Intestinal metabolism of weaned piglets fed a typical United States or European diet with or without...

Fiorentini, J. Wolinski, R. Zabielski and J. A. PattersonA. Piva, E. Grilli, L. Fabbri, V. Pizzamiglio, P. P. Gatta, F. Galvano, M. Bognanno, L.

diet with or without supplementation of tributyrin and lactitolIntestinal metabolism of weaned piglets fed either a typical United States or European

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Intestinal metabolism of weanling piglets1

2

3

4

5

Intestinal metabolism of weaned piglets fed either a typical United States or European diet with 6

or without supplementation of tributyrin and lactitol7

8

9

10

A. Piva,*1 E. Grilli,* L. Fabbri,* V. Pizzamiglio,* P. P. Gatta,* F. Galvano,† M. Bognanno,‡ L. 11

Fiorentini,§ J. Woliński,# R. Zabielski,║ and J. A. Patterson¶12

13

14

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*DIMORFIPA, University of Bologna, 40064, Ozzano Emilia, Bologna, Italy; †Department of 16

Biological Chemistry, Medical Chemistry and Molecular Biology, University of Catania, 95125, 17

Catania, Italy; ‡Department of Agro-forestry, Environmental Science and Technology, Mediterranean 18

University of Reggio Calabria, 89061, Reggio Calabria, Italy; §ISAN, Università Cattolica S. Cuore, 19

29100, Piacenza, Italy; #The Kielanowski Institute of Animal Physiology and Nutrition, 05110, 20

Jablonna, Poland, ║Department of Physiological Sciences, Warsaw Agricultural University, 02787, 21

Warsaw, Poland; and, ¶Department of Animal Sciences, Purdue University, West Lafayette, IN 47907.22

1Corresponding author: [email protected]

Page 1 of 32 Journal of Animal Science



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ABSTRACT: Aim of the study was to investigate the effect of supplementation of a 23

microencapsulated blend of tributyrin and lactitol (TL) to a standard European (EU) diet without 24

antibiotic growth promoters on intestinal metabolism and mucosa development of weaned piglets, and 25

to compare it to a standard US diet containing animal proteins, zinc oxide, copper sulphate, and 26

carbadox. Ninety piglets weaned at 21 d were divided into 3 dietary groups consisting of 5 replicates 27

each: 1) US diet supplemented with 55 ppm carbadox, and 2.5% each of plasma proteins and spray 28

dried blood cells in the first phase, 3,055 ppm Zn in the first and second phases, and 180 ppm Cu in the 29

third phase; 2) EU diet based on vegetable proteins and no antibiotics; and 3) the same EU diet 30

supplemented with 3,000 ppm microencapsulated TL. The study was divided into 3 phases: 0 to 7, 8 to 31

21, and 22 to 35 d. On d 7, 21, and 35, animals were weighed, and feed consumption and efficiency 32

were determined. On d 14 and 35, one pig per pen was sacrificed, and the intestinal content and mucosa 33

from proximal, middle, distal jejunum, and ileum were sampled. Intestinal wall sections were fixed for 34

hystological analysis, and intestinal content was used for VFA, ammonia, and polyamines analysis. 35

Throughout the study (d 0 to 35), the US diet had greater ADG and ADFI than the EU diet (P < 0.05). 36

The EU diet supplemented with TL tended to have 11% greater ADG (P = 0.17). Feeding the EU diet 37

caused a reduction in proximal and middle jejunum villi length by 10% (P < 0.05) and an increase in 38

crypt size in proximal jejunum (P < 0.05) compared with the US diet, probably due to an increased rate 39

of cell loss and crypt cell production. The TL supplementation resulted in longer villi along the 40

jejunum and less deep crypts in the proximal jejunum (+15.9 and –8.9%, respectively; P < 0.05) than 41

the unsupplemented EU diet. The TL diet increased the concentrations of cadaverine and putrescine in 42

the small intestine (P < 0.05), and seemed to increase cadaverine, histamine, putrescine, and spermine 43

in the large intestine by 1.5- to 10-fold compared with the US or EU diet. In conclusion, although the 44

US diet had a greater effect on growth performance and mucosal trophic status than the EU diets, the 45

Page 2 of 32Journal of Animal Science



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supplementation with slowly-released TL seemed to be an effective tool to partially overcome the46

adverse effects of vegetable protein diets.47

Key words: lactitol, metabolism, piglets, tributyrin, weaning48

49

INTRODUCTION50

The ban of antibiotics as a growth promoter from animal feed in the European Union (EU) has 51

motivated research on alternative ways to optimize the digestive process and increase nutrient 52

availability. Diet formulation may include feed supplements, such as probiotic cultures, organic acids 53

