Dietary fructooligosaccharides and transgalactooligosaccharides can affect fermentation characteristics in gut contents and portal plasma of growing pigs

Livestock Production Science 73 (2002) 175–184 www.elsevier.com / locate / livprodsci

Dietary fructooligosaccharides and transgalactooligosaccharides can affect fermentation characteristics in gut contents and portal plasma of growing pigs a, a a Jos G.M. Houdijk *, Martin W.A. Verstegen , Marlou W. Bosch , Katrien J.M. van Laere b a

Animal Nutrition Group, Wageningen Institute of Animal Sciences ( WIAS), Wageningen Agricultural University, P.O. Box 338, 6700 AH Wageningen, The Netherlands b Food Chemistry Group, Advanced Studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences ( VLAG), Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands Received 6 March 2000; received in revised form 7 June 2001; accepted 7 June 2001

Abstract We studied whether dietary non-digestible oligosaccharides (NDOs) affected pH and volatile fatty acids (VFAs) in gastrointestinal contents and in portal plasma of young pigs. Five groups of five 57-day-old pigs received for 44 days either a corn-based control diet or this diet with 7.5 or 15 g / kg fructooligosaccharides (FOSs, Raftilose P95  ) or the control diet with 10 or 20 g / kg transgalactooligosaccharides (TOSs, Oligostroop  ). The pigs weighed on average 45.561.3 kg during dissection, which took place 3 h after feeding. Dietary NDOs tended to lower the pH of the stomach content from 4.5 to 4.2 (P 5 0.06). Pigs fed the high TOS diet had more caecal VFAs than the control pigs (30.4 vs. 15.6 mmol, P , 0.05). Compared to TOS-fed pigs, FOS-fed pigs had a higher proximal colon pH (6.5 vs. 6.2, P , 0.01), lower proximal colon VFA concentration (131 vs. 166 mmol / l, P , 0.01) and lower portal VFA concentration (0.9 vs. 1.6 mmol / l, P , 0.05), with the control pigs being intermediate. However, the amount of colonic VFAs was similar across diets ( | 40 mmol). The results support the view that dietary FOSs and TOSs may have different effects on fermentation characteristics of gut contents of pigs.  2002 Elsevier Science B.V. All rights reserved. Keywords: Fructooligosaccharides; Transgalactooligosaccharides; Pigs; Gut contents; Portal plasma; Volatile fatty acids

1. Introduction Certain dietary oligosaccharides are not digested *Corresponding author. Current address: Scottish Agricultural College, Animal Biology Division, Bush Estate, Penicuik, Penicuik EH26 0PH, UK. E-mail address: [email protected] (J.G.M. Houdijk).

in the small intestine, since the porcine endogenous secretions lack the enzymes to hydrolyze them. Therefore, these carbohydrates are referred to as non-digestible oligosaccharides (NDOs) and are more likely degraded by the gastrointestinal microflora. It has been suggested that certain NDOs may selectively stimulate beneficial microbial activity (Gibson and Roberfroid, 1995). These NDOs are

0301-6226 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 01 )00250-0

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referred to as prebiotics and are increasingly being used as additives in animal feedstuffs. Fructooligosaccharides (FOSs) are b-linked fructose monomers and are found in barley and wheat (Henry and Saini, 1989). Transgalactooligosaccharides (TOSs) are b-linked galactose units and are not found in feedstuffs. These NDOs can be found in fermented products like yoghurts, as the result of bacterial activity on milk sugars (Toba et al., 1983). When fed to pigs, FOSs and TOSs could not be recovered in the faeces (Houdijk et al., 1998a). Thus, these NDOs must had been degraded, and therefore may have affected characteristics of the gastrointestinal contents, including pH and the concentration of volatile fatty acids (VFAs). From the scarce information available, one would conclude that dietary NDOs hardly affect physical– chemical characteristics of the gastrointestinal contents in pigs. The inclusion of FOSs at 15 g / kg feed did not affect VFA concentrations in the content of the colon of growing pigs (Farnworth et al., 1992). Similarly, the inclusion of a range of NDOs at 2 g / kg feed did not affect pH, VFA and lactic acid concentrations in the content of stomach, ileum and colon of weanling pigs (Bolduan et al., 1993; Gabert et al., 1995). These NDOs included FOSs, TOSs, galactooligosaccharides, isomaltooligosaccharides and b-glucooligosaccharides. However, the control diets used in these studies were composed mainly of barley, wheat and soybean meal. Such ingredients contain considerable levels of NDOs, including FOSs (Henry and Saini, 1989) and a-galactooligosaccharides (Saini, 1989). Therefore, the use of such control diets may have diluted or masked the effects of NDOs added. A better approach to elucidate effects of dietary NDOs on physical–chemical characteristics of the gastrointestinal contents might be via adding NDOs to control diets which are not based on NDO-rich feedstuffs. Corn-based diets may be suitable for this approach, since corn contains no FOSs and only traces of a-galactooligosaccharides (Houdijk et al., 1998a). Therefore, in the present study, we used a corn-based diet to assess effects of FOSs and TOSs on gastrointestinal pH and VFAs, as well as on portal plasma VFA concentrations in growing pigs. Since NDOs are likely to be fermented, it was hypothesized that the inclusion of NDOs in a cornbased control diet results in a decreased pH and

increased VFA concentrations in the caecum and colon, as well as in an increased VFA concentration in portal plasma.

