfeeds International Ltd.). b Lignobond 2X (Lignotech, Norway). c Pigmv No. 2 (Devenish Feed Supplements, Belfast), supplying (per kg diet): vitamin A 8000 i.u.; vitamin D 3 2000 i.u.; vitamin E 40 mg; vitamin K 1 mg; vitamin B 2 2 mg; vitamin B 12 0.012 mg; iodine 0.85 mg; selenium 0.2 mg; cobalt 0.54 mg; iron 58 mg; zinc 72 mg; manganese 42 mg; copper 15 mg; calcium 500 mg; anti-oxidant 10 mg. d TiO 2 (1 g / kg) and Cr 2 O 3 (2 g / kg) were added to each diet.

ible markers, were cold-pelleted (3 mm die) and fed dry. In order to minimise between-pig variation the experiment was based on a cross-over design with each pig undergoing four collection periods. This meant that each of the eight dietary treatments was replicated six times. Pigs were randomly allocated to individual pens according to the initial randomisation of the treatments.

2.1.2. Feeding regimen and sampling of faeces and ileal digesta Pigs were fed daily at 08:00 and 17:00 h (20:00 h during the faeces and ileal digesta collection periods). They were given a fixed allowance which was kept constant during each experimental period. The amount given was the intake of the pig with the lowest consumption during the first 2 days of the adjustment period. Feed intakes were 1400, 1533, 1713 and 1838 g / d in periods 1, 2, 3 and 4. After a 5 day adaptation period, 7 day faecal collections were

2.2.2. Feeding regimen and sampling of ileal digesta and blood Feed intake was 500 g / d for each diet and the pigs were fed in two equal portions at 07:30 and 18:30 h during the entire experimental period. In order to compensate for by-passing the caecum and colon, the diets (Table 1) were additionally supplemented with sodium chloride (4 g / kg diet), sodium bicarbonate (4 g / kg diet) and a vitamin mix (1 g / kg diet). Two days after insertion of the catheters, a 10 day continuous infusion of 15 N-leucine (95% 15 N-enrichment) via the jugular catheter was initiated. On the day prior to the start of the 10 day infusion period, ileal digesta and blood samples (10 ml) were taken from each animal to determine the background 15 Nenrichment of the trichloroacetic acid (TCA)-soluble fraction of blood plasma and ileal digesta. Each animal was infused with 50 ml / d of sterile 0.9%

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NaCl solution containing 1.5 mg 15 N-leucine (95% 15 N labelled). An infusion pump (Harvard 22, Harvard instruments, Cambridge, USA) was used to infuse the solution. From the first day of infusion of the 15 N-leucine to the last day, blood samples (10 ml) were taken twice a day (8:00 and 19:00) from the carotid catheter. Immediately after sampling, the blood was centrifuged (10 min 3000 3 g, temperature 48C) and the plasma was removed. The plasma protein was precipitated by mixing 5 ml plasma with 5 ml 10% TCA and centrifuging at a speed of 4000 g at a temperature of 48C. The supernatant fluid (TCA-soluble fraction) from the samples taken from each animal per day were stored at 2 208C for further analyses. Ileal digesta were collected for 5 days from the 6th day after infusion to the last day of the experiment. At the end of each experiment a last blood sample was taken from each animal for 15 N-enrichment analysis and the animals were killed by euthanasia with Hexobarbital-Na (Hexobarbital Natrium AWD, VEB Arzneimittelwerke, Dresden).

trated phosphoric acid, using a Pye Unicam 304 gas chromatograph with a 10 m Hewlett Packard (HP20M) Carbowax column.

2.4. Calculation and statistical analysis The results obtained from the PVTC method (Belfast) were analysed using the ANOVA procedure of the statistical package Genstat 5 (1993) corresponding to the 12 pigs 3 4 periods partially balanced change over design (six replicates per treatment). This removed the effects of pigs and periods and tested for the main effect of diets and xylanase and their interaction. The amount of endogenous N (five replicates per treatment) was calculated from the ratio of 15 Nenrichment in ileal digesta (Ed) to that in the TCAsoluble blood plasma of the corresponding day using the following formula according to Souffrant et al. (1981) and de Lange et al. (1990): Ne 5 Nd 3 [(Ed–Enf) /(Epl–Enpl)]