(Partanen and Mroz, 1999), botanicals, and non-digestible oligosaccarides. 54

Volatile fatty acids play a role in modulating the digestive process and can be supplied by direct 55

feed supplementation or properly promoting the intestinal microbial production. Butyric acid is 56

produced by bacterial fermentation of carbohydrates, and it serves as a primary source of energy for 57

colonocytes and a strong mitosis promoter and a differentiation agent in the gastrointestinal tract in 58

vivo (Salminen et al., 1998). In a previous work, it has been shown that the synergistic effect of a 59

fermentable substrate, lactitol, and a precursor of butyric acid, tributyrin, can improve the trophic status 60

of the intestinal mucosa in the gut of nursery piglets and control intestinal histamine (Piva et al., 2002).61

The purpose of the present study was to investigate the effect of supplementation of a 62

microencapsulated blend of tributyrin and lactitol (TL) to a standard EU diet without antibiotic growth 63

promoters on intestinal metabolism and mucosa development of weaned piglets, and to compare it to a 64

standard US diet containing animal proteins, zinc oxide, copper sulphate, and carbadox. 65

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MATERIALS AND METHODS67




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Animals, Diets and Facilities68

At 21 d after birth, the piglets (Landrace × Large White; 6.14 ± 1.38 kg BW) were randomly 69

allocated to 3 experimental dietary groups (5 pens/diet with 6 piglets/pen) according to their initial 70

weight, gender (females and castrated males), and litter, and were fed ad libitum the following 71

experimental diets: 1) typical US diet containing animal proteins in the first phase (2.5% plasma 72

proteins and 2.5% spray dried blood cells), 3,055 ppm zinc (3,800 ppm ZnO) in the first and the second 73

phase, 180 ppm copper (800 ppm CuSO4) in the third phase, and 55 ppm carbadox during the whole 74

experimental period (Table 1); 2) typical EU diet based on vegetable protein without growth-promoters 75

(Table 2); and 3) the same EU diet supplemented with 3,000 ppm of microencapsulated blend of TL 76

(Luchansky and Piva, 2001; Vetagro S.R.L., Reggio Emilia, Italy). The feeding study was subdivided 77

into 3 phases: phase 1 (0 to 7 d), 2 (8 to 21 d), and 3 (22 to 35 d). On d 7, 21, and 35 after the 78

beginning of the study, pigs were individually weighed, and feed consumption and efficiency were 79

determined.80

On d 14 and 35, 1 pig per pen was sacrificed, and, within 15 min, intestinal content and mucosa 81

were sampled for morphometric, VFA, ammonia, and polyamines analyses. Intestinal content 82

collected from the same sections was divided in aliquots and frozen at -20°C. The present study was 83

conducted at Purdue University facilities with approval of Purdue University Animal Care and Use 84

Committee.85

Chemical Analyses of Feed and Intestinal Contents86

The DM, CP, ether extract, crude fiber, ash, and starch contents of the feed were determined 87

according to the AOAC (2000) methods. Volatile fatty acids were determined using a gas 88

chromatograph (Hewlett Packard 3790) and a packed column (SP-1200; Hewlett Packard, Palo Alto, 89

CA) according to the method described by Playne (1985). Before injection, samples were diluted with 90

sterile deionized water (1:1), and, then, 0.8 mL of samples were mixed with 0.2 mL of 25% o-91




5

phosphoric acid (Fischer Chemical, Fair Lawn, NJ) and, after 30 min, centrifuged at 13,000 × g for 12 92

min at 4°C. The supernatants were frozen at -20°C overnight, thawed, centrifuged again, filtered, and, 93

then, injected into the gas chromatograph for determination of propionic, acetic, butyric, valeric, 94

isobutyric, and isovaleric acid concentrations. Ammonia concentration was determined (Chaney and 95

Marbach, 1962) after centrifuging intestinal liquor samples.96

Polyamine Determination97

Mono-, di-, and polyamines were separated and quantified by HPLC with fluorimetric detection as 98

described in a previous work (Piva et. al., 2002). The limits of detection for the various amines were: 99

putrescine 0.023 pM; cadaverine 0.022 pM; histamine 0.48 pM ; tyramine 0.068 pM; spermidine 0.012 100

pM ; and spermine 0.007 pM.101

Morphometry Analysis102

The small intestine from jejunum to the ileal cecal valve was subdivided into 4 segments as 103

proximal, middle, distal jejunum, and ileum. The segment from the pylorus to the ligament of Trietz 104

was considered as duodenum, the proximal segment of the rest of the small intestine was considered 105

the jejunum, and a distal segment 10 cm proximal to the ileocecal junction was considered the ileum106

(Tang et al., 1999). Intestinal wall sections were sampled from jejunum (proximal, middle, and distal) 107

and ileum, fixed in Bouin’s fluid (Ricca Chemical Company, Arlington, TX), and preserved in 70% 108

ethanol for hystological analysis. Pig intestinal mucosa was sampled from proximal, middle, and distal 109

jejunum (25, 50, and 75% of the total length, respectively), and ileum. Whole thickness segments of 110

the gut were collected, fixed in Bouin’s solution (Sigma, St. Louis, MO) and stored in alcohol for 111

morphometry analysis. The fixed samples were dehydrated in 96 and 99.8% ethanol and embedded 112

into the xylene and paraffin (ParaPlast Regular, P-3558, Sigma). Serial, histological sections of 5 µm 113

thickness were cut (Microm HM 355S; MICROM GmbH, Walldorf, Germany) and stained with 114

hematoxylin and eosin for morphometry analysis. After staining, the depth of crypts, length of villi, 115