2. Materials and methods

2.1. Diets, animals and housing Table 1 shows the composition of the experimental control diet. Four other experimental diets were Table 1 Composition and proximate analysis of the control diet Control diet Ingredients (g / kg fresh) Corn a Glucose Cellulose Protein sources b Soy oil Fumaric acid Mineral mix c Amino acids d Premix e

634.6 117.8 30.0 150.0 10.0 10.0 32.4 5.2 10.0

Proximate analysis (g / kg fresh) Dry matter Inorganic matter Crude protein Ether extract Crude fibre N-free extract Calculated ileal digestible crude protein (g / kg) Calculated metabolizable energy (MJ / kg)

893.8 54.4 170.4 43.8 36.1 589.1 132.7 14.2

a

Half of the corn was pressurized toasted ( . 1008C), and then flaked and pelleted, in order to increase accessibility of starch for digestive enzymes (Cornax  , Schouten / Giessen, The Netherlands). b Casein, fish meal, and animal meal: 50 g / kg diet each. c This mineral mix provided (per kg feed) 2.0 g NaCl, 8.0 g CaCO 3 , 9.4 g CaHPO 4 , 2.0 g MgO and 11.0 g KHCO 3 . d Added synthetic amino acids (per kg feed) were 2.6 g L-lysine HCl, 1.1 g DL-methionine, 0.8 g L-threonine and 0.7 g Ltryptophan. e The vitamin / mineral mix provided (per kg feed): 9000 IU vitamin A, 1800 IU vitamin D3, 40 mg vitamin E, 3 mg vitamin K, 2 mg thiamine, 5 mg riboflavin, 12 mg D-panthothenic acid, 1 mg folic acid, 3 mg pyridoxine, 30 mg niacin, 40 mg cobalamin, 1000 mg choline chloride, 50 mg vitamin C, 0.1 mg biotin, 2.5 mg CoSO 4 ? 7H 2 O, 0.2 mg Na 2 SeO 3 ? 5H 2 O, 0.5 mg KI, 400 mg FeSO 4 ? 7H 2 O, 60 mg CuSO 4 ? 5H 2 O, 70 mg MnO 2 and 300 mg ZnSO 4 .

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formulated by including NDO-rich products in this control diet. FOS-rich Raftilose P95  (Orafti, Tienen, Belgium) was included at 7.5 g / kg (FOS-L) and 15.0 g / kg (FOS-H) and TOS-rich Oligostroop  (Borculo Whey Products, Borculo, The Netherlands) at 10.0 g / kg (TOS-L) and 20.0 g / kg (TOS-H). We used slightly different inclusion levels since earlier research has shown that an improved growth performance in weaned pigs was achieved with somewhat lower levels for FOS than for TOS (Hidaka et al., 1985; Katta et al., 1993). The NDOs were included in the control diet at the expense of purified cellulose. The diets did not contain antibiotics or additional copper. The acid buffer capacity of the diets was estimated at | 90 mequiv. / 0.5 g dry matter, based on tabulated acid buffer capacity of feedstuff ingredients (Jasaitis et al., 1987). The five diets were fed to five groups of five, individually housed, 57-day-old castrated pigs (Great Yorkshire 3 Landrace)? 3 (Great Yorkshire)/ . The mean body weight of the pigs was 15.960.6 kg at day 0, and the diets were offered for 44 days. The pigs were fed ad libitum for the first 3 weeks and restrictedly from day 21 until day 44. The allowances of the diets during the restricted feeding were calculated to provide 2.6 times the energy requirement for maintenance, which was assumed to be 459 kJ metabolizable energy per kg 0.75 . The pigs were fed twice daily at | 08.00 and | 16.00. The diets were mixed with three parts of water, prepared 15 min before feeding, since no other means of water supply was available. Between day 21 and day 44, apparent total tract and ileal nutrient digestion was assessed; these results have been reported in detail elsewhere (Houdijk et al., 1999). We used the slaughter technique for the assessment of apparent ileal nutrient digestion; the pigs were dissected on average on day 44 (actually between day 42 and day 46, one pig of each group per day). This provided the opportunity to assess the effects of dietary NDO on fermentation characteristics in the gut contents throughout the gastrointestinal tract.

