Published December 5, 2014
Intestinal metabolism of weaned piglets fed a typical United States
or European diet with or without supplementation
of tributyrin and lactitol
A. Piva,*1,2 E. Grilli,* L. Fabbri,* V. Pizzamiglio,* P. P. Gatta,* F. Galvano,† M. Bognanno,‡
L. Fiorentini,§ J. Woliński,# R. Zabielski,║ and J. A. Patterson¶
*DIMORFIPA, University of Bologna, 40064, Ozzano Emilia, Bologna, Italy; †Department of Biological
Chemistry, Medical Chemistry and Molecular Biology, University of Catania, 95125, Catania, Italy;
‡Department of Agro-forestry, Environmental Science and Technology, Mediterranean University
of Reggio Calabria, 89061, Reggio Calabria, Italy; §ISAN, Università Cattolica S. Cuore, 29100, Piacenza, Italy;
#The Kielanowski Institute of Animal Physiology and Nutrition, 05110, Jablonna, Poland,
║Department of Physiological Sciences, Warsaw Agricultural University, 02787, Warsaw, Poland;
and ¶Department of Animal Sciences, Purdue University, West Lafayette, IN 47907
ABSTRACT: The aim of the study was to investigate
the effects of supplementation of a microencapsulated
blend of tributyrin and lactitol (TL) to a standard European (EU) diet without antibiotic growth promoters
on intestinal metabolism and mucosa development of
weaned piglets and to compare it with a standard US
diet containing animal proteins, zinc oxide, copper sulfate, and carbadox. Ninety piglets weaned at 21 d were
divided into 3 dietary groups consisting of 5 replicates
each: 1) US diet supplemented with 55 mg/kg of carbadox, and 2.5% each of plasma proteins and spray-dried
blood cells in the first phase, 3,055 mg/kg of Zn in the
first and second phases, and 180 mg/kg of Cu in the
third phase; 2) EU diet based on vegetable proteins and
no antibiotics; and 3) the same EU diet supplemented
with 3,000 mg/kg of microencapsulated TL. The study
was divided into 3 phases: 0 to 7, 8 to 21, and 22 to
35 d. On d 7, 21, and 35, animals were weighed, and
feed consumption and efficiency were determined. On
d 14 and 35, one pig per pen was killed, and the intestinal contents and mucosa from the proximal, middle,
distal jejunum and the ileum were sampled. Intestinal
wall sections were fixed for histological analysis, and
intestinal content was used for VFA, ammonia, and
polyamine analysis. Throughout the study (d 0 to 35),
the US diet had greater ADG and ADFI than the EU
diet (P < 0.05). The EU diet supplemented with TL
tended to have 11% greater ADG (P = 0.17). Feeding
the EU diet caused a reduction in proximal and middle
jejunum villi length by 10% (P < 0.05) and an increase
in crypt size in proximal jejunum (P < 0.05) compared
with the US diet, probably due to an increased rate of
cell loss and crypt cell production. The TL supplementation resulted in longer villi along the jejunum and
less deep crypts in the proximal jejunum (+15.9 and
–8.9%, respectively; P < 0.05) than the unsupplemented EU diet. The TL diet increased the concentrations
of cadaverine and putrescine in the small intestine (P
< 0.05) and seemed to increase cadaverine, histamine,
putrescine, and spermine in the large intestine by 1.5to 10-fold compared with the US or EU diet. In conclusion, although the US diet had a greater effect on
growth performance and mucosal trophic status than
the EU diets, the supplementation with slowly released
TL seemed to be an effective tool to partially overcome
the adverse effects of vegetable protein diets.
Key words: lactitol, metabolism, piglet, tributyrin, weaning
©2008 American Society of Animal Science. All rights reserved.
INTRODUCTION
The ban of antibiotics as a growth promoter from animal feed in the European Union (EU) has motivated
1
Corresponding author: [email protected]
Patent No.: U.S. 6,217,915 (Luchansky and Piva, 2001).
Received July 4, 2007.
Accepted May 7, 2008.
2
J. Anim. Sci. 2008. 86:2952–2961
doi:10.2527/jas.2007-0402
research on alternative ways to optimize the digestive
process and increase nutrient availability. Diet formulation may include feed supplements, such as probiotic
cultures, organic acids (Partanen and Mroz, 1999), botanicals, and nondigestible oligosaccharides.
Volatile fatty acids play a role in modulating the
digestive process and can be supplied by direct feed
supplementation or properly promoting the intestinal
2952
Intestinal metabolism of weanling piglets
microbial production. Butyric acid is produced by bacterial fermentation of carbohydrates, and it serves as a
primary source of energy for colonocytes and a strong
mitosis promoter and a differentiation agent in the
gastrointestinal tract in vivo (Salminen et al., 1998).