2.3. Chemical analysis Samples of faeces and ileal digesta were freezedried and pooled within period and pig for each dietary treatment. The proximate analysis was carried out according to AOAC (1984). Amino acid (AA) analyses of diets and ileal digesta were carried out on an LKB 4400 Amino Acid Analyser using norleucine as the internal standard, after hydrolysis for 22 h in 6N HCl. Gross energy of diets, ileal digesta and faeces was determined using an adiabatic bomb calorimeter (Gallenkamp, Model CBA-305). TiO 2 was measured according to Leone (1973). Cr 2 O 3 was measured according to the method of Saha and Gilbreath (1991) by atomic absorption spectrophotometry. Soluble and insoluble NSP sugars and uronic acids and total NSP in diets were determined by gas chromatography (Pye Unicam 304) according to Englyst and Cummings (1984). 15 N-enrichment of the ileal digesta and TCA-soluble blood plasma samples were analysed by the method of Souffrant et al. (1982). Volatile fatty acids (VFA) in fresh ileal digesta were determined after centrifugation and treatment of the supernatant with concen-

where Ne is the endogenous N loss (g / d or g / kg DM intake); Nd is the total amount of N in the ileal digesta (g / d or g / kg DMI); Ed is the 15 N-enrichment in ileal digesta; Enf is the background 15 Nenrichment in the digesta; Epl is the 15 N-enrichment in the TCA-soluble blood plasma; and Enpl is the background 15 N-enrichment in the TCA-soluble blood plasma. The factor [(Ed–Enf) /(Epl–Enpl)], referred to as the dilution factor, was calculated for each animal for each individual day of ileal digesta collection. Daily values for the 15 N-enrichment excess were subjected to non-linear regression (Proc. NLIN, modified Gauss–Newton method, SAS, 1990) according to the formula given by Souffrant et al. (1993), to calculate the steady state 15 N-enrichment of total N in the chosen precursor pool. The results obtained from the IRA method (Rostock) were analysed by analysis of variance according to the 3 (diets)32 (enzymes) factorial design. In contrast to the changeover design in Experiment 1, the treatments were compared using between-pig variation.

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3. Results The analyses of a number of chemical constituents of the diets, including amino acids are shown in Table 2. The CP was highest for diet WM (232 g / kg) and lowest for diets W and WR (219 and 218 g / kg), whereas the crude ash and oil were highest for WB (67, 30 g / kg) and lowest for diets W (51, 22 g / kg) and WR (53, 23 g / kg). The gross energy content was higher for the WM and WB diets than for the wheat diets (W and WR) in keeping with the higher levels of protein and oil. The total AA contents were slightly higher for diet WM and diet WB (192 g / kg) than for diets W and WR (183 g / kg). The lysine concentration was also higher in diets WM and WB (9.9 g / kg) than in diet W (8.7 g / kg) or WR (8.6 g / kg).

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Conversely, the total NSP content was highest for diet WB (193.5 g / kg), followed by diet WM (151.6 g / kg), diet W and diet WR (82.8, 83.2 g / kg). Of this 9.89, 9.88, 8.36, 8.41 g / kg were soluble. The arabinoxylans comprised 76.3, 72.0, 69.3, 69.1% of the total insoluble NSP for diets WB, WM, W and WR respectively. There were no significant diet*enzyme interactions for any of the parameters reported throughout the paper. The inclusion of both high fibre products (wheat middlings and bran) in the diets significantly decreased (P,0.001) the dry matter content of the faeces, relative to diets W and WR, the values being lowest for diet WB. The total faecal output was highest (P,0.001) for diet WB, but the ileal digesta output measured with the PVTC method (Experiment 1) for diet WB was significantly lower than for

Table 2 Proximate analysis, amino acid and NSP analysis of diets (DM basis) Diet

W

WR

WM

WB

Crude protein (g / kg) Crude ash (g / kg) Oil (acid hydrolysis) (g / kg) Crude fibre (g / kg) Gross energy (MJ / kg)

219 51 22 28.9 18.1

218 53 23 29 18.1

232 62 28 50.2 18.8

227 67 30 63.8 18.5

Essential AA ( g /kg) Threonine Valine Cystine Methionine Isoleucine Leucine Phenylalanine Lysine Histidine Non-essential AA ( g /kg) Arginine Tyrosine Aspartic acid Serine Glutamic acid Proline Glycine Alanine Total AA Insoluble NSP Soluble NSP