2.2. Dissection protocol From 4 days before dissection onwards, feeding times were tailored to the estimated time of dissection, allowing 1 h per pig. The pigs were fed in the

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morning and injected with azaperone (Stresnil, Janssen Animal Health, Tilburg, The Netherlands) approximately 20 min before being weighed and being anaesthetized using an inhalation mask providing O 2 / N 2 O and halothane. We used Stresnil to ensure that the pigs were calm at times of anaesthesia, in order to minimise gut contents movement before the actual start of the dissection. It has been shown that | 5% and | 65% of the water-soluble food components may have reached the hindgut by, respectively, 2 and 4 h after feeding (Clemens et al., 1975). In this experiment, gut contents were collected 3 h after the last meal. Therefore, we expected that a considerable proportion of the water-soluble food components, which would include FOSs and TOSs, could have reached the proximal colon at the time of dissection. The abdominal wall was opened and a blood sample taken from the portal vein. Before removal, the gastrointestinal tract was divided into seven sections using clamps and / or plastic strips to further prevent gut contents movement. Firstly, the stomach was isolated. Next, the caecum was located and isolated. Then, 7 m anterior to the ileo–caecal valve, the small intestine was divided into two parts (proximal and distal small intestine). The colon ascendens was divided into two parts, the descending and the ascending part of the ansa spiralis (proximal and mid colon). The remainder was called distal colon.

2.3. Measurements and chemical analysis The separated gut sections were weighed. The gut sections were emptied by stripping gently to obtain a representative sample of the gut contents. The gut sections were rinsed, dried with paper towels and weighed again, in order to obtain the weight of gut contents. The pH was measured with a digital pH meter, immediately after collection. Gut contents were then stored at 2 208C pending analysis. The concentration of dry matter (g / kg) was determined via oven drying (1038C to constant weight) in the contents of each gut section. The concentration of crude protein (g / kg) was measured as 6.25 ? Kjeldahl-N in the contents from the distal small intestine, caecum and proximal colon. The supernatants of the later two, as well as the portal plasma, were analyzed for VFAs, using the gas chromatog-

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raphy method as described in detail previously (Schutte et al., 1992). Sample preparation for gut contents and plasma samples was essentially the same, though plasma samples were deproteinized prior to VFA analysis. Furthermore, the glass column used for analysis of plasma VFAs had a smaller internal diameter (0.5 mm) compared to that used for VFA analysis in gut contents (2 mm). Freeze–dried contents of the stomach, distal small intestine, proximal colon and distal colon were analyzed for the presence of FOSs or TOSs as described previously for freeze–dried faeces (Houdijk et al., 1998a). The high-performance liquid chromatography (HPLC) technique used at the time of the study was carried out, did not allow for a quantitative measurement of FOSs or TOSs, though the detection level of NDOs could be assumed to be at least 100 ppm.

2.4. Statistical analysis An analysis of variance was conducted using the diet as a main effect. Predetermined orthogonal contrasts were used to locate effects of dietary NDOs. These were the effect of type of dietary NDOs (FOSs vs. TOSs), level of dietary NDOs (L vs. H), interaction between type and level of dietary NDOs and the effect of dietary NDOs per se. We have considered effects to be significant at P , 0.05; effects at 0.05 , P , 0.10 have been reported as

tendencies with reference given to the exact P-value. Multiple comparisons were carried out post hoc in order to locate effects within contrasts. The statistical analysis was performed using the SAS GLM procedure (SAS, 1989).

3. Results Table 2 shows the mean growth performance and total tract and ileal dry matter digestion. On average, the NDO pigs were 1.7 and 3.1 kg lighter than the control pigs at day 25 and during dissection, respectively, but these differences were not significant. Dietary NDOs did not significantly affect daily weight gain and feed conversion ratio between day 25 and day 44, which averaged 692625.5 g / day and 1.8660.045, respectively. Dietary NDO did not significantly affect total tract and ileal dry matter digestion. Dietary NDOs did not significantly affect the weight of any gut section, though on average, the NDO pigs tended to have a heavier stomach than the control pigs (6.9 vs. 6.1 g / kg body weight, SED 0.42, P 5 0.06). Table 3 shows the mean gut contents, dry matter concentrations and pH throughout the gastrointestinal tract. The feed intake at the morning of the dissection averaged 683615 g. Significant effects of dietary NDOs on the amount of

Table 2 Performance and dry matter digestion of pigs fed diets with or without non-digestible oligosaccharides Diets a,b CON

Body weight Day 25 Day 44 Daily feed intake (g / day) Daily body weight gain (g / day) Feed conversion Dry matter digestion c Faecal ( | day 30) Ileal ( | day 44)

SED FOS

TOS

Level: L

Level: H

Level: L

Level: H

33.7 47.7 1314 740 1.80

32.3 45.2 1262 683 1.86

33.2 46.3 1279 693 1.89

32.1 44.5 1230 650 1.97

29.5 42.4 1199 679 1.78

2.47 3.63 68.3 72.0 0.123

86.6 74.4

86.9 71.8

86.8 73.7

86.5 73.6

86.9 72.9

0.81 2.85

a CON: Corn-based control diet, no added non-digestible oligosaccharides; FOS: Raftilose P95  at 7.5 (L) and 15.0 (H) g / kg; TOS: Oligostroop  at 10.0 (L) and 20.0 (H) g / kg. Raftilose P95  and Oligostroop  contain fructooligosaccharides and transgalactooligosaccharides, respectively. b Least-square means with standard error of the difference. c From Houdijk et al. (1999).