In a previous work, it has been shown that the synergistic effect of a fermentable substrate, lactitol, and a
precursor of butyric acid, tributyrin, can improve the
trophic status of the intestinal mucosa in the gut of
nursery piglets and control intestinal histamine (Piva
et al., 2002).
The purpose of the present study was to investigate
the effects of supplementation of a microencapsulated
blend of tributyrin and lactitol (TL) to a standard EU
diet without antibiotic growth promoters on intestinal
metabolism and mucosa development of weaned piglets and to compare it with a standard US diet containing animal proteins, zinc oxide, copper sulfate, and
carbadox.
MATERIALS AND METHODS
The present study was conducted at Purdue University facilities with approval of the Purdue University
Animal Care and Use Committee.
Animals, Diets, and Facilities
At 21 d after birth, the piglets (Landrace × Large
White; 6.14 ± 1.38 kg of BW) were randomly allocated
to 3 experimental dietary groups (5 pens/diet with 6
piglets/pen) according to their initial BW, sex (females
and castrated males), and litter and were fed ad libitum
the following experimental diets: 1) typical US diet containing animal proteins in the first phase (2.5% plasma
proteins and 2.5% spray-dried blood cells), 3,055 mg/kg
of zinc (3,800 mg/kg of ZnO) in the first and the second
phase, 180 mg/kg of copper (800 mg/kg of CuSO4) in
the third phase, and 55 mg/kg of carbadox during the
whole experimental period (Table 1); 2) typical EU diet
based on vegetable protein without growth-promoters
(Table 2); and 3) the same EU diet supplemented with
3,000 mg/kg of microencapsulated blend of TL (Luchansky and Piva, 2001; Vetagro SpA, Reggio Emilia,
Italy). The feeding study was subdivided 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 beginning of the study, pigs
were individually weighed, and feed consumption and
efficiency were determined.
On d 14 and 35, 1 pig per pen was killed, and within
15 min, intestinal contents and mucosa were sampled
for morphometric, VFA, ammonia, and polyamine
analyses. Intestinal contents collected from the same
sections were divided in aliquots and frozen at −20°C.
Chemical Analyses of Feed
and Intestinal Contents
The DM, CP, ether extract, crude fiber, ash, and
starch contents of the feed were determined accord-
2953
ing to the AOAC (2000) methods. Volatile fatty acids
were determined using a gas chromatograph (Hewlett
Packard 3790) and a packed column (SP-1200; Hewlett Packard, Palo Alto, CA) according to the method
described by Playne (1985). Before injection, samples
were diluted with sterile deionized water (1:1; vol/vol),
and then 0.8 mL of samples was mixed with 0.2 mL of
25% o-phosphoric acid (Fisher Chemical, Fair Lawn,
NJ), and after 30 min, centrifuged at 13,000 × g for
12 min at 4°C. The supernatants were frozen at −20°C
overnight, thawed, centrifuged again, filtered, and
then injected into the gas chromatograph for determination of propionic, acetic, butyric, valeric, isobutyric,
and isovaleric acid concentrations. Ammonia concentration was determined (Chaney and Marbach, 1962)
after centrifuging intestinal liquor samples.
Polyamine Determination
Mono-, di-, and polyamines were separated and
quantified by HPLC with fluorimetric detection as described in a previous work (Piva et al., 2002). The limits of detection for the various amines were putrescine
0.023 pM, cadaverine 0.022 pM, histamine 0.48 pM,
tyramine 0.068 pM, spermidine 0.012 pM, and spermine 0.007 pM.
Morphometry Analysis
The small intestine from jejunum to the ileal cecal
valve was subdivided into 4 segments as proximal,
middle, distal jejunum, and ileum. The segment from
the pylorus to the ligament of Treitz was considered
the duodenum; the proximal segment of the rest of the
small intestine was considered the jejunum; and a distal segment 10 cm proximal to the ileocecal junction
was considered the ileum (Tang et al., 1999). Intestinal wall sections were sampled from jejunum (proximal, middle, and distal) and ileum, fixed in Bouin’s
fluid (Ricca Chemical Company, Arlington, TX), and
preserved in 70% ethanol for histological analysis. Pig
intestinal mucosa was sampled from proximal, middle,
and distal jejunum (25, 50, and 75% of the total length,
respectively) and ileum. Whole thickness segments of
the gut were collected, fixed in Bouin’s solution (Sigma,
St. Louis, MO), and stored in alcohol for morphometry
analysis. The fixed samples were dehydrated in 96 and
99.8% ethanol and embedded into the xylene and paraffin (ParaPlast Regular, P-3558, Sigma). Serial histological sections of 5-μm thickness were cut (Microm
HM 355S; Microm GmbH, Walldorf, Germany) and
stained with hematoxylin and eosin for morphometry
analysis. After staining, the depth of crypts, length of
villi, and thickness of tunica mucosa and muscolaris
mucosae were measured in the small intestine preparations. In each slide, about 20 well-oriented villi and
crypts located outside the area with Peyer’s patches
were measured at small magnification with an optical
binocular microscope coupled to the personal computer
2954
Piva et al.