6.7 8.3 8.4 4.0 7.5 1.34 8.9 8.7 4.3

11.1 5.9 14.9 9.2 42.7 12.9 8.9 7.7 182.8 74.5 8.36

6.6 8.7 8.7 4.0 7.7 1.37 9.2 8.6 4.4

11.0 5.7 14.8 9.2 41.4 13.0 8.8 7.9 183.4 74.8 8.41

7.2 9.4 8.9 3.9 8 1.39 9.0 9.9 4.8

7.2 9.1 9.6 4.1 7.6 13.9 9.0 9.9 4.9

12.9 6.1 16.3 9.2 41.1 13.2 9.8 8.7 192.5

13 6.1 16.5 9.4 41.1 12.2 9.9 8.7 191.9

141.7 9.88

183.6 9.89

Y.-L. Yin et al. / Livestock Production Science 62 (2000) 119 – 132

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WM. The ileal digesta values (g / kg DM intake) for diet WR tended to be lower than for diet W and the effect was significant (P,0.05) for fresh digesta weight. Enzyme inclusion only significantly reduced the fresh and dried ileal digesta output measured with the IRA method (Table 3), though there also was a trend towards reduced faecal output in Experiment 1. There were significant differences between diets in the digestibility of all measured nutrients (P,0.001) including amino acids (Tables 4–7) with values being highest for diets W and WR and lowest for WB, except for the ileal results from the PVTC total collection method (Table 4). In general, there were no significant differences in ileal or overall digestibility of nutrients between diets W and WR. Although the pattern of the results was similar for the PVTC TiO 2 and PVTC Cr 2 O 3 methods, the absolute values for IAD of DM, gross energy, CP and AA in all diets measured with PVTC Cr 2 O 3 were lower than those measured with PVTC TiO 2 (Tables 4–6). The IAD of DM, CP, energy and AA in all diets measured by the IRA method were in good agreement with those measured by the PVTC TiO 2 method. Enzyme inclusion improved overall apparent digestibility (OAD) of DM, energy and CP (P,0.05) measured by the PVTC method and IAD of DM, CP

and several AA (P,0.05) calculated with the PVTC TiO 2 method (Tables 4,5). Enzyme addition also significantly improved the IAD of DM and gross energy measured with the IRA method. The improvements in ileal digestibility were mainly due to increased digestibility coefficients for diet WB, increases being 4.7% (0.607 vs. 0.580), 5.8% (0.619 vs. 0.585), 3.3% (0.732 vs. 0.708) and 4.6% (0.799 vs. 0.764) for DM, energy, CP and total amino acids, respectively. Enzyme inclusion did not significantly improve the IAD of CP and AA measured with the IRA method (Tables 4,7) or any IAD parameters measured with PVTC Cr 2 O 3 (Tables 4,6), except for threonine, aspartic acid, serine and glycine. In Experiment 2, wheat middlings or wheat bran inclusion significantly (P,0.001) increased the ileal total N, endogenous N (g / d) and endogenous N (g / kg dry matter intake, DMI) compared with diet W, but no differences were observed between diets WB and WM (Table 8). There were no statistically significant effects of enzyme addition on ileal total N or endogenous N flow but in each case the values were numerically lower with enzyme addition, particularly with diet WB, where the endogenous N flow was reduced by 19% (2.35 vs. 2.80 g / d). The ileal total apparent VFA production (expressed g / d per kg DM intake) for WM and WB were significantly (P,0.05) greater than for W and

Table 3 Outputs (g / kg DM intake) of faeces and ileal digesta and their DM concentrations (g / kg) measured with PVTC and IRA methods Diet W Faeces weight ( g /kg DM intake) Fresh 371 c Dry 110 c DM (g / kg) 298 a Ileal digesta ( g /kg DM intake) Fresh (PVTC) 2288 c Dry (PVTC) 232 b DM (g / kg) 102 Fresh (IRA) 2135 c Dry (IRA) 237 c DM (g / kg) 111 a,b,c,d

Enzyme WR

WM

WB

s.e.d.

P

2

1

s.e.d.

P

395 c 114 c 289 a,b

672 b 184 b 275 b

1030 a 243 a 236 c

19.3 4.1 8.8

,0.001 ,0.001 ,0.001

630 167 279

605 159 270

13.3 8.1 6.3

NS NS NS

2203 d 215 b,c 98 ND ND ND

2902 a 312 a 107 3327 b 376 b 113

2583 b 277 a,b 107 3789 a 413 a 109

20.4 23.5 3.2 15.9 10.4 2.3

,0.001 ,0.001 NS ,0.001 ,0.001 NS

252 261 104 3178 356 112

247 258 104 2955 328 111

14.0 16.1 2.3 10.8 8.38 1.8

NS NS NS 0.01 0.01 NS

Values within the same row without a common superscript are significantly different (P,0.05). ND, not determined.

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Table 4 Ileal and overall apparent digestibility of DM, CP and gross energy (E) Diet

Enzyme

W

WM

WB

s.e.d.

P

2

1

s.e.d.