6

and thickness of tunica mucosa and muscolaris mucosae were measured in the small intestine 116

preparations. In each slide, about 20 well-oriented villi and crypts located outside the area with Peyer’s 117

patches were measured at small magnification with an optical binocular microscope coupled to the 118

personal computer with a planimetry software (LSM 5 PASCAL 3.2 SP2; Carl Zeiss, Göttingen, 119

Germany). The villus length was considered as the distance from the crypt opening to the tip of the 120

villus, while the depth of crypt was measured from the base of the crypt to the level of the crypt 121

opening (Kotunia et al., 2004).122

Statistical Analysis123

All data were analyzed using the GraphPad Prism program (GraphPad Prism v. 4.0, GraphPad 124

Software, San Diego, CA). The pen was the experimental unit for growth performance data, while the 125

animal was the experimental unit for chemical and morphological intestinal data. Animal performance 126

was analyzed with repeated measures 1-way ANOVA with Bartlett’s test for equal variances and the 127

Newman-Keuls post test; whereas, ammonia, VFA, and amine concentration data were subjected to 128

simple 1-way ANOVA with Bartlett’s test for equal variances and the Newman-Keuls post test. For 129

morphometric data, nonparametric Kruskal-Wallis test and Dunn's multiple comparison test were used 130

to compare the effect of diets. In all statistical analyses, P < 0.05 was considered as the level of 131

significance. An unpaired t-student test was performed whenever comparisons between treatments 132

were necessary to discuss results.133

134

RESULTS135

Animal Performance136

During the phase 1 of the study (d 0 to 7), no differences in growth performance were observed 137

(Table 3). During phase 2 (d 8 to 21), animals fed the US diet had greater (P < 0.05) ADG and ADFI 138

than animals fed the EU or TL diet. The statistically significant advantage in ADG obtained by the US 139




7

diet at the end of phase 2 were no longer present by the end of phase 3. However, during the entire140

study (d 0 to 35), piglets fed the US diet supplemented with carbadox, ZnO, and CuSO4 had greater (P141

< 0.05) ADG and ADFI than those fed the EU diet . The EU diet supplemented with TL resulted in 142

intermediate ADG between the US and EU diets.143

Morphometry Analysis144

Mucosa thickness did not differ among treatments in the proximal jejunum, while in the middle and 145

distal jejunum, it increased by TL supplementation (P < 0.05; Table 4). Villi length was the highest (P146

< 0.05) for the US diet in the proximal jejunum, while in the distal jejunum, the EU diets had longer 147

villi than the US diet (P < 0.05; Figure 1a). Moreover, TL supplementation produced longer villi 148

throughout the jejunum (P < 0.05; Figure 1a) and less deep crypts in the proximal jejunum (P < 0.05; 149

Figure 1b) than the unsupplemented EU diet. Muscolaris mucosae thickness for the EU diets was 150

greater than the US diet in proximal and middle jejunum (P < 0.05), while the US and TL diets had the 151

same thickness in the distal jejunum. Ileal samples were damaged during transport and preparation, 152

and an appropriate statistical analysis could not be conducted.153

Amines and Ammonia in Jejunum, Ileum, Cecum and Colon154

Gut tyramine and spermidine concentrations were not affected by any dietary treatments (Table 5). 155

Piglets receiving the TL diet had greater concentrations of cadaverine in jejunum, ileum, cecum, and 156

colon than piglets fed the US or the EU diet (P < 0.05). Similarly, the TL diet resulted in a greater157

putrescine concentration in ileum of piglets than those fed the US or EU diet. In cecum, the TL diet 158

resulted in greater concentrations of cadaverine, histamine, putrescine, and spermine than the US diet 159

(P < 0.05). The same effect was observed in the colon for cadaverine, histamine, putrescine, and 160

spermine (P < 0.05). There were no effects of diets on gut ammonia concentrations (Table 6).161




8

Volatile Fatty Acids in the Cecum and Colon162

At d 14 of the study, the cecal contents showed no differences in VFA concentrations (Table 7). At 163

the same time, the TL diet had greater colonic acetic acid and total VFA concentrations than the US 164

diet (P < 0.05; Table 8). No difference were observed in propionic, isobutyric, butyric, isovaleric, and 165

valeric acids.166

At d 35, cecal valeric acid concentration was greater for the EU based diets compared to the US 167

diet (P < 0.05); whereas, colonic valeric acid concentration was greater for piglets fed the TL diet than 168

those fed the US diet (P < 0.05). Cecal acetic and propionic acid proportions were the greatest and the 169

least (P < 0.05), respectively, for the US diet.170

171

DISCUSSION172

Animals fed a typical US diet supplemented with plasma proteins, carbadox, copper sulfate, and 173

zinc oxide had the best growth performance from 8 to 21 d post-weaning. No appreciable difference 174

was observed thereafter. However, during the entire 35-d study, piglets fed with a typical EU diet with 175

soybean protein supplemented with microencapsulated tributyrin and lactitol tended to have improved176

growth performance than piglets fed with the same diet without supplementation. In fact, final weights 177

were 4.8 and 6.2% higher for piglets fed the TL diet than the EU diet after 21 and 35 d, respectively, 178

and overall ADG, ADFI, and G:F tended to be 11.1, 2.2, and 8.0% greater, respectively.179