Table 1. Composition of US diet for phases 1 to 3
Item
Ingredient, % (as-fed basis)
Corn
Soybean meal, 48% CP
Dicalcium phosphate (CaHPO4)
Limestone
Salt
Animal fat
Soybean oil
l-Lysine⋅HCl
dl-Methionine
Swine vitamin premix1
Swine trace mineral premix2
Dried whey
Fish meal
Plasma protein
Spray-dried blood cells
Zinc oxide (ZnO)
Copper sulfate (CuSO4)
Carbadox
Banmith dewormer3
Phytase, 600 PU4/g
Chemical composition, DM basis (other than DM)
CP, %
Ether extract, %
Crude fiber, %
Ash, %
Starch, %
GE, MJ/kg
Phase 1,
d 0 to 7
Phase 2,
d 8 to 21
Phase 3,
d 22 to 35
39.25
20.00
1.10
0.60
0.30
5.00
—
0.15
0.15
0.15
0.17
25.00
2.50
2.50
2.50
0.38
—
0.25
—
—
53.12
27.15
0.74
0.39
0.25
—
3.00
0.15
0.05
0.25
0.17
10.00
4.00
—
—
0.38
—
0.25
—
0.10
68.56
26.98
1.29
0.72
0.35
1.00
—
0.15
—
0.25
0.17
—
—
—
—
—
0.08
0.25
0.10
0.10
22.69
8.23
1.99
6.20
34.74
18.28
23.56
5.26
2.55
6.00
38.16
18.05
19.88
3.76
2.95
5.36
48.42
17.42
1
Provided per kilogram of complete diet: 11,026 IU of vitamin A; 1,654 IU of vitamin D3; 44 IU of vitamin
E; 4.4 mg of vitamin K (menadione sodium bisulfite); 55.1 mg of niacin, 33.1 mg of pantothenic acid (as calcium pantothenate); 9.9 mg of riboflavin; and 0.044 mg of B12.
2
Provided per kilogram of complete diet: 39.7 mg of Mn (oxide), 165.4 mg of Fe (sulfate), 165 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 selenite).
3
Anthelmintic (Pfizer Inc., New York, NY).
4
PU = phytase unit; Natuphos (BASF Corporation, Parsippany, NJ).
with a planimetry software (LSM 5 PASCAL 3.2 SP2;
Carl Zeiss, Göttingen, Germany). The villus length was
considered as the distance from the crypt opening to
the tip of the villus, whereas the depth of crypt was
measured from the base of the crypt to the level of the
crypt opening (Kotunia et al., 2004).
significance. An unpaired Student t-test was performed
whenever comparisons between treatments were necessary to discuss results.
Statistical Analysis
During the phase 1 of the study (d 0 to 7), no differences in growth performance were observed (Table 3).
During phase 2 (d 8 to 21), animals fed the US diet had
greater (P < 0.05) ADG and ADFI than animals fed the
EU or TL diet. The statistically significant advantage
in ADG obtained by the US diet at the end of phase 2
was no longer present by the end of phase 3. However,
during the entire study (d 0 to 35), piglets fed the US
diet supplemented with carbadox, ZnO, and CuSO4 had
greater (P < 0.05) ADG and ADFI than those fed the
EU diet. The EU diet supplemented with TL resulted
in intermediate ADG between the US and EU diets.