P

DM CP E

d

0.773 0.807 d 0.787 d

d

0.766 0.802 d 0.771 d

e

0.673 0.757 e 0.683 e

f

0.594 0.720 f 0.595 f

0.0098 0.0050 0.0091

,0.001 ,0.001 ,0.001

0.694 0.765 0.704

0.709 0.778 0.714

0.0066 0.0034 0.0062

,0.05 ,0.05 NS

Ileal b

DM CP E

0.741 d 0.779 d 0.757 d

0.752 d 0.790 d 0.758 d

0.632 e 0.727 e 0.644 e

0.558 f 0.695 f 0.560 f

0.0107 0.0082 0.0097

,0.001 ,0.001 ,0.001

0.668 0.744 0.679

0.674 0.752 0.680

0.0072 0.0056 0.0066

NS NS NS

Ileal c

DM CP E

0.763 d 0.811 d 0.784 d

ND g ND ND

0.624 e 0.747 e 0.656 e

0.587 f 0.745 e 0.603 f

0.0104 0.0123 0.0111

,0.001 ,0.001 ,0.001

0.644 0.760 0.670

0.672 0.776 0.692

0.0084 0.0099 0.0091

,0.01 NS ,0.05

Overall a

DM CP E

0.890 d 0.903 d 0.897 d

0.885 d 0.903 d 0.886 d

0.816 e 0.864 e 0.819 e

0.758 f 0.831 f 0.759 f

0.0038 0.0043 0.0039

,0.001 ,0.001 ,0.001

0.833 0.872 0.837

0.841 0.878 0.843

0.0027 0.0030 0.0031

,0.05 ,0.05 ,0.05

Ileal

a

WR

a

Measured with the PVTC TiO 2 method. Measured with PVTC Cr 2 O 3 method. c Measured with IRA method. d,e,f Values within the same row without a common superscript are significantly different (P,0.05). g ND, not determined. b

Table 5 Ileal apparent digestibility of amino acids measured with the PVTC TiO 2 method Diet

Enzyme WR

WM

WB

s.e.d.

P

2

1

s.e.d.

P

Essential amino acids Threonine 0.770 a Valine 0.766 a Methionine 0.898 a Isoleucine 0.831 a Leucine 0.848 a Phenylalanine 0.935 a Lysine 0.843 a Histidine 0.865 a

0.767 a 0.777 a 0.894 a 0.836 a 0.841 a 0.934 a 0.832 a 0.862 a

0.743 b 0.749 b 0.861 b 0.803 b 0.807 b 0.918 b 0.820 b 0.847 b

0.708 c 0.701 c 0.851 b 0.767 c 0.779 c 0.904 c 0.786 c 0.824 c

0.0064 0.0073 0.0108 0.0044 0.0102 0.0028 0.0051 0.0052

,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001

0.738 0.741 0.871 0.805 0.812 0.920 0.815 0.844

0.756 0.756 0.881 0.814 0.826 0.926 0.821 0.855

0.0084 0.0069 0.0051 0.0057 0.0075 0.0083 0.0043 0.0043

,0.05 ,0.05 ,0.05 ,0.05 NS NS NS ,0.05

Non-essential amino acids Cystine 0.814 a Arginine 0.887 Tyrosine 0.849 a Aspartic acid 0.777 a Serine 0.834 a Glutamic acid 0.909 a Proline 0.768 b Glycine 0.749 a Alanine 0.737 a Total 0.834 b

0.817 a 0.882 0.852 a 0.779 a 0.836 a 0.907 a 0.881 a 0.739 a 0.749 a 0.854 a

0.780 b 0.885 0.833 b 0.751 b 0.800 b 0.879 b 0.763 b 0.719 b 0.719 b 0.810 c

0.784 b 0.876 0.789 c 0.716 c 0.778 c 0.873 b 0.709 c 0.674 c 0.665 c 0.783 d

0.0100 0.0054 0.0044 0.0066 0.0052 0.0055 0.0268 0.0138 0.0087 0.0053

,0.01 NS ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001

0.789 0.878 0.828 0.749 0.805 0.889 0.773 0.706 0.714 0.815

0.808 0.887 0.834 0.763 0.819 0.895 0.787 0.734 0.722 0.826

0.0079 0.0042 0.0093 0.0051 0.0085 0.0054 0.0184 0.0099 0.0057 0.0050

,0.05 ,0.05 NS ,0.05 ,0.05 NS NS ,0.05 NS ,0.05

W

a,b,c,d

Values within the same row without a common superscript are significantly different (P,0.05).

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Table 6 Ileal apparent digestibility of amino acids measured with the PVTC Cr 2 O 3 method Diet

Essential amino acids Threonine Valine Cystine Methionine Isoleucine Leucine Phenylalanine Lysine Histidine

Enzyme

W

WR

WM

WB

s.e.d.

P

2

1

s.e.d.