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Table 3 Amount of gut contents and contents pH and dry matter from pigs fed diets with or without non-digestible oligosaccharides Diets a,b CON

SED FOS

TOS

Orthogonal contrasts c NDO

Type

Level

Type 3

Level: L

Level: H

Level: L

Level: H

Gut contents (g fresh) ST d 1506 PSI 215 DSI 473 CC 111 CD PC 397 MC 241 DC 46

1527 193 535 168 BC 313 211 45

1554 52 485 161 BC 371 183 35

1430 132 570 82 D 307 200 28

1415 75 419 204 AB 369 172 22

177 95 87 31 72 81 25

NS NS NS 0.07 NS NS NS

NS NS NS NS NS NS NS

NS NS NS * NS NS NS

NS NS NS * NS NS NS

pH of gut contents ST d 4.5 A PSI 5.9 DSI 6.7 CC 6.0 PC 6.3 B MC 6.4 DC 6.4

4.1 B 5.7 6.8 5.9 6.5 A 6.4 5.9

4.3 AB 5.9 6.6 5.8 6.5 A 6.7 6.5

4.3 AB 5.7 6.7 6.0 6.3 AB 6.3 6.0

4.4 AB 5.9 6.8 5.8 6.2 B 6.4 6.3

0.15 0.40 0.11 0.19 0.10 0.18 0.23

0.06 NS NS NS NS NS NS

NS NS NS NS ** NS NS

NS NS NS NS NS NS 0.07

NS NS NS NS NS NS NS

196 120 143 171 AB 278 AB 316 305

213 125 117 139 BC 252 B 305 355

18.3 29.6 19.5 19.2 18.1 22.8 –

NS NS NS * 0.08 NS NS

NS NS NS NS NS NS NS

NS NS NS NS 0.09 NS NS

NS NS NS * NS NS NS

Dry matter ST d PSI DSI CC PC MC DC

concentration of gut contents (g / kg) 213 189 196 111 123 138 145 137 153 130 C 162 ABC 190 A 252 B 293 A 276 AB 297 316 300 308 307 383

Level

CON: Corn-based control diet, no added non-digestible oligosaccharides; FOS: Raftilose P95  at 7.5 (L) and 15.0 (H) g / kg; TOS: Oligostroop  at 10.0 (L) and 20.0 (H) g / kg. Raftilose P95  and Oligostroop  contain fructooligosaccharides and transgalactooligosaccharides, respectively. b Least-square means with standard error of the difference. Values with different superscripts differ significantly according to post hoc multiple comparisons (P , 0.05). c *: P , 0.05 and **: P , 0.01. P-values between 0.05 and 0.10 have been given. d ST: Stomach; PSI: proximal small intestine; DSI: distal small intestine; CC: caecum; PC: proximal colon; MC: mid colon; DC distal colon (see Materials and methods for more details). a

gut contents and its dry matter concentration were restricted to the caecum. The TOS-H pigs had more caecal contents than the TOS-L pigs (P , 0.05), and caecal contents of the NDO pigs had a greater dry matter concentration than that of the control pigs (P , 0.05). Within the NDO pigs, an increase in FOS increased caecal dry matter concentration, whilst the opposite was observed for the TOS pigs (P , 0.05). The NDO pigs tended to a lower stomach pH than the control pigs. Dietary NDO did not affect the pH in the small intestine and caecum contents, but the FOS pigs had higher pH in the proximal colon

contents (P , 0.01) than the TOS pigs; that for the control pigs was intermediate. The qualitative HPLC analysis revealed that FOSs and TOSs were present in the distal small intestine contents, but not in the stomach and hindgut contents. Table 4 shows the mean VFA and crude protein concentrations as well as the size of the VFA and crude protein pool in the caecum and proximal colon. Dietary NDOs did not significantly affect the caecal VFA concentration, though the TOS-H pigs had a larger caecal VFA pool than the TOS-L pigs (P , 0.05) and the control pigs. That of the FOS pigs

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180

Table 4 Volatile fatty acids (VFAs) and crude protein in the caecum and proximal colon contents of pigs fed diets with or without non-digestible oligosaccharides Diets a,b CON

SED FOS Level: L

TOS Level: H

Level: L

Orthogonal contrasts c NDO

Type

Level

Level: H

Type 3 level

VFA concentration in gut contents (mmol / l supernatants) Caecum 134 164 165 140 Colon 160 A 120 B 142 AB 164 A

174 168 A

30.2 13.8

NS NS

NS **

NS NS

NS NS

VFA pool (mmol in total gut section) Caecum 15.6 B 22.9 AB Colon 48.5 31.2

10.9 BC 33.4

30.4 A 46.8

5.11 11.44

NS NS

NS NS

* NS

* NS

Crude protein concentration in gut contents (g / kg) Caecum 34.7 39.8 46.1 Colon 59.6 B 66.3 AB 67.5 AB