All data were analyzed using the GraphPad Prism
program (GraphPad Prism v. 4.0, GraphPad Software,
San Diego, CA). The pen was the experimental unit for
growth performance data, whereas the animal was the
experimental unit for chemical and morphological intestinal data. Animal performance was analyzed with
repeated measures 1-way ANOVA with Bartlett’s test
for equal variances and the Newman-Keuls post test,
whereas ammonia, VFA, and amine concentration data
were subjected to simple 1-way ANOVA with Bart­
lett’s test for equal variances and the Newman-Keuls
post test. For morphometric data, the nonparametric
Kruskal-Wallis test and Dunn’s multiple comparison
test were used to compare the effect of diets. In all statistical analyses, P < 0.05 was considered as the level of
RESULTS
Animal Performance
Morphometry Analysis
Mucosa thickness did not differ among treatments
in the proximal jejunum, whereas in the middle and
2955
Intestinal metabolism of weanling piglets
Table 2. Composition of European diets for phases 1 to 3
Phase 1, d 0 to 7
Item
Ingredient, % (as-fed basis)
Whey
Steam-rolled barley
Steam-rolled corn
Corn
Dextrose
Extruded soybeans
Soy protein concentrate
Soybean meal, 48% CP
Skim milk
Potato protein1
Wheat bran
Soy oil
Swine trace mineral premix2
Swine vitamin premix3
Monocalcium phosphate (CaH2PO4)
l-Lysine⋅HCl
Calcium sulfate (CaSO4)
dl-Methionine
Salt
l-Threonine
l-Tryptophan
Herbiotic HB4
Trilac5
Chemical composition, DM basis (other than DM)
CP, %
Ether extract, %
Crude fiber, %
Ash, %
Starch, %
GE, MJ/kg
Phase 2, d 8 to 21
Phase 3, d 22 to 35
EU
TL
EU
TL
EU
TL
20.13
25.16
10.07
9.74
7.05
5.03
5.03
3.32
3.02
3.02
2.83
2.52
0.13
0.25
0.85
0.54
0.30
0.29
0.24
0.22
0.07
0.20
—
20.13
25.16
10.07
9.14
7.05
5.03
5.03
3.32
3.02
3.02
2.83
2.52
0.13
0.25
0.85
0.54
0.30
0.29
0.24
0.22
0.07
0.20
0.30
9.99
29.96
4.99
28.05
3.00
3.00
3.00
7.57
2.00
2.50
0.14
2.50
0.13
0.25
1.41
0.54
0.30
0.23
—
0.21
0.06
0.20
—
9.99
29.96
4.99
27.65
3.00
3.00
3.00
7.58
2.00
2.50
0.14
2.50
0.13
0.25
1.42
0.54
0.30
0.23
—
0.21
0.06
0.20
0.30
7.03
35.15
—
31.99
—
5.02
2.01
9.36
—
2.01
—
3.42
0.13
0.25
1.90
0.45
0.30
0.19
0.37
0.18
0.04
0.20
—
7.03
35.15
—
31.38
—
5.02
2.01
9.36
—
2.01
—
3.42
0.13
0.25
1.90
0.45
0.30
0.19
0.37
0.18
0.04
0.20
0.30
19.31
5.94
2.95
5.09
30.24
17.88
19.13
5.88
3.08
4.83
33.84
17.96
18.69
5.83
2.75
4.33
40.32
18.05
19.19
6.03
3.08
4.51
44.46
18.07
19.63
6.55
2.67
4.82
42.12
18.23
19.25
7.27
3.45
4.98
43.20
18.15
1
Protastar (AVEBE Feed, Veendam, the Netherlands).
Provided per kilogram of complete diet: 11,026 IU of vitamin A; 1,654 IU of vitamin D3; 44 IU of vitamin E; 4.4 mg of vitamin K (menadione
sodium bisulfite); 55.1 mg of niacin, 33.1 mg of pantothenic acid (as calcium pantothenate); 9.9 mg of riboflavin; and 0.044 mg of B12.
3
Provided per kilogram of complete diet: 39.7 mg of Mn (oxide), 165.4 mg of Fe (sulfate), 165 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 selenite).
4
Flavorant (Agri-Life Sciences HB, Souderton, PA).
5
Supplying Trilac at 3,000 mg/kg (Vetagro SpA, Reggio Emilia, Italy).
2
distal jejunum, it increased by TL supplementation
(P < 0.05; Table 4). Villi length was the greatest (P <
0.05) for the US diet in the proximal jejunum, whereas in the distal jejunum, the EU diets had longer villi
than the US diet (P < 0.05; Figure 1a). Moreover, TL
supplementation produced longer villi throughout the
jejunum (P < 0.05; Figure 1a) and less deep crypts in
the proximal jejunum (P < 0.05; Figure 1b) than the
unsupplemented EU diet. Muscolaris mucosae thickness for the EU diets was greater than the US diet in
proximal and middle jejunum (P < 0.05), whereas the
US and TL diets had the same thickness in the distal
jejunum. Ileal samples were damaged during transport
and preparation, and an appropriate statistical analysis could not be conducted.
Amines and Ammonia in Jejunum, Ileum,
Cecum, and Colon
Gut tyramine and spermidine concentrations were
not affected by any dietary treatments (Table 5). Piglets
receiving the TL diet had greater concentrations of cadaverine in jejunum, ileum, cecum, and colon than piglets fed the US or the EU diet (P < 0.05). Similarly, the
TL diet resulted in a greater putrescine concentration
in ileum of piglets than those fed the US or EU diet. In
cecum, the TL diet resulted in greater concentrations of
cadaverine, histamine, putrescine, and spermine than
the US diet (P < 0.05). The same effect was observed
in the colon for cadaverine, histamine, putrescine, and
spermine (P < 0.05). There were no effects of diets on
gut ammonia concentrations (Table 6).