P

0.737 a 0.733 b 0.786 a,b 0.884 a 0.808 b 0.827 a 0.830 b 0.811 a 0.845 a

0.753 a 0.763 a 0.805 a 0.888 a 0.827 a 0.831 a 0.846 a 0.821 a 0.854 a

0.710 b 0.718 b 0.750 c 0.843 b 0.778 c 0.783 b 0.797 c 0.797 a 0.827 b

0.683 c 0.674 c 0.765 b,c 0.838 b 0.747 d 0.760 c 0.777 d 0.768 b 0.809 c

0.0091 0.0089 0.0142 0.0060 0.0074 0.0111 0.0061 0.0076 0.0059

,0.001 ,0.001 0.006 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001

0.714 0.717 0.769 0.859 0.788 0.796 0.811 0.799 0.830

0.728 0.727 0.784 0.867 0.792 0.805 0.815 0.800 0.838

0.0057 0.0093 0.0110 0.0098 0.0085 0.0080 0.0083 0.0075 0.0042

,0.05 NS NS NS NS NS NS NS NS

0.875 0.844 a 0.723 b 0.826 a 0.902 a 0.874 a 0.726 a 0.735 a 0.846 a

0.871 0.813 c 0.720 b 0.775 b 0.863 b 0.733 c 0.679 b 0.683 b 0.785 c

0.865 0.771 d 0.688 c 0.759 c 0.862 b 0.683 d 0.645 c 0.636 c 0.764 d

0.0050 0.0054 0.0039 0.0068 0.0066 0.0276 0.0132 0.0110 0.0060

NS ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001

0.867 0.813 0.875 0.787 0.879 0.748 0.680 0.688 0.798

0.874 0.815 0.881 0.798 0.883 0.762 0.701 0.689 0.805

0.0093 0.0074 0.0028 0.0047 0.0093 0.0190 0.0093 0.0080 0.0084

NS NS ,0.05 ,0.05 NS NS ,0.05 NS NS

Non-essential amino acids Arginine 0.871 Tyrosine 0.828 b Aspartic acid 0.760 a Serine 0.810 a Glutamic acid 0.897 a Proline 0.731 b Glycine 0.712 a Alanine 0.700 b Total 0.810 b a,b,c,d

Values within the same row without a common superscript are significantly different (P,0.05).

Table 7 Ileal apparent digestibility of amino acids measured with IRA method Diet W

s.e.d.

P

Threonine Valine Isoleucine Leucine Phenylalanine Lysine Histidine Tryptophan

a

WM

0.764 0.805 a 0.843 a 0.851 a 0.876 a 0.844 a 0.869 a 0.774

b

WB

0.732 0.769 b 0.815 b 0.82 a 0.851 b 0.833 a 0.865 a 0.766

b

0.702 0.756 b 0.800 b 0.807 b 0.834 b 0.807 b 0.848 b 0.749

0.0177 0.0115 0.0112 0.0096 0.0096 0.0107 0.0082 0.0152

,0.01 ,0.001 ,0.01 ,0.001 ,0.001 ,0.01 ,0.05 NS

Tyrosine Arginine Aspartic acid Serine Glutamic acid Proline Alanine Glycine Total

0.851 0.908 0.799 a 0.836 a 0.912 a 0.891 a 0.770 a 0.784 a 0.856 a

0.858 0.901 0.769 b 0.801 b 0.873 b 0.858 b 0.741 a 0.740 b 0.825 b

0.834 0.895 0.750 b 0.786 c 0.879 b 0.854 b 0.714 b 0.732 b 0.814 b

0.0111 0.0061 0.0149 0.0112 0.0078 0.0083 0.0151 0.0179 0.0097

NS NS ,0.01 ,0.001 ,0.001 ,0.001 ,0.01 ,0.05 ,0.001

a,b,c Values within the same row without a common superscript are significantly different (P,0.05). No significant effects (P. 0.05) of enzyme addition occurred.

WR, respectively, but there were no significant differences between diet WB and WM (Table 9). The increase in total VFA on diets WB and WM was reflected in the individual VFAs. Xylanase supplementation had no significant effect on ileal VFA apparent production, although there were numerical increases with supplementation of 16, 14, 17, 21% on average for total, acetic, propionic and butyric acids respectively and of 38% (6.46 vs. 4.66 g / per kg DM intake), 38, 27 and 82% for diet WB. The extent of fermentation (Ferm) occurring in the large intestine assessed in various ways is shown in Table 9. The difference between overall and ileal digestibility coefficients (i.e. extent of hindgut fermentation) was expressed as Ferm 1. Ferm 2 was the proportion of hindgut fermentation (Ferm 1) expressed as a fraction of the overall digestibility. Ferm 3 was Ferm 1 expressed as a proportion of the undigested residue flowing through the terminal ileum. The results showed that when the fermentation was expressed as Ferm 1 or Ferm 2, the extent

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Table 8 Nitrogen (N) flows at the terminal ileum measured with IRA method Diet

Enzyme

W Total N (g / d) Total N (g / kg of DMI) Endogenous N (g / d) Endogenous N (g / kg of DMI) a,b

WM b

WB

a

2.77 6.7 b 1.71 b 4.14 b

a

4.27 9.47 a 2.79 a 6.42 a

4.03 9.37 a 2.57 a 5.98 a

s.e.d.

