Valeric acid glyceride esters in feed promote broiler performance and reduce

the incidence of necrotic enteritis

Lonneke Onrust,∗,1 Karolien Van Driessche,∗ Richard Ducatelle,∗ Koen Schwarzer,†

Freddy Haesebrouck,∗ and Filip Van Immerseel∗
∗
Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University,
Salisburylaan 133, B-9820, Merelbeke, Belgium; and † Perstorp BV, Industrieweg 8, NL-5165NH, Waspik, The
Netherlands

ABSTRACT Valeric acid is a C5 fatty acid, naturally
produced in low concentrations by specific members of
the microbiota of the lower intestinal tract. Effects of
valeric acid on intestinal health have been poorly in-
vestigated. Valeric acid derivatives can be produced as
glyceride esters and added to broiler feed. In the cur-
rent study, experiments were carried out to evaluate
the effect of valeric acid glycerides (GVA) on growth
performance, on the morphology of the small intesti-
nal mucosa and on protection against necrotic enteri-
tis. In a first feeding trial, Ross-308 chicks were ran-
domly divided into 2 dietary treatment groups and
fed either a non-supplemented diet or a diet supple-
mented with GVA (1.5 g/kg). In the GVA supple-
mented group, the feed conversion ratio was signifi-
cantly decreased during the entire trial period (D1–37).
In a second trial, gut wall morphology was evalu-
ated. In broilers fed a GVA-containing diet at 5 g/kg,
Key words: valeric acid, broiler, necrotic enteritis, intestinal health
the villus height/crypt depth ratio in the jejunum
was significantly increased (P ≤ 0.05), and the crypt
depth was significantly decreased at 28 d. In a third
trial, immunohistochemistry showed that the density
of glucagon-like peptide-2 immunoreactive cells in je-
junal and ileal villi from broilers supplemented with
GVA (5 g/kg) was significantly increased (P ≤ 0.05)
on d 10. In a necrotic enteritis challenge model, a sig-
nificant reduction of the number of birds with necrotic
lesions was found at d 21, using in-feed supplementa-
tion of low and high regimen of GVA. These data show
that GVA supplementation to broiler feed can decrease
the feed conversion, positively affect the morphology
of the small intestinal mucosa, increase the density
of glucagon-like peptide-2 producing enteroendocrine
cells, and reduce the incidence of necrotic enteritis,
making GVA a valuable candidate feed additive for
broilers.
2018 Poultry Science 97:2303–2311
http://dx.doi.org/10.3382/ps/pey085

INTRODUCTION in the intestinal tract (Teirlynck et al., 2011). In recent

Intestinal health is a major issue in broiler produc-
tion, especially since the ban on antibiotic growth
promoters in animal feed in the European Union
on January 1, 2006 (EC Regulation No. 1831/2003;
http://eurlex.europa.eu/LexUriServ/LexUriServ.do?
uri=OJ:L:2003:268:0029:0043:EN:PDF). Without the
growth promoters and with ever-increasing levels of
feed intake, broilers tend to develop an unfavorable
intestinal microbiota composition, commonly known as
dysbiosis or dysbacteriosis. In general terms, dysbiosis
can be defined as a loss of richness and evenness of the
microbiota composition, resulting in dominance of a
limited number of species, some of which can be strong
triggers of inflammation (Chan et al., 2013). Dysbiosis
in chickens indeed has been linked with inflammation

C 2018 Poultry Science Association Inc.
Received August 10, 2017.
1
Corresponding author: lonneke.onrust@ugent.be
years, major research efforts have focused on the
development of alternative feed additives to replace the
growth promoting antibiotics for maintaining eubiosis
in the intestinal tract of broilers (Huyghebaert et al.,
2011). The goal is to support the growth of beneficial
microbes and/or to provide the host with microbe- or
feed-derived signaling molecules that support intestinal
health. Tools available to reinforce the beneficial
microbes include probiotics and prebiotics leading to
the production of metabolites that are sensed by the
host as favorable (Kim et al., 2011; Rahimi et al.,
2011; Tellez et al., 2012; Han et al., 2013). Among
the microbe- and feed-derived signaling molecules,
a lot of attention has been paid to intestinal health
promoting effects of microbe-derived short-chain fatty
acids (SCFA) and plant-derived medium-chain fatty
acids (MCFA). The SCFA with documented beneficial
effects are propionic (C3) and butyric acid (C4),
which are added to the feed mostly in a fat coated
form or as glyceride esters, in order to promote a

2303
2304 ONRUST ET AL.