Feeding the US diet resulted in differences in small intestine histology compared with the EU diet 180

(Table 4). Feeding the EU diet caused a reduction in villi length by about 20% and an increase in crypt 181

depth. This implied that the gut absorptive surface was reduced in piglets fed with the standard EU diet 182

in comparison with the US diet, and this might be due to either reduction in mitosis or in the increase 183

of apoptosis ratio or both (Godlewski et al., 2005). However, in the distal jejunum of piglets fed the 184

EU diet, an increased mucosa thickness and longer villi, in comparison with piglets fed the US diet, 185




9

were observed. This might have compensated for the reduction of the absorptive surface (short villi) in 186

the proximal jejunum. The muscular wall in the proximal, middle, and distal jejunum was thicker in 187

piglets fed the EU diet than those fed the US diet. Supplementation of the EU diet with TL improved 188

the mucosa thickness and the villi length in the middle and distal jejunum, thereby, increasing the 189

absorptive area of the gut, while muscolaris thickness was reduced in the proximal and distal jejunum. 190

The effect on the crypts was observed only in the proximal jejunum where the depth had been reduced 191

by TL supplementation in neonatal piglets, and sodium butyrate reduced the crypt depth in the jejunum 192

(Kotunia et al., 2004).193

Along the length of the jejunum, the downstream reduction of villous length observed in piglets fed 194

the US diet could be associated with a decreased enteral nutrient availability (Pluske, 2001). Such 195

downstream reduction of villi length did not occur in piglets fed the EU diet that had shorter villi at 196

proximal and middle jejunum than those fed the US diet; whereas, in distal jejunum, piglets fed the EU 197

diet had 15.2% longer villi than those fed the US diet, which is likely due to greater nutrient 198

availability at distal jejunum.199

The villous shortening, observed in piglets fed the EU diet compared to those fed the US diet, is 200

often associated with an increased rate of cell loss, increased crypt-cell production, and increased crypt 201

depth (Pluske, 2001). Such increased crypt-depth was, in fact, found in proximal jejunum of piglets 202

fed the EU diet compared to those fed the US diet. The reduction in crypt-depth in the US diet, as 203

compared to EU diet, is presumably the result of anti-secretory action of ZnO in the US diet. Tributyrin 204

and lactitol supplementation increased villous length over piglets fed the EU diet in the proximal, 205

middle, and distal jejunum by 14.2, 22.5, and 11%, respectively, and villus/crypt ratios for piglets fed 206

the US, EU, and TL diets were 2.3, 1.4, and 1.7 for proximal jejunum, 1.7, 1.5, and 1.8 for middle 207

jejunum, and 1.5, 1.5, and 1.6 for distal jejunum, respectively. This is certainly the result of 208

stimulation of mitosis as well as reduction of programmed cell death type I (apoptosis) via butyrate 209




10

(Kotunia et al., 2004). Morover, Kotunia et al. (2004) demonstrated a 4-fold increase in plasma 210

cholecystokinin in piglets fed the butyrate supplemented diet. This regulatory peptide is known for its 211

secretion, as well as growth-promoting effects, in the gastrointestinal tract of young mammals (Biernat 212

et al., 1999). Similarly, increased mucosa and muscolaris mucosae thickness in piglets fed the EU and 213

TL diets, compared to those fed the US diet was observed along the jejunum.214

Overall, morphometric measurements matched growth performance data, which showed the best 215

growth performance with the US diet, with the intestinal mucosa architecture supporting that the diet 216

would be highly digestible in proximal and middle jejunum. Conversely, the longer villi in distal 217

jejunum implied that the EU diet was slowly or less digestible than the US diet. Tributyrin and lactitol 218

treatment increased villous length in jejunum and tended to improve growth performance.219

Tyramine and spermidine concentrations did not show any differences associated with diets in any 220

of the gastrointestinal tract section analyzed. Tributyrin and lactitol increased the concentrations of 221

cadaverine and putrescine in the small intestine and cadaverine, histamine, putrescine, and spermine in 222

the large intestine by 1.5- to 10-fold. The TL diet increased putrescine and its metabolite spermine 223

concentrations by approximatively 1.5-fold compared to the EU diet in the large intestine; whereas, in 224

the small intestine, a 2-fold increase was observed only for putrescine. Putrescine can act as growth 225

factor for the gut (Seidel et al., 1985), while its metabolite spermine can induce maturation of the small 226

intestine primarily in suckling but not in weaned animals (Peulen et al., 2004; Powroznik et al, 2004). 227