VFA in the Cecum and Colon
At d 14 of the study, the cecal contents showed no differences in VFA concentrations (Table 7). At the same
time, the TL diet had greater colonic acetic acid and
total VFA concentrations than the US diet (P < 0.05;
Table 8). No differences were observed in propionic,
isobutyric, butyric, isovaleric, and valeric acids.
2956
Piva et al.
Table 3. Growth performance of piglets fed a medicated US diet or a nonmedicated
European diet
Treatment1
Item
US
Pens/treatment
Initial BW, kg
Phase 1 (d 0 to 7)
ADG, g
ADFI, g
G:F, g/kg
BW, kg (d 7)
Phase 2 (d 8 to 21)
ADG, g
ADFI, g
G:F, g/kg
BW, kg (d 21)
Phase 3 (d 22 to 35)
ADG, g
ADFI, g
G:F, g/kg
BW, kg (d 35)
Overall (d 0 to 35)
ADG, g
ADFI, g
G:F, g/kg
EU
TL
SEM
P-value
5
6.56
5
6.21
5
6.15
0.86
0.94
98.6
161.2
612
7.25
68.2
152.6
447
6.69
88.1
164.0
537
6.77
26.36
20.14
343.42
0.91
0.82
0.92
0.66
0.90
437.9b
740.1b
592
13.38
196.8a
555.7a
354
9.42
221.0a
559.9a
395
9.87
26.42
32.65
50.74
1.28
0.001
0.006
0.06
0.12
552.4
1,044.0b
529
21.11
480.5
833.6a
576
16.15
520.8
855.9a
608
17.16
40.78
30.74
39.33
1.54
0.28
0.002
0.67
0.11
415.7b
724.1b
574
284.3a
569.0a
500
315.8a
581.5a
543
14.84
23.62
24.51
0.003
0.003
0.47
a,b
Within a row, means without a common superscript letter differ (P < 0.05).
US = United States diet; EU = European standard diet; and TL = European standard diet plus Trilac at
3,000 mg/kg (Vetagro SpA, Reggio Emilia, Italy).
1
At d 35, cecal valeric acid concentration was greater
for the EU-based diets compared with the US diet (P <
0.05), whereas colonic valeric acid concentration was
greater for piglets fed the TL diet than those fed the US
diet (P < 0.05). Cecal acetic and propionic acid proportions were the greatest and the least (P < 0.05), respectively, for the US diet.
DISCUSSION
Animals fed a typical US diet supplemented with
plasma proteins, carbadox, copper sulfate, and zinc oxide had the best growth performance from 8 to 21 d
postweaning. No appreciable difference was observed
thereafter. However, during the entire 35-d study, piglets fed with a typical EU diet with soybean protein
supplemented with microencapsulated tributyrin and
lactitol tended to have improved growth performance
than piglets fed with the same diet without supplementation. In fact, final BW were 4.8 and 6.2% greater for
piglets fed the TL diet than the EU diet after 21 and 35
d, respectively, and overall ADG, ADFI, and G:F tended to be 11.1, 2.2, and 8.0% greater, respectively.
Feeding the US diet resulted in differences in small
intestine histology compared with the EU diet. Feeding
the EU diet caused a reduction in villi length by about
20% and an increase in crypt depth. This implied that
the gut absorptive surface was reduced in piglets fed
with the standard EU diet in comparison with the US
diet, and this might be due to reduction in mitosis or
in the increase of apoptosis ratio, or both (Godlewski
et al., 2005). However, in the distal jejunum of piglets
fed the EU diet, an increased mucosa thickness and
longer villi, in comparison with piglets fed the US diet,
were observed. This might have compensated for the
reduction of the absorptive surface (short villi) in the
proximal jejunum. The muscular wall in the proximal,
middle, and distal jejunum was thicker in piglets fed
the EU diet than those fed the US diet. Supplementation of the EU diet with TL improved the mucosa
thickness and the villi length in the middle and distal
jejunum, thereby increasing the absorptive area of the
gut, whereas muscularis thickness was reduced in the
proximal and distal jejunum. The effect on the crypts
was observed only in the proximal jejunum where the
depth had been reduced by TL supplementation in neonatal piglets, and sodium butyrate reduced the crypt
depth in the jejunum (Kotunia et al., 2004).
Along the length of the jejunum, the downstream
reduction of villous length observed in piglets fed the
US diet could be associated with a decreased enteral
nutrient availability (Pluske, 2001). Such downstream
reduction of villi length did not occur in piglets fed the
EU diet that had shorter villi at proximal and middle
jejunum than those fed the US diet, whereas in distal
jejunum, piglets fed the EU diet had 15.2% longer villi
than those fed the US diet, which is likely due to greater nutrient availability at the distal jejunum.