46.8 70.4 A

40.1 67.3 AB

5.50 4.83

0.07 *

NS NS

NS NS

NS NS

Crude protein pool (g in total gut section) Caecum 4.4 CD 6.5 ABC 7.4 AB Colon 23.5 20.8 24.7

3.9 D 19.7

8.1 A 24.6

0.98 4.26

* NS

NS NS

** NS

* NS

22.1 AB 38.9

CON: Corn-based control diet, no added non-digestible oligosaccharides; FOS: Raftilose P95  at 7.5 (L) and 15.0 (H) g / kg; TOS: Oligostroop  at 10.0 (L) and 20.0 (H) g / kg. Raftilose P95  and Oligostroop  contain fructooligosaccharides and transgalactooligosaccharides, respectively. b Least-square means with standard error of the difference. Values with different superscripts differ significantly according to post hoc multiple comparisons (P , 0.05). c *: P , 0.05 and **: P , 0.01. P-values between 0.05 and 0.10 have been given. a

was intermediate. In contrast, dietary NDO significantly affected the colon VFA concentration, but did not affect the size of the colonic VFA pool; the FOS pigs had lower VFA concentrations in the proximal colon than the TOS pigs (P , 0.01). That of the control pigs was intermediate. Dietary NDO did not significantly affect the composition of VFAs in both caecal and proximal colon contents. The percentage of acetic, propionic, butyric, valeric, isobutyric and isovaleric acids averaged 60, 27, 9, 2, 1 and 1% for caecal VFAs and 59, 23, 11, 3, 2 and 2% for colonic VFAs, respectively. The NDO pigs tended to have higher crude protein concentrations in caecal contents than the control pigs (P 5 0.07). This was significant for the proximal colon contents (P , 0.05). Dietary NDOs affected the pool of crude protein in the caecum but not in the colon; TOS-H pigs, and to a lesser extent FOS-H pigs, contained more crude protein in the caecum than the control pigs (P , 0.05). Dietary NDOs did not affect the crude protein concentration and pool in

the distal small intestine, which averaged 40.462.51 g / kg and 19.962.06 g, respectively. Table 5 shows VFA mean concentration and composition in the portal plasma. The TOS pigs had a higher portal VFA concentration than the FOS pigs (P , 0.05). Dietary NDO affected the portal VFA composition (P , 0.05). The FOS pigs had relatively more acetic and isovaleric acids and less propionic, butyric and valeric acids compared to the TOS pigs and the control pigs.

4. Discussion The results of this study support the view that dietary NDOs can affect fermentation characteristics of the gut contents of growing pigs. The possibility of observing such effects may have been limited in other studies, where the effects of NDOs from the control diet may have masked or diluted the effects of the NDOs added (Farnworth et al., 1992; Bolduan

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Table 5 Volatile fatty acids (VFAs) in the portal plasma of pigs fed diets with or without non-digestible oligosaccharides Diets a,b CON

SED FOS Level: L

TOS

Orthogonal contrasts c NDO

Type

Level

Type 3

Level: H

Level: L

Level: H

Total VFA (mmol / l supernatants) Plasma 1.2 AB 1.2 AB

0.6 B

1.3 AB

1.8 A

0.36

NS

*

NS

0.06

Composition of VFA (%) Acetic 65.0 AB Propionic 20.0 AB Butyric 6.7 A Valeric 2.8 AB Isobutyric 2.2 AB Isovaleric 3.3 B

76.0 A 12.3 B 2.6 B 1.6 B 2.1 AB 5.4 A

62.1 B 22.4 A 5.7 AB 3.6 A 2.7 A 3.5 B

63.3 B 22.8 A 6.4 AB 2.9 AB 1.8 B 2.8 B

5.06 3.68 1.77 0.75 0.35 0.74

NS NS NS NS NS NS

* 0.06 * * NS *

NS NS NS NS NS NS

NS NS NS NS * 0.07

69.2 AB 20.8 AB 2.4 B 2.0 AB 1.8 B 3.8 AB

level

CON: Corn-based control diet, no added non-digestible oligosaccharides; FOS: Raftilose P95  at 7.5 (L) and 15.0 (H) g / kg; TOS: Oligostroop  at 10.0 (L) and 20.0 (H) g / kg. Raftilose P95  and Oligostroop  contain fructooligosaccharides and transgalactooligosaccharides, respectively. b Least-square means with standard error of the difference. Values with different superscripts differ significantly according to post hoc multiple comparisons (P , 0.05). c *: P , 0.05. P-values between 0.05 and 0.10 have been given. a