Such increase in putrescine concentrations in the small intestine can be associated with the longer 228

villi measured in piglets fed the TL diet. However, the increased availability of putrescine might have 229

been linked primarily to an increased large intestine microbial production due to lactitol contained in 230

TL as described for other indigestible oligosaccharides or inuline (Noack et al., 1998; Deizenne et al., 231

2000; Bailey et al., 2002) and to a subsequent transport to the small intestine via the portal circulation 232

and bilary duct (Osborne and Seidel, 1990).233




11

The enterohepatic circulation of polyamines can also justify the increased cadaverine 234

concentrations in the small intestine by 5- to 10-fold observed in piglets fed the TL diet compared to 235

those fed the EU diet. Cadaverine and its metabolite piperidine are originated from microbial 236

decarboxylation of lysine and have shown to specifically inhibit Shigella flexneri-induced 237

transepithelial migration of polymorphonuclear leucocytes in case of dysentery in humans (McCormik 238

et al., 1999; Fernandez et al., 2001) and to prevent the invasion of Salmonella typhimurium into 239

intestinal epithelium (Kohler et al., 2002). In our study, we did not measure piperidine; however, the 240

increase of its precursor cadaverine may be useful in down-regulating active inflammation at mucosal 241

surface (Kohler et al., 2002).242

The profound changes associated with the TL diet were not observed in a previous study despite the 243

greater dose of tributyrin and lactitol used (Piva et al., 2002). The explanation for such difference may 244

be a consequence that in the current study, tributyrin and lactitol were microencapsulated in a lipid 245

matrix that has been shown to allow a slow-release of material along the gut (Piva et al., 2007) and that 246

might have prevented a substantial loss of lactitol in the small intestine, which has been shown to be 247

fermented to lactic acid (Piva et al., 2002).248

Cecal and colonic ammonia concentrations were not different among any of the tested diets. 249

Conversely, the ratio of acetic to propionic acids was decreased in cecum of piglets fed the EU and TL 250

diets compared to those fed the US diet, indicating that a greater amount of indigested starch could be 251

present in piglets fed the EU and TL diets in the second phase, but such difference was not evident at d 252

14 of the study. Overall, total VFA concentrations were not different in the cecum after d 14 and 35 253

and in the colon d 35.254

Piglets fed the TL diet did not show any difference in VFA when compared to those fed the EU 255

diet, with the exception of a relevant increase in acetic acid in the colon after the first phase diet, 256




12

despite the fact that the non-microencapsulated TL promoted propionic rather than acetic acid 257

production (Piva et al. 1996).258

The US diet nutrition plan showed the best growth performance until 21 d post-weaning, which was 259

associated with longer villi in the proximal and middle jejunum. The plain vegetable protein (EU diet)260

was less effective and showed an intestinal architecture indicating that a diet would be digested more 261

slowly. The TL supplementation to the EU diet increased villous length through an increase in 262

putrescine and cadaverine that, along with the promitotic and antiapoptotic effect of butyric acid, have 263

resulted in improved digestion and in a tendency toward increased growth rate. In conclusion, the use 264

of slowly-released tributyrin and lactitol seemed to be an effective tool to partially overcome the lack 265

of animal proteins, carbadox, zinc oxide, and copper sulphate in the EU vegetable diets for piglets. 266

267

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Tang, M., B. Laarveld, A. G. Van Kessel, D. L. Hamilton, A. Estrada and J. F. Patience. 1999. Effect 327

of segregated early weaning on postweaning small intestinal development in pigs. J. Anim. Sci. 328

77:3191-3200.329




16

Table 1. Composition of US diet for phases 1 to 3330

Item

Phase 1,

d 0 to 7

Phase 2,

d 8 to 21

Phase 3,

d 22 to 35

Ingredients, % (as-fed basis)

Corn 39.25 53.12 68.56

Soybean meal, 48% CP 20.0 27.15 26.98

Dicalcium phosphate (CaHPO4) 1.10 0.74 1.29

Limestone 0.60 0.39 0.72

Salt 0.30 0.25 0.35

Animal fat 5.00 - 1.0

Soybean oil - 3.00 -

L-lysine•HCl 0.15 0.15 0.15

DL-methionine 0.15 0.05 -

Swine vitamin premix1 0.15 0.25 0.25

Swine trace mineral premix2 0.17 0.17 0.17

Dried whey 25.0 10.0 -

Fish meal 2.50 4.00 -

Plasma protein 2.50 - -

Spray dried blood cells 2.50 - -

Zinc oxide (ZnO) 0.38 0.38 -

Copper sulfate (CuSO4) - - 0.08

Carbadox 0.25 0.25 0.25

Banmith dewormer3 - - 0.10

Phytase4, 600 PU/g - 0.1 0.10

Chemical composition, DM basis (other than DM)