The villous shortening observed in piglets fed the EU
diet compared with those fed the US diet is often associated with an increased rate of cell loss, increased cryptcell production, and increased crypt depth (Pluske,
2957
Intestinal metabolism of weanling piglets
Table 4. Morphometry analysis (thickness, μm) of the jejunal intestinal mucosa and
muscolaris at d 35 of piglets fed a medicated US diet or a nonmedicated European
diet
Treatment1
Item
Experimental unit/treatment
Proximal
Mucosa
Muscolaris
Middle
Mucosa
Muscolaris
Distal
Mucosa
Muscolaris
US
EU
5
5
TL
SEM
P-value
5
624
97a
601
140c
627
117b
27.957
63.529
0.562
<0.001
585a
119a
586a
135b
654b
156b
150.466
58.686
<0.001
0.003
496a
172a
596b
195b
626c
169a
233.452
50.398
<0.001
<0.001
a–c
Within a row, means without a common superscript letter differ (P < 0.05).
US = United States diet; EU = European standard diet; and TL = European standard diet supplemented
with Trilac at 3,000 mg/kg (Vetagro SpA, Reggio Emilia, Italy).
1
Figure 1. Morphometry analysis (μm) of the small intestinal mucosa villi length (a) and crypt depth (b) in growing pigs fed the US, EU, or TL diet. Data are shown as means ± SEM. US = United States diet; EU = European
standard diet; and TL = European standard diet plus Trilac at 3,000 mg/kg (Vetagro SpA, Reggio Emilia, Italy).
a–c
Within an intestinal segment, means without a common letter differ (P < 0.05).
2958
Piva et al.
Table 5. Amines concentrations (μmol/g) along the intestinal tract of piglets fed a
medicated US diet or a nonmedicated European diet at d 35
Treatment1
Variable
Experimental unit/treatment
Jejunum
Tyramine
Cadaverine
Histamine
Putrescine
Spermidine
Spermine
Ileum
Tyramine
Cadaverine
Histamine
Putrescine
Spermidine
Spermine
Cecum
Tyramine
Cadaverine
Histamine
Putrescine
Spermidine
Spermine
Colon
Tyramine
Cadaverine
Histamine
Putrescine
Spermidine
Spermine
US
EU
TL
SEM
P-value
5
5
5
0.003
0.004a
0
0.015a
0.094
0.072
0.014
0.027a
0
0.035ab
0.035
0.071
0.016
0.275b
0.024
0.064b
0.137
0.107
0.009
0.025
0.009
0.011
0.031
0.019
0.397
<0.001
0.128
0.011
0.324
0.605
0.009
0.007a
0
0.023a
0.138
0.109
0.009
0.045a
0.008
0.039a
0.120
0.143
0.004
0.229b
0.020
0.080b
0.169
0.133
0.005
0.035
0.014
0.013
0.026
0.026
0.715
<0.001
0.538
0.014
0.703
0.808
0.024
0.097a
0a
0.059a
0.273
0.026a
0.054
0.128a
0.030ab
0.229ab
0.252
0.051ab
0.036
0.426b
0.062b
0.372b
0.27
0.078b
0.013
0.118
0.011
0.059
0.040
0.007
0.130
0.019
0.006
0.013
0.812
0.020
0.046
0.345a
0.000a
0.229a
0.466
0.016a
0.053
0.567a
0.042ab
0.446ab
0.482
0.026ab
0.053
1.457b
0.106b
0.662b
0.556
0.067b
0.015
0.324
0.021
0.06
0.06
0.008
0.945
0.020
0.015
0.056
0.540
0.045
a,b
Within a row, means without a common superscript letter differ (P < 0.05).
US = United States diet; EU = European standard diet; and TL = European standard diet plus Trilac at
3,000 mg/kg (Vetagro SpA, Reggio Emilia, Italy).
1
2001). Such increased crypt-depth was, in fact, found
in the proximal jejunum of piglets fed the EU diet compared with those fed the US diet. The reduction in crypt
depth in the US diet, as compared with the EU diet, is
presumably the result of antisecretory action of ZnO in
the US diet. Tributyrin and lactitol supplementation
increased villous length over piglets fed the EU diet
in the proximal, middle, and distal jejunum by 14.2,
22.5, and 11%, respectively, and villus/crypt ratios for
piglets fed 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
jejunum; and 1.5, 1.5, and 1.6 for distal jejunum, respectively. This is certainly the result of stimulation of
mitosis as well as reduction of programmed cell death
Table 6. Ammonia concentrations (mg/L) along the intestinal tracts of piglets fed a
medicated US diet or a nonmedicated European diet at d 14 and 35
Treatment1
Variable
Experimental unit/treatment
d 14
Ileum
Cecum
Colon
d 35
Ileum
Cecum
Colon
US
EU
TL
SEM
P-value
5
5
5
220.93
192.33
155.25
151.50
200.52
133.20
231.03
268.44
300.89
55.35
39.59
21.57
0.66
0.36
0.69
202.13
202.87
223.47
64.90
75.25
104.63
216.16
167.42
201.35
41.60
23.70
39.72
0.94
0.34
0.68
1
US = United States diet; EU = European standard diet; and TL = European standard diet plus Trilac at
3,000 mg/kg (Vetagro SpA, Reggio Emilia, Italy).