et al., 1993; Gabert et al., 1995). In this study, the control diet contained hardly any NDOs, and the inclusion of NDOs in this diet resulted in changes in gut fermentation characteristics. However, in contrast to other studies (Hidaka et al., 1985; Katta et al., 1993), dietary NDOs did not affect pig performance. This was probably due to the relatively high performance observed of the pigs offered the control diet, which may have reduced the scope for growth performance improvement. In our experiment, samples were taken and measurements were made 3 h after the last morning meal. Although it may take more than 12 h before indigestible water-soluble food components have quantitatively reached the hindgut in pigs (Clemens et al., 1975), it was expected that a significant proportion of the water-soluble FOSs and TOSs could have reached the hindgut at sampling time. Similarly, the majority of lactulose (Levitt et al., 1987) and FOSs (Stone-Dorshow and Levitt, 1987) in man may be fermented 2–3 h after ingestion. However, it should be noted that small intestinal transit time in man is considerably less than in pigs (Clemens et al., 1975). The inclusion of soluble, non-digestible carbohydrates, such as FOSs and TOSs, may result in an increase in osmotic pressure

in the stomach and small intestine contents, which can consequently reduce oral–caecal transit time (Wiggins, 1984). Calculation on gut contents distribution indicated that the latter may have occurred to some extent in our study; on average, the distal small intestine and caecum of the NDO pigs tended to have relatively more dry matter and water than those gut sections of the control pigs (18 vs. 12%, respectively, P 5 0.08). However, conclusions on the latter should be supported by results obtained from the inclusion of both insoluble and soluble markers in the diets (Clemens et al., 1975). The slightly lowered pH in the stomach content of the NDO pigs may have indicated that to some extent, fermentation of dietary NDO had taken place in the stomach. The porcine stomach mucosa may harbour up to 10 6 colony forming units of lactobacilli per cm 2 (Henriksson et al., 1995). This microflora could have been able to ferment NDOs; Jerusalem artichoke fructan, a polymer of b-linked fructose units, was degraded up to 50% in the ˚ porcine stomach (Graham and Aman, 1986). It might be argued that the absence of FOSs and TOSs in stomach contents was in agreement with dietary NDOs being fermented in the stomach. However, NDO disappearance by fermentation or by stomach

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emptying could not be distinguished in this study. We cannot rule out that NDO is not fermented in the stomach. However, since at least some FOSs and TOSs were found in the contents of the distal small intestine, NDO disappearance from the stomach via emptying did take place to some extent. The effect on stomach content pH may have been small due to the relatively high acid buffering capacity of the diets. In addition, pH changes may have resulted from VFA absorption; it has been shown that VFAs are readily absorbed by the gastric mucosa of pigs (Argenzio and Southworth, 1975). Furthermore, the observed effects on stomach contents pH may have derived from fermentation of any dietary organic matter; some part of the food may be retained in the porcine stomach for many hours (Clemens et al., 1975). The NDO pigs may have had elevated concentrations of VFAs in the hind gut, if NDOs were fermented in this part of the gastrointestinal tract. Hindgut VFAs may inhibit stomach motility (Cuche and Malbert, 1997). Therefore, NDOs may have indirectly led to a prolonged retention of dietary organic matter in the stomach. The question whether dietary NDOs can be fermented in the porcine stomach deserves further attention. To further elucidate NDO fermentation in the porcine stomach, additional measures such as bacterial counts and the concentration of VFAs and other fermentation end products may be taken. The VFAs measured in the gut contents and portal plasma did not derive from dietary NDOs only. In addition to VFAs arising from fermentation in the stomach, it can be estimated from Table 2 that | 27% of the ingested dry matter was potentially available for fermentation in the hindgut, and that | 50% of this amount was apparently degraded. In addition to NDOs, dietary substrates such as cell walls, starch (especially from the unprocessed corn) and undigested protein may have contributed to the VFAs measured. The VFAs formed during fermentation of carbohydrates are acetic, propionic and butyric acids. Valeric acid and the branched chain isobutyric acid and isovaleric acid are considered to originate mainly from protein fermentation, though the latter also yields acetic propionic and butyric acids (Mortensen et al., 1990). The mean concentration of VFAs in the caecum and proximal colon contents were similar to those

earlier observed (Bolduan et al., 1993; Clemens et al., 1975). The NDO pigs tended to have a lower pH and higher VFA and crude protein concentrations in the caecal contents, compared to the control pigs, but these effects were not significant. However, the VFA and the crude protein pool in the caecal contents of the TOS-H pigs was elevated compared to the control pigs. A larger pool of crude protein may indicate that more nitrogen is present in the form of biomass. This indicates that fermentation in the caecum may have been more pronounced in especially the TOS-H pigs at times of sampling than in the other pigs. It is not clear whether the substrates subjected to this fermentation were TOS or whether other organic matter may have been present at larger amounts, as result of a correlated response to dietary TOS more proximal in the gut. The higher pH in the proximal colon contents of the FOS pigs compared to the TOS pigs was in agreement with differences observed in the concentration of VFA (Table 4). However, dietary NDOs did not affect the size of the VFA pool present in the proximal colon contents. Bolduan et al. (1993) also observed similar colonic VFA pool sizes between control and FOS pigs, although the latter may also have been due to a combination of a low inclusion level (2 g FOS / kg) and the wheat / barley based control diet. The discrepancy between effect of dietary NDOs on VFA concentration in colonic water and absence of effect on the size of colonic VFA pool was probably caused by a combination of changes in amount of gut contents and gut contents dry matter concentration. However, the contrast ‘type of dietary NDO’ was not significant for either of these (Table 3). Although dietary NDOs may have reached the hindgut at times of sampling, no NDOs were recovered from the proximal colon contents. This may indicate that NDOs that escaped fermentation in the stomach, are fermented at a very fast rate in, or to some extent prior to, the caecum. The potential fermentation in the stomach has already been addressed (see above). In addition, the ileum also harbours an active microflora (Jensen and Jørgensen, 1994), which may have been capable of fermenting NDOs. This does not necessarily contradict with the presence of NDOs in the distal small intestine contents; the qualitative HPLC analysis may have