17

CP, %22.69 23.56 19.88

Ether extract, % 8.23 5.26 3.76

Crude fiber, %1.99 2.55 2.95

Ash, %6.20 6.00 5.36

Starch, %34.74 38.16 48.42

GE, MJ/kg18.28 18.05 17.42

1Provided per kilogram of complete diet: 11,026 IU of vitamin A; 1,654 IU of vitamin D3; 44 IU 331

of vitamin E; 4.4 mg of vitamin K (menadione sodium bisulfite); 55.1 mg of niacin, 33.1 mg of 332

pantothenic acid (as calcium pantothenate); 9.9 mg of riboflavin; and 0.044 mg of B12.333

2Provided per kilogram of complete diet: 39.7 mg of Mn (oxide), 165.4 mg of Fe (sulfate), 165 334

mg of Zn (oxide), 16.5 mg of Cu (sulfate), 0.30 mg of I (as Ca iodate), and 0.30 mg of Se (as Na 335

selenite).336

3Anthelmintic (Pfizer, Inc., New York, NY).337

4PU = phytase unit; Natuphos (BASF Corporation, Parsippany, NJ).338




18

Table 2. Composition of European diets for phases 1 to 3339

Phase 1,

d 0 to 7

Phase 2,

d 8 to 21

Phase 3,

d 22 to 35

Item EU TL EU TL EU TL

Ingredients, % (as-fed basis)

Whey 20.13 20.13 9.99 9.99 7.03 7.03

Steam rolled barley 25.16 25.16 29.96 29.96 35.15 35.15

Steam rolled corn 10.07 10.07 4.99 4.99 - -

Corn 9.74 9.14 28.05 27.65 31.99 31.38

Dextrose 7.05 7.05 3.00 3.00 - -

Extruded soybeans 5.03 5.03 3.00 3.00 5.02 5.02

Soy protein

concentrate 5.03 5.03 3.00 3.00 2.01 2.01

Soybean meal, 48%

CP 3.32 3.32 7.57 7.58 9.36 9.36

Skim milk 3.02 3.02 2.00 2.00 - -

Potato protein1 3.02 3.02 2.50 2.50 2.01 2.01

Wheat bran 2.83 2.83 0.14 0.14 - -

Soy oil 2.52 2.52 2.50 2.50 3.42 3.42

Swine trace mineral

premix2 0.13 0.13 0.13 0.13 0.13 0.13

Swine vitamin

premix3 0.25 0.25 0.25 0.25 0.25 0.25

Monocalcium

phosphate 0.85 0.85 1.41 1.42 1.90 1.90




19

(CaH2PO4)

L-lysine•HCl 0.54 0.54 0.54 0.54 0.45 0.45

Calcium sulphate

(CaSO4) 0.30 0.30 0.30 0.30 0.30 0.30

DL-methionine 0.29 0.29 0.23 0.23 0.19 0.19

Salt 0.24 0.24 - - 0.37 0.37

L-threonine 0.22 0.22 0.21 0.21 0.18 0.18

L-tryptophan 0.07 0.07 0.06 0.06 0.04 0.04

Herbiotic HB4 0.20 0.20 0.20 0.20 0.20 0.20

TRILAC5 - 0.30 - 0.30 - 0.30

Chemical composition, DM basis (other than DM)

CP, %19.31 19.13 18.69 19.19 19.63 19.25

Ether extract, %5.94 5.88 5.83 6.03 6.55 7.27

Crude fiber, %2.95 3.08 2.75 3.08 2.67 3.45

Ash, %5.09 4.83 4.33 4.51 4.82 4.98

Starch, %30.24 33.84 40.32 44.46 42.12 43.20

GE, MJ/kg17.88 17.96 18.05 18.07 18.23 18.15

1Protastar (AVEBE Feed, Veendam, The Netherlands).340

2Provided per kilogram of complete diet: 11,026 IU of vitamin A; 1,654 IU of vitamin D3; 44 341

IU of vitamin E; 4.4 mg of vitamin K (menadione sodium bisulfite); 55.1 mg of niacin, 33.1 mg of 342

pantothenic acid (as calcium pantothenate); 9.9 mg of riboflavin; and 0.044 mg of B12.343

3Provided per kilogram of complete diet: 39.7 mg of Mn (oxide), 165.4 mg of Fe (sulfate), 165 344

mg of Zn (oxide), 16.5 mg of Cu (sulfate), 0.30 mg of I (as Ca iodate), and 0.30 mg of Se (as Na 345

selenite).346

4Flavorant (Agri-Life Sciences HB, Souderton, PA).347

5Supplying tributyrin and lactitol at 3,000 ppm (Vetagro, Reggio Emilia, Italy).348




20

Table 3. Growth performance of piglets fed either a medicated US diet or a non-medicated 349

European diet350

Treatment1

Item US EU TL SEM P- value

Pens/treatment 5 5 5

Initial Weight, kg 6.56 6.21 6.15 0.86 0.94

Phase 1 (d 0 to 7)