2959
Intestinal metabolism of weanling piglets
Table 7. Cecal VFA concentrations along the intestinal tracts of piglets fed a medicated US diet or a nonmedicated
European diet at d 14 and 35
Acetic acid
Treatment1
d 14
US
EU
TL
Pooled SEM
P-value
d 35
US
EU
TL
SEM
P-value
Propionic acid
Butyric acid
Valeric acid
Total2
μmol/L
%
μmol/L
%
μmol/L
%
μmol/L
%
μmol/L
%
38.79
36.38
30.62
5.33
0.62
66.33
57.33
56.82
5.21
0.28
11.64
20.18
16.49
4.59
0.20
19.12
29.20
30.56
4.23
0.11
7.51
7.46
5.95
1.33
0.056
13.65
11.42
11.10
2.33
0.65
0.42
1.32
0.92
0.60
0.38
0.60
2.05
1.52
0.96
0.46
58.36
65.34
53.99
9.70
0.67
100
100
100
47.60
43.09
38.65
3.36
0.25
64.88b
52.94a
49.25a
1.34
<0.001
18.64
28.14
29.36
2.33
0.07
25.79a
34.55b
37.08b
1.74
0.002
6.19
7.81
7.76
1.15
0.53
0.53a
2.62b
2.98b
0.38
0.01
0.80
3.18
3.77
0.44
0.001
72.97
81.66
78.75
5.59
0.64
100
100
100
8.54
9.33
9.90
1.17
0.178
a,b
Within a column, means without a common superscript letter differ (P < 0.05).
US = United States diet; EU = European standard diet; and TL = European standard diet plus Trilac at 3,000 mg/kg (Vetagro SpA, Reggio
Emilia, Italy).
2
Total short-chain fatty acids not including lactic acid.
1
type I (apoptosis) via butyrate (Kotunia et al., 2004).
Moreover, Kotunia et al. (2004) demonstrated a 4-fold
increase in plasma cholecystokinin in piglets fed the
butyrate-supplemented diet. This regulatory peptide is
known for its secretion, as well as growth-promoting
effects, in the gastrointestinal tract of young mammals
(Biernat et al., 1999). Similarly, increased mucosa and
muscularis mucosae thickness in piglets fed the EU
and TL diets, compared with those fed the US diet, was
observed along the jejunum.
Overall, morphometric measurements matched
growth performance data, which showed the best
growth performance with the US diet, with the intestinal mucosa architecture supporting that the diet
would be highly digestible in the proximal and middle
jejunum. Conversely, the longer villi in distal jejunum
implied that the EU diet was slowly or less digestible
than the US diet. Tributyrin and lactitol treatment
increased villous length in jejunum and tended to improve growth performance.
Tyramine and spermidine concentrations did not
show any differences associated with diets in any of the
gastrointestinal tract section analyzed. Tributyrin and
lactitol increased the concentrations of cadaverine and
putrescine in the small intestine and cadaverine, histamine, putrescine, and spermine in the large intestine
by 1.5- to 10-fold. The TL diet increased putrescine and
its metabolite spermine concentrations by approximatively 1.5-fold compared with the EU diet in the large
intestine, whereas in the small intestine, a 2-fold increase was observed only for putrescine. Putrescine
can act as growth factor for the gut (Seidel et al., 1985),
whereas its metabolite spermine can induce maturation of the small intestine primarily in suckling but not
in weaned animals (Peulen et al., 2004; Powroznik et
al., 2004).
Such increase in putrescine concentrations in the
small intestine can be associated with the longer vil-
li measured in piglets fed the TL diet. However, the
increased availability of putrescine might have been
linked primarily to an increased large intestine microbial production due to lactitol contained in TL as
described for other indigestible oligosaccharides or inuline (Noack et al., 1998; Deizenne et al., 2000; Bailey et
al., 2002) and to a subsequent transport to the small
intestine via the portal circulation and biliary duct
(Osborne and Seidel, 1990).