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recovered only traces of NDOs, and these may have originated from the more proximal part of what was identified as the distal small intestine. It should be noted that portal VFA measurements, as all other measurements in this study, are taken at one time point, and may vary substantially over time. Nevertheless, it has been shown that the difference in portal flow rate did not differ between pigs fed diets with substantially different amounts of resistant starch (Van der Meulen et al., 1997). On average, our observations on plasma VFA concentration and composition were in close agreement with those reported in the latter study. The FOS pigs had a different VFA composition that the TOS pigs. These differences may indicate that the FOS pigs had a relatively higher degree of microbial proteolytic activity than the TOS pigs. Dietary FOS and TOS may specifically affect the type of amino acids being fermented when needed as a source for energy. Isobutyric, isovaleric, and valeric acids originate from valine, leucine / isoleucine, and proline / hydroxyproline, respectively (Mortensen et al., 1990). The true NDO contents of the NDO-rich products used differed; Raftilose P95  contains | 900 g FOS / kg whilst Oligostroop  contains | 400 g TOS / kg. Therefore, to some extent, effects of type and level of dietary NDO were confounded; a more fair comparison would be between control pigs, FOS-L pigs and TOS-H pigs. The size of the caecal VFA pool of the TOS-H pigs, but not of the FOS-L pigs, was elevated compared to the control pigs. This was reflected to some extent in different portal plasma VFA concentrations; portal VFA concentrations were similar for control- and FOS-L pigs and tended to be higher for the TOS-H pigs (P 5 0.10). These differences point towards the suggestion that porcine microflora may fermented FOS at a higher rate than TOS. The latter has been confirmed in in vitro studies (Houdijk et al., 1998b). The changes observed in gastrointestinal contents characteristics and in portal plasma VFA concentrations have been discussed in relation to the last meal. However, to some extent, effects observed may actually have been related to previous meals. Solid parts of the food may be retained in the gastrointestinal tract, especially in the hindgut, for many days in pigs (Clemens et al., 1975). For example, since both FOSs and TOSs are potential

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laxatives (Schneeman, 1999), total retention time of previous meals in the gastrointestinal tract may have been reduced. This could have affected gut contents characteristics of especially the large intestine. An approach to distinguish between last and previous meals may include the use of particulate markers in previous meals, but not in the last meal.

5. Conclusion This study showed that dietary NDOs can affect fermentation characteristics in gut contents and portal plasma of growing pigs. The data support the view that dietary TOSs may be fermented at a slower rate than dietary FOSs, and that fermentation of NDOs may take place to some extent prior to the caecum. However, since the data were obtained at one time point after feeding (3 h), the conclusions should be confirmed by a serial slaughter experiment, or by sampling of the same animal over time.

Acknowledgements The authors thank Erik Berenpas, Tamme Zandstra, Peter van der Togt, Piet van Leeuwen, Casper Deuring and Dick van Kleef for taking care of the pigs and the collection of the samples. Margaret Bosveld is thanked for the NDO analysis, and Dick Bongers and Henry Leuvenink for the VFA analysis. We thank Barbara Williams and Seerp Tamminga for their comments on earlier versions of the manuscript. This work was supported by the Netherlands Ministry of Agriculture, Nature Management and Fisheries, the Dutch Foundation on Nutrition and Health, AVEBE, Nutreco (all The Netherlands), and ORAFTI (Belgium).

References Argenzio, R.A., Southworth, M., 1975. Sites of organic acid production and absorption in the gastrointestinal tract of the pig. Am. J. Physiol. 228, 454–460. Bolduan, G., Beck, M., Schubert, C., 1993. Zur werking von oligosacchariden beim ferkel. Arch. Anim. Nutr. 44, 21–27. Cuche, G., Malbert, C.H., 1997. Ileal short chain fatty acids inhibit gastric motility via an humoral pathway. In: Laplace,