ADG, g 98.6 68.2 88.1 26.36 0.82

ADFI, g 161.2 152.6 164.0 20.14 0.92

G:F, g/kg 612 447 537 343.42 0.66

Weight, kg (d 7) 7.25 6.69 6.77 0.91 0.90

Phase 2 (d 8 to 21)

ADG, g 437.9b 196.8a 221.0a 26.42 0.001

ADFI, g 740.1b 555.7a 559.9a 32.65 0.006

G:F, g/kg 592 354 395 50.74 0.06

Weight, kg (d 21) 13.38 9.42 9.87 1.28 0.12

Phase 3 (d 22 to 35)

ADG, g 552.4 480.5 520.8 40.78 0.28

ADFI, g 1,044.0b 833.6a 855.9a 30.74 0.002

G:F, g/kg 529 576 608 39.33 0.67

Weight, kg (d 35) 21.11 16.15 17.16 1.54 0.11

Overall (d 0 to 35)

ADG, g 415.7b 284.3a 315.8a 14.84 0.003

ADFI, g 724.1b 569.0a 581.5a 23.62 0.003

G:F, g/kg 574 500 543 24.51 0.47

a,bWithin a row, means without a common superscript letter differ (P < 0.05).351




21

1US = United States diet; EU = European standard diet; and TL = European standard diet plus 352

tributyrin and lactitol at 3,000 ppm (Vetagro, Reggio Emilia, Italy).353

354




22

Table 4. Morphometry analysis (thickness, µm) of the jejunal intestinal mucosa and muscolaris at d 355

35 of piglets fed either a medicated US diet or a non-medicated European diet356

Treatment1

Item US EU TL SEM P-value

Experimental unit/treatment 5 5 5

Proximal

Mucosa2 624 601 627 27.957 0.562

Muscolaris 97a 140c 117b 63.529 < 0.001

Middle

Mucosa 585a 586a 654b 150.466 < 0.001

Muscolaris 119a 135b 156b 58.686 0.003

Distal

Mucosa 496a 596b 626c 233.452 < 0.001

Muscolaris 172a 195b 169a 50.398 < 0.001

a-cWithin a row, means without a common superscript letter differ (P < 0.05).357

1US = United States diet; EU = European standard diet; and TL = European standard diet 358

supplemented with tributyrin and lactitol at 3,000 ppm (Vetagro, Reggio Emilia, Italy).359

360




23

Table 5. Amines concentrations (µmol/g) along the intestinal tract of piglets fed either a medicated 361

US diet or a non-medicated European diet at d 35362

Treatment1

Variable US EU TL SEM P-value


Jejunum

Tyramine 0.003 0.014 0.016 0.009 0.397

Cadaverine 0.004a 0.027a 0.275b 0.025 < 0.001

Histamine 0 0 0.024 0.009 0.128

Putrescine 0.015a 0.035ab 0.064b 0.011 0.011

Spermidine 0.094 0.035 0.137 0.031 0.324

Spermine 0.072 0.071 0.107 0.019 0.605

Ileum

Tyramine 0.009 0.009 0.004 0.005 0.715

Cadaverine 0.007a 0.045a 0.229b 0.035 < 0.001

Histamine 0 0.008 0.02 0.014 0.538

Putrescine 0.023a 0.039a 0.080b 0.013 0.014

Spermidine 0.138 0.12 0.169 0.026 0.703

Spermine 0.109 0.143 0.133 0.026 0.808

Cecum

Tyramine 0.024 0.054 0.036 0.013 0.13

Cadaverine 0.097a 0.128a 0.426b 0.118 0.019




24

Histamine 0.000a 0.030ab 0.062b 0.011 0.006

Putrescine 0.059a 0.229ab 0.372b 0.059 0.013

Spermidine 0.273 0.252 0.27 0.04 0.812

Spermine 0.026a 0.051ab 0.078b 0.007 0.02

Colon

Tyramine 0.046 0.053 0.053 0.015 0.945

Cadaverine 0.345a 0.567a 1.457b 0.324 0.02

Histamine 0.000a 0.042ab 0.106b 0.021 0.015

Putrescine 0.229a 0.446ab 0.662b 0.06 0.056

Spermidine 0.466 0.482 0.556 0.06 0.54

Spermine 0.016a 0.026ab 0.067b 0.008 0.045

a,bWithin a row, means without a common superscript letter differ (P < 0.05).363






25

Table 6. Ammonia concentrations (mg/L) along the intestinal tracts of piglets fed either a 366

medicated US diet or a non-medicated European diet at d 14 and 35367

Treatment1

Variable US2 EU TL SEM P-value


d 14

Ileum 220.93 151.50 231.03 55.35 0.66

Cecum 192.33 200.52 268.44 39.59 0.36

Colon 155.25 133.20 300.89 21.57 0.69

d 35

Ileum 202.13 64.90 216.16 41.60 0.94

Cecum 202.87 75.25 167.42 23.70 0.34

Colon 223.47 104.63 201.35 39.72 0.68






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Table 7. Cecal volatile fatty acids concentrations along the intestinal tracts of piglets fed either a medicated US diet or a non-medicated European 370