The enterohepatic circulation of polyamines can also
justify the increased cadaverine concentrations in the
small intestine by 5- to 10-fold observed in piglets fed
the TL diet compared with those fed the EU diet. Cadaverine and its metabolite piperidine are originated
from microbial decarboxylation of lysine and have been
shown to specifically inhibit Shigella flexneri-induced
transepithelial migration of polymorphonuclear leucocytes in case of dysentery in humans (McCormick et
al., 1999; Fernandez et al., 2001) and to prevent the
invasion of Salmonella Typhimurium into intestinal
epithelium (Kohler et al., 2002). In our study, we did
not measure piperidine; however, the increase of its
precursor cadaverine may be useful in downregulating
active inflammation at the mucosal surface (Kohler et
al., 2002).
The profound changes associated with the TL diet
were not observed in a previous study despite the
greater dose of tributyrin and lactitol used (Piva et al.,
2002). The explanation for such difference may be a
consequence that in the current study, tributyrin and
lactitol were microencapsulated in a lipid matrix that
has been shown to allow a slow release of material
along the gut (Piva et al., 2007) and that might have
prevented a substantial loss of lactitol in the small intestine, which has been shown to be fermented to lactic
acid (Piva et al., 2002).
Cecal and colonic ammonia concentrations were not
different among any of the tested diets. Conversely, the
100
100
100
1
a,b
Within a column, means without a common superscript letter differ (P < 0.05).
US = United States diet; EU = European standard diet; and TL = European standard diet plus Trilac at 3,000 mg/kg (Vetagro SpA, Reggio Emilia, Italy).
2
Total short-chain fatty acids not including lactic acid.
61.66
78.63
76.01
7.81
0.14
1.56a
2.63ab
4.00b
0.60
0.03
9.14
8.20
7.92
1.20
0.71
61.85
56.57
55.29
2.51
0.14
38.55
44.16
41.40
4.33
0.45
15.56
26.03
25.38
3.75
0.07
25.50
32.61
32.44
2.61
0.10
0.72
0.00
0.23
0.26
0.25
1.29
0.00
0.35
0.39
0.11
5.63
6.33
5.93
1.06
0.31
0.34
0.00
0.00
0.13
0.23
0.66
0.00
0.00
0.28
0.15
0.85a
2.11ab
3.06b
0.48
0.004
100
100
100
36.26a
46.12ab
64.24b
5.66
0.02
0.80
0.94
1.22
0.79
0.92
9.96
8.19
10.13
1.51
0.51
62.46
62.69
57.43
4.97
0.68
d 14
US
EU
TL
SEM
P-value
d 35
US
EU
TL
SEM
P-value
22.00a
28.94ab
36.34b
2.90
0.02
9.49
12.40
20.71
3.29
0.14
24.43
27.29
31.22
4.57
0.55
0.33
0.43
0.00
0.32
0.78
0.65
0.88
0.00
0.55
0.56
3.42
3.87
6.38
0.70
0.07
0.74
0.00
0.00
0.27
0.31
1.70
0.00
0.00
0.69
0.19
0.31
0.49
0.82
0.39
0.48
%
μmol/L
%
μmol/L
%
μmol/L
%
μmol/L
%
μmol/L
%
μmol/L
%
μmol/L
Treatment1
Propionic acid
Isobutyric acid
Butyric acid
Isovaleric acid
Valeric acid
Total2
Piva et al.
Acetic acid
Table 8. Colonic VFA concentrations along the intestinal tracts of piglets fed a medicated US diet or a nonmedicated European diet at d 14 and 35
2960
ratio of acetic to propionic acids was decreased in cecum of piglets fed the EU and TL diets compared with
those fed the US diet, indicating that a greater amount
of indigested starch could be present in piglets fed the
EU and TL diets in the second phase, but such difference was not evident at d 14 of the study. Overall, total
VFA concentrations were not different in the cecum after d 14 and 35 and in the colon d 35.
Piglets fed the TL diet did not show any difference in
VFA when compared with those fed the EU diet, with
the exception of a relevant increase in acetic acid in the
colon after the first phase diet, despite the fact that the
nonmicroencapsulated TL promoted propionic rather
than acetic acid production (Piva et al., 1996).
The US diet nutrition plan showed the best growth
performance until 21 d postweaning, which was associated with longer villi in the proximal and middle jejunum. The plain vegetable protein (EU diet) was less
effective and showed an intestinal architecture indicating that a diet would be digested more slowly. The TL
supplementation to the EU diet increased villous length
through an increase in putrescine and cadaverine that,
along with the promitotic and antiapoptotic effect of
butyric acid, have resulted in improved digestion and
in a tendency toward increased growth rate. In conclusion, the use of slowly released tributyrin and lactitol
seemed to be an effective tool to partially overcome the
lack of animal proteins, carbadox, zinc oxide, and copper sulfate in the EU vegetable diets for piglets.
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