P

2

1

s.e.d.

P

0.288 0.444 0.231 0.527

,0.001 ,0.001 ,0.001 ,0.001

3.82 8.67 2.46 5.75

3.56 8.36 2.25 5.28

0.223 0.357 0.185 0.424

NS NS NS NS

Values within the same row without a common superscript are significantly different (P,0.05).

Table 9 Ileal VFA apparent production (g / per kg DM intake) and estimates of hindgut fermentation (Ferm) measured with PVTC method Diet W

WR e

Total VFA Acetic Propionic Butyric

WM e

d

WB d

s.e.d.

P

3.64 2.78 e 0.51 e 0.35 e

3.57 2.81 e 0.46 e 0.3 e

5.32 3.83 d 1.02 d 0.47 d

5.56 3.82 d 1.28 d 0.46 d

0.6138 0.4177 0.1589 0.0789

0.021 0.01 ,0.001 0.07

Ferm 1 a

DM CP GE

0.117 f 0.096 0.110 f

0.120 f 0.102 0.116 f

0.143 e 0.107 0.136 e

0.173 d 0.108 0.162 d

0.0101 0.0047 0.0088

,0.001 0.074 ,0.001

Ferm 2 b

DM CP GE

0.131 f 0.106 e,f 0.123 f

0.135 f 0.113 e 0.131 f

0.175 e 0.123 d,e 0.165 e

0.229 d 0.131 d 0.214 d

0.0124 0.0052 0.0104

,0.001 ,0.001 ,0.001

Ferm 3 c

DM CP GE

0.514 d 0.495 d 0.515 d

0.510 d 0.512 d 0.506 d

0.435 e 0.436 e 0.426 e

0.426 e 0.386 f 0.401 e

0.0223 0.0179 0.0217

,0.001 ,0.001 ,0.001

a

Fermentation expressed as the difference between overall and ileal digestibility coefficients (5overall digestibility–ileal digestibility). As a proportion of overall digestibility (5Ferm 1 / overall digestibility). c Fermentation as a proportion of undigested material reaching the hindgut (5Ferm 1 / 1–ileal digestibility). d,e,f Values within the same row without a common superscript are significantly different (P,0.05). No significant effects (P.0.05) of enzyme occurred. b

of DM and energy fermentation were highest for diet WB (P,0.001), followed by diet WM, except for CP (Ferm 1). However, when fermentation was considered as the proportion of undigested residue flowing through the terminal ileum which disappeared in the hindgut (Ferm 3), the extent of disappearance of DM, energy and CP was significantly (P,0.001) lower for diets WM and WB relative to diets W and WR. Enzyme addition had no significant effect on hindgut fermentation.

4. Discussion

4.1. Effects of dietary NSP on ileal apparent digestibility of nutrients, ileal endogenous N flow and microbial fermentation There were no significant differences in digestibility of nutrients or other parameters measured in this experiment between diets W and WR. This indicated that physical separation of the wheat milled

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Fig. 1. Relationship between ileal apparent digestibility of energy and dietary total NSP.

products does not affect digestion and absorption. The well-established negative influence of dietary fibre on digestibility is demonstrated in Fig. 1, using the results for ileal apparent digestibility of gross energy, measured by the TiO 2 method, as an example. Similar relationships were seen for DM, crude protein and amino acids and the slopes for the regression equations differed from zero (P,0.05). For example the negative relationship with crude protein digestibility was described by the equation: IAD (CP) 5 0.873 2 0.00078 X (r 5 0.999), where X5total dietary NSP (g / kg DM). The ileal apparent digestibility of gross energy decreased by 32%, while that for CP and total AA decreased by 12 and 6%, respectively, when the dietary total NSP increased from 83 to 193 g / kg. These results are similar to those of McClean (1993) who studied the effects of wheat middlings on digestibility of diets for weaned piglets. In her study, increasing dietary NDF from 0 to 309 g / kg, by inclusion of wheat middlings, led to reductions in the overall apparent digestibilities of energy and CP, from 0.959 to 0.738 for energy and from 0.943 to 0.750 for CP. The effect of dietary NSP on nutrient digestibility may partly be explained by an increased rate of passage through the digestive tract (Varel et al., 1982; Jørgensen et al., 1996). Using data from the IRA method as an example, the ileal DM outflow