184

J.G.M. Houdijk et al. / Livestock Production Science 73 (2002) 175 – 184

´ J.-P., Fevrier, C., Barbeau, A. (Eds.), Digestive Physiology in Pigs. EAAP Publication No. 88. EAAP, Saint Malo, pp. 13–17. Clemens, E.T., Stevens, C.E., Southworth, M., 1975. Sites of organic acid production and pattern of digesta movement in the gastrointestinal tract of swine. J. Nutr. 105, 759–768. Farnworth, E.R., Modler, H.W., Jones, J.D., Cave, N., Yamazaki, H., Rao, A.V., 1992. Feeding Jerusalem artichoke flour rich in fructooligosaccharides to weanling pigs. Can. J. Anim. Sci. 72, 977–980. Gabert, V.M., Sauer, W.C., Mosenthin, R., Schmitz, M., Ahrens, F., 1995. The effect of oligosaccharides and lactitol on the ileal digestibilities of amino acids, monosaccharides and bacterial populations and metabolites in the small intestine of weanling pigs. Can. J. Anim. Sci. 75, 99–107. Gibson, G.R., Roberfroid, B.M., 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125, 1401–1412. ˚ Graham, H., Aman, P., 1986. Composition and digestion in the pig gastrointestinal tract of Jerusalem artichoke tubers. Food Chem. 22, 67–76. Henry, R.J., Saini, H.S., 1989. Characterization of cereal sugars and oligosaccharides. Cereal Chem. 66, 362–365. Henriksson, A., Andre, L., Conway, P.L., 1995. Distribution of lactobaccili in the porcine gastrointestinal tract. FEMS Microbiol. Ecol. 16, 55–60. Hidaka, H., Eida, T., Hashimoto, T., Nakazawa, T., 1985. Feeds for domestic animals and method for breeding them. Eur. Pat. Appl. 133547A2. Houdijk, J.G.M., Bosch, M.W., Verstegen, M.W.A., Berenpas, H.J., 1998a. Effects of dietary oligosaccharides on the growth performance and faecal characteristics of young growing pigs. Anim. Feed Sci. Technol. 71, 35–48. Houdijk, J.G.M., Williams, B.A., Tamminga, S., Verstegen, M.W.A., 1998b. Effect of non-digestible oligosaccharides in diets for weaner pigs on in vitro fermentation. Proc. Br. Soc. Anim. Sci. 114, 30. Houdijk, J.G.M., Bosch, M.W., Tamminga, S., Verstegen, M.W.A., Berenpas, E.H., Knoop, H., 1999. Apparent ileal and total tract nutrient digestion by pigs as affected by dietary nondigestible oligosaccharides. J. Anim. Sci. 77, 148–158. Jasaitis, D.K., Wohlt, J.E., Evans, J.L., 1987. Influence of feed ion

content on buffering capacity of ruminant feedstuffs in vitro. J. Dairy Sci. 70, 1391–1403. Jensen, B.B., Jørgensen, H., 1994. Effect of dietary fiber on microbial activity and microbial gas production in various regions of the gastrointestinal tract of pigs. Appl. Environ. Microbiol. 60, 1897–1904. Katta, Y., Ohkuma, K., Satouchi, M., Takahashi, R., Yamamoto, T., 1993. Feed for livestock. Eur. Pat. Appl. 549478A1. Levitt, M.D., Hirsch, P., Fetzer, C.A., Sheanan, M., Levine, A.S., 1987. H 2 excretion after ingestion of complex carbohydrates. Gastroenterology 92, 383–389. ´ H., Clausen, M.R., 1990. Mortensen, P.B., Holtug, K., Bonnen, The degradation of amino acids, proteins, and blood to shortchain fatty acids in colon is prevented by lactulose. Gastroenterology 98, 353–360. Saini, H.S., 1989. Legume seed oligosaccharides. In: Huisman, J., Van der Poel, A.F.B., Liener, I.E. (Eds.), Recent Advances of Research in Antinutritional Factors in Legume Seeds. Pudoc, Wageningen, pp. 329–341. SAS, 1989. SAS User’s Guide: Statistics, 5th Edition. SAS Institute, Cary, NC. Schneeman, B.O., 1999. Fiber, inulin and oligofructose: similarities and differences. J. Nutr. 129 (Suppl.), 1424S–1427S. Schutte, J.B., Jong, J., de Weerden, E.J., van Tamminga, S., 1992. Nutritional implications of L-arabinose in pigs. Br. J. Nutr. 68, 195–207. Stone-Dorshow, T., Levitt, M.D., 1987. Gaseous response to ingestion of a poorly absorbed fructooligosaccharide sweetener. Am. J. Clin. Nutr. 46, 61–65. Toba, T., Watanabe, A., Adachi, S., 1983. Quantitative changes in sugars, especially oligosaccharides, during fermentation and storage of yogurt. J. Dairy Sci. 66, 17–20. Van der Meulen, J., Bakker, G.C.M., Bakker, J.G.M., De Visser, H., Jongbloed, A.W., Everts, H., 1997. Effect of resistant starch on net portal-drained viscera flux of glucose, volatile fatty acids, urea, and ammonia in growing pigs. J. Anim. Sci. 75, 2697–2704. Wiggins, H.S., 1984. Nutritional value of sugars and related compounds undigested in the small gut. Proc. Nutr. Soc. 43, 69–75.