diet at d 14 and 35371

Treatment1 Acetic acid Propionic acid Butyric acid Valeric acid Total2

µmol/L % µmol/L % µmol/L % µmol/L % µmol/L %

d 14

US 38.79 66.33 11.64 19.12 7.51 13.65 0.42 0.60 58.36 100

EU 36.38 57.33 20.18 29.20 7.46 11.42 1.32 2.05 65.34 100

TL 30.62 56.82 16.49 30.56 5.95 11.10 0.92 1.52 53.99 100

Pooled SEM 5.33 5.21 4.59 4.23 1.33 2..33 0.60 0.96 9.70

P-value 0.62 0.28 0.20 0.11 0.056 0.65 0.38 0.46 0.67

d 35

US 47.60 64.88b 18.64 25.79a 6.19 8.54 0.53a 0.80 72.97 100

EU 43.09 52.94a 28.14 34.55b 7.81 9.33 2.62b 3.18 81.66 100

TL 38.65 49.25a 29.36 37.08b 7.76 9.90 2.98b 3.77 78.75 100

SEM 3.36 1.34 2.33 1.74 1.15 1.17 0.38 0.44 5.59

P-value 0.25 < 0.001 0.07 0.002 0.53 0.178 0.01 0.001 0.64

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a,bWithin a column, means without a common superscript letter differ (P < 0.05).373

1US = United States diet; EU = European standard diet; and TL = European standard diet plus tributyrin and lactitol at 3,000 ppm (Vetagro, 374

Reggio Emilia, Italy).375

2Total short chains fatty acids not including lactic acid.376



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Table 8. Colonic volatile fatty acids concentrations along the intestinal tracts of piglets fed either a medicated US diet or a non-medicated European 377

diet at d 14 and 35378

a,bWithin a column, means without a common superscript letter differ (P < 0.05).379

Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Valeric acid Total2

Treatment1

µmol/L % µmol/L % µmol/L % µmol/L % µmol/L % µmol/L % µmol/L %

d 14

US 22.00a 62.46 9.49 24.43 0.33 0.65 3.42 9.96 0.74 1.70 0.31 0.80 36.26a 100

EU 28.94ab 62.69 12.40 27.29 0.43 0.88 3.87 8.19 0.00 0.00 0.49 0.94 46.12ab 100

TL 36.34b 57.43 20.71 31.22 0.00 0.00 6.38 10.13 0.00 0.00 0.82 1.22 64.24b 100

SEM 2.90 4.97 3.29 4.57 0.32 0.55 0.70 1.51 0.27 0.69 0.39 0.79 5.66

P-value 0.02 0.68 0.14 0.55 0.78 0.56 0.07 0.51 0.31 0.19 0.48 0.92 0.02

d 35

US 38.55 61.85 15.56 25.50 0.72 1.29 5.63 9.14 0.34 0.66 0.85a 1.56a 61.66 100

EU 44.16 56.57 26.03 32.61 0.00 0.00 6.33 8.20 0.00 0.00 2.11ab 2.63ab 78.63 100

TL 41.40 55.29 25.38 32.44 0.23 0.35 5.93 7.92 0.00 0.00 3.06b 4.00b 76.01 100

SEM 4.33 2.51 3.75 2.61 0.26 0.39 1.06 1.20 0.13 0.28 0.48 0.60 7.81

P-value 0.45 0.14 0.07 0.10 0.25 0.11 0.31 0.71 0.23 0.15 0.004 0.03 0.14



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1US = United States diet; EU = European standard diet; and TL = European standard diet plus tributyrin and lactitol at 3,000 ppm (Vetagro, 380

Reggio Emilia, Italy).381

2Total short chains fatty acids not including lactic acid.382



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Figure 1a. 383

384

385

386

Proximal jejunum Middle jejunum Distal jejunum

100

200

300

400

500 US EU TLc

ab b

a

b

ab

c

Vil

li le

nght

, µm




31

Figure 1b.387

388

389

Proximal jejunum Middle jejunum Distal jejunum

100

200

300

400

500US EU TL

a

c ba

ab b

Cry

pt d

epth

, µm




32

CAPTIONS390

Figure 1: Morphometry analysis (µm) of the small intestinal mucosa villi length (a) and crypts 391

depth (b) in growing pigs fed US, EU or TL diet. Data are shown as means ± SEM. US = United 392

States diet; EU = European standard diet; and TL = European standard diet plus tributyrin and 393

lactitol at 3,000 ppm (Vetagro, Reggio Emilia, Italy). a,bWithin an intestinal segment, means 394

without a common superscript letter differ (P < 0.05), and a-cwithin an intestinal segment, means 395

without a common superscript letter differ (P < 0.05).396




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