(g / kg DM intake) increased by 1.7-fold when the dietary total NSP increased from 83 g / kg (diet, W) to 193 g / kg (diet, WB). This result is similar to those of Schulze (1994) and Jørgensen et al. (1996) who also observed a significant influence of dietary NSP on ileal DM output. For example, Schulze (1994) reported that the ileal DM output (g / kg DM intake) increased 2-fold when the dietary NDF increased from 83 g / kg to 195 g / kg. Dietary fibre has an influence on microbial fermentation not only in the large intestine, but also in the small intestine (Fuller, 1991). In this study microbial activity in the small intestine was assessed in terms of the apparent production of VFA. The term ‘‘apparent’’ has been used since it is recognised that some absorption or utilisation of VFA, or both, may have occurred within the small intestine. With this proviso it is clear that increased microbial activity occurred in the small intestine with added NSP in accordance with the observations of Schutte et al. (1991) and Schulze (1994). Quantitatively, when compared with hindgut microbial fermentation (Table 9), the total small intestinal fermentation (about 1% of DM intake) is small. However, the increased microbial activity leads to increased competition for nutrients with the host and increased levels of harmful bacterial metabolites e.g. ammonia, amines, secondary bile acids and bacterial enterotox-

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ins, all of which, in high concentrations, have a damaging effect on the mucosa of the small intestine and, therefore, may reduce the host digestion and absorption rate for nutrients, and increase metabolic demand (Just et al., 1979, 1981; McKinnon, 1985; Chesson, 1994). Dietary fibre also affects the endogenous N flow and apparent digestibility of protein. An increase in endogenous ileal N and AA with higher levels of fibre in diets has been demonstrated (Sauer et al., 1977; Taverner et al., 1981; Schulze, 1994). The additional ileal endogenous N was 1.84 g / kg DM intake as the dietary total NSP increased from 83 g / kg (diet W) to 193 g / kg (diet WB). This result is somewhat higher than that of Schulze (1994) who reported that the additional ileal endogenous N flow was 1.13 g / kg DM intake when the dietary NDF content was increased from 60 to 177 g / kg by inclusion of wheat bran. Sauer (1976) suggested that the increase in endogenous output caused by increased fibre intake was due to increased cellular losses of epithelial cells or increased mucus secretion or both. He suggested also that increasing dietary fibre levels resulted in increased water-holding capacity of the digesta, causing an increase in physical abrasion of epithelial cells by the digesta. In the present experiment, however, whereas there was a significant increase in ileal DM flow from diet WM (152 g NSP/ kg) to diet WB (196 g NSP/ kg), this had no effect on the ileal N flow, suggesting that this relationship may not be linear. This agrees with the results of Taverner et al. (1981) who reported that endogenous N ileal output increased with dietary fibre up to approximately 100 g NDF / kg, but not thereafter. The total quantity of nitrogenous material passing the terminal ileum is dependent on both the undigested dietary N and the endogenous N that is not re-absorbed. The present experiment shows that the ileal endogenous N flow is very high, being over half of the total N. It is important that both quantities are known (Huisman et al., 1993). However, in order to distinguish between the endogenous and exogenous N fractions, specific experimental and analytical methods are required. Although not entirely above criticism (Fuller, 1991; de Lange et al., 1992), the 15 N-isotope dilution method is one of the most used methods to determine the endogenous N flow at the

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terminal ileum of pigs fed diets containing protein (de Lange et al., 1990; Souffrant, 1991; Souffrant et al., 1993). The present results clearly show that the depressed ileal apparent digestibility of protein with increased dietary NSP is mainly due to increased ileal endogenous N. When dietary total NSP was increased from 83 g / kg (diet W) to 193 g / kg (diet WB), the calculated total N excreted in ileal digesta was increased by 1.3 g / d, while the increased ileal endogenous N flow was 0.9 g / d. The calculated true CP digestibility was 2% higher for diet W than diet WB (93 vs. 91%).

4.2. Effects of enzyme addition on ileal digestibility of nutrients, and growth performance As discussed above, there is a strong negative relationship between dietary NSP and ileal digestibility and absorption of nutrients. It has been suggested that xylanase addition could cause improvements through disruption or solubilization of cell wall polysaccharides, resulting in reduction or elimination of the encapsulating effects of the cell wall and a consequent shift of digestion of energy and protein towards the proximal region of the small intestine (Dierick and Decuypere, 1994). The present study and others (Dierick and Decuypere, 1994; Schulze et al., 1996) have shown that the main source of dietary NSP in wheat is arabinoxylans and in barley, arabinoxylans plus glucans. These groups comprise over 50% of the total NSP in the whole grain. Although the observed improvements in overall and ileal apparent digestibility of DM, CP and energy were statistically significant (P,0.05), the effects of xylanase supplementation with wheatbased diets were small when compared with the ˚ results for poultry (Pettersson and Aman, 1989; Preston, 1997). The improvements of overall digestibility of DM, energy and CP were only of the order of 1% and those at the ileum less than 2%. However, the effects were greater for the high fibre diet WB where the mean ileal apparent digestibilities of CP and energy were improved by 3 and 5% respectively with enzyme addition. This, taken with the results of Choct et al. (1994) and Knudsen and Hansen (1991) indicates that the influence of feed enzyme addition on nutrient digestibility is depen-

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dent on the substrate available to the exogenous enzyme. For diets of high fibre content, the digestibility is relatively low and, therefore, there is more scope for enzyme action (Collier and Hardy, 1986; Knudsen and Hansen,1991). The present study shows that there was a numerical trend of increased apparent ileal VFA production with xylanase addition, especially for diet WB where ileal apparent VFA production was increased by 38%. This suggests that enzyme addition increased the availability of nutrients to the microorganisms and that part of the energy digestibility at the distal ileum for diet WB was due to fermentation. However, as discussed earlier, this effect would account for less than 1% of DM intake and, therefore, the increased ileal digestibility of gross energy by enzyme addition would appear to be mainly due to increased host enzyme digestion. Inborr et al. (1994), Li et al. (1994) and Baidoo et al. (1997) suggested that the increased apparent ileal digestibility of CP and AA with enzyme supplemented barley-based diets for pigs was due to reduction in the physical barrier created by the gel-forming property of bglucans, therefore providing an ideal environment for the interaction between endogenous digestive enzymes and respective substrates. However, the numerical increase in apparent ileal digestibility of CP, measured with the IRA method, can be mainly attributed to the reduced ileal endogenous N secretion. The calculated mean values of the total ileal N flow of the diets W, WM and WB were reduced by 0.26 g / d by enzyme addition, while the ileal endogenous N flow was reduced by 0.21 g / d and the true ileal digestibility of CP with or without enzyme was the same (92%). Contrary to the results with poultry (Pettersson ˚ and Aman, 1988, 1989; Choct et al., 1994), the present results clearly indicate that the effects of enzyme preparations containing xylanase on nutrient utilization of wheat or its by-products in diets for growing pigs are small. The main reason for the difference in results may be the anatomical differences between pigs and poultry. In pigs, the stomach acts as the primary reservoir for food (Kidder and Manners, 1978) and, unlike the crop of poultry, gastric pH may decline to low levels that are detrimental to enzyme activity. In order for exogenous enzymes to have beneficial effects, they must be

protected in the pre-gastric and gastric region. Enzyme stability is therefore an important factor in determining efficacy (Baas and Thacker, 1996). The pH in the stomach of pigs fed ad libitum rarely rises above pH 3.0 (Kidder and Manners, 1978). Although low pH levels may also be reached in the gizzard of poultry (Hill, 1971), the duration of exposure to the low pH is much shorter than with pigs. Therefore, a greater proportion of enzyme activity would be expected to survive in the small intestine of chickens compared with pigs. However, Baas and Thacker (1996) found that, although the low gastric pH observed in the stomach of the pig has a detrimental effect on b-glucanase activity, enzyme activity was still present in the small intestine of the pig and enzyme addition significantly improved the ileal digestibility of nutrients in barley-based diet compared with the control group. The feed enzyme xylanase used in this study has been selected to be active under the conditions encountered in the stomach and in the small intestine and to effectively degrade targeted polysaccharides (optimum pH range 3.5–6.5, according to Finnfeeds International Ltd.). In addition, the amount of xylanase activity included was about 30% above the guaranteed minimum. Therefore, it seems unlikely that the modest responses in digestibility of nutrients are due to poor survival of enzyme. It may be that the exogenous xylanase is not fully effective in solubilizing NSP under the conditions prevailing in the small intestine or that there is already significant hydrolysis by microbial enzymes. Quantitatively the contribution of microbial fermentation in the small intestine is small but microbial xylanase activity and its effects on NSP degradation may be biologically significant. The relatively long small intestine in the pig (18 m vs. 120 cm for poultry) increases transit time and provides increased opportunity for microbial colonisation of the small intestine. In retrospect it would have been interesting to examine the microflora in various regions of the small intestine in the absence or presence of enzyme.

5. Conclusions It is concluded that dietary NSP from wheat has a significantly negative effect on ileal and overall

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digestibility resulting in increased DM output, endogenous N secretion and microbial fermentation (apparent VFA production and overall–ileal differences in digestibility) in pigs. There were small, but significant improvements in overall digestibility of DM, CP and energy and ileal digestibility of DM, CP, energy and some AA associated with xylanase inclusion. The increased apparent ileal digestibility of gross energy by xylanase addition observed in this study may be mainly due to the increased host endogenous enzyme digestion and partly due to the increased fermentation in the small intestine. The increased ileal apparent crude protein digestibility appears to be mainly due to the reduced endogenous nitrogen reaching the ileum rather than to improved true digestibility of protein.

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