gradual release of the acids along the intestinal tract Table 1. Nutrient composition of the diet of the first
(Fernandez-Rubio et al., 2009; Namkung et al., 2011; feeding trial, evaluating the effects of valeric acid glyc-
eride esters on performance.
Khan and Iqbal, 2016). The MCFA include caproic
(C6), caprylic (C8), capric (C10), and lauric (C12) Nutrient (%) Starter, Grower, Finisher,
acid. Coconut oil and palm kernel oil constitute d 1–11 d 21–31 d 32–37
rich sources of MCFA. MCFA also have documented ME (kcal/kg) 2,800 2,925 3,000
beneficial effects on intestinal health (Dierick et al., Crude protein 21.0 19.5 18.0
2004; Bertevello et al., 2012; Zentek et al., 2012). Lysine 1.17 1.06 0.98
Methionine + Cysteine 0.85 0.80 0.74
Valeric acid is a C5 fatty acid which is sometimes Methionine 0.57 0.54 0.50
classified with the SCFA and other times with the Threonine 0.72 0.69 0.65
MCFA (Høverstad et al., 1984). It is naturally present Tryptophan 0.21 0.18 0.16
Calcium 0.80 0.65 0.50
in the lower intestinal tract, albeit at an approximately Phosphorus 0.38 0.33 0.30
5 times lower concentration than butyric acid. It is pro-
duced by certain members of the intestinal microbiota,
mostly those belonging to the Oscillibacter genus (Lino BV, Waspik, The Netherlands). The ester composition
et al., 2007). As valeric acid in the intestine is of mi- consisted of 45% monovalerin (26% VA), 24% divalerin
crobial origin, it may be more appropriate to classify it (18.8% VA), 3.2% trivalerin (2.9% VA) and glycerol.
with the SCFA, which are all of microbial origin. Unlike
the other SCFA, however, very little is known about
the effects of valeric acid on intestinal health and on Experimental Procedures
health in general. Nevertheless, there are indirect in- The procedures for temperature, light, and feed
dications from epidemiological observational studies in schedule were the same for each conducted experiment.
humans suggesting that valeric acid might beneficially Temperature for broilers was maintained during the tri-
support intestinal health (Mondot et al., 2011; Cai als according to the recommendations in the breeder’s
et al., 2016). Indeed, the valeric acid-producing Oscil- manual, and light schedule was set to 18 h light and 6 h
libacter valericigenes is found to be more abundant in darkness from d 7 onwards. Until d 6, 1 h darkness per
the stools of healthy people as compared to the stools d was applied. Birds were allowed ad libitum access to
of patients with Crohn’s disease (Mondot et al., 2011). feed and water.
Moreover, valeric acid is found together with propionic
acid and butyric acid in significantly higher concentra-
tions in the stools of healthy centenarians compared Effects of Valeric Acid Glyceride Esters on
to elderly people (Cai et al., 2016). Conversely, Oscil- Performance
libacter spp. are associated with diet-induced obesity
in mice (Lam et al., 2012), suggesting that valeric acid Experimental Design and Diet A completely ran-
might promote energy harvest in the intestinal tract. domized design was used with 2 dietary treatment
This can be of benefit for food-producing animals. To groups. GVA were included as feed additive for one
the best of our knowledge, no feeding trials using va- group, and the other group was a control group with-
leric acid or any of its derivatives have been carried out feed additives. Both treatment groups were repli-
out in any animal species. Therefore, the aim of the cated 8 times. Inclusion level of the GVA was 1.5 g/kg
present study was to investigate the effects of valeric in a commercial broiler feed formula (Quartes, Deinze,
acid glyceride esters on intestinal health in broilers. Five Belgium). Analyzed nutrient composition is given in
different experiments, focusing on different aspects of Table 1.
intestinal health, were carried out with different con- Animals and Experimental Procedures A total of
centrations of GVA. One study was a trial in which per- 640 day-of-hatch Ross 308 broiler chickens were ran-
formance was measured over the whole rearing period, domly divided in 16 pens, 8 pens of 43 female birds and
while 2 trials, using higher dosages of GVA, were carried 8 pens of 37 male birds. Each pen had a surface of 2.45
out to study gut wall morphology and structure. Two m2 with a solid floor covered with wood shavings.
necrotic enteritis trials were carried out with multiple On d 1, 11, 32, and 39, all broilers and feed were
dose regimens. weighed per pen to calculate the weight gain and feed
conversion ratio (FCR).

MATERIALS AND METHODS

Feed Additive
Because of the rapid absorption of SCFAs in the in-
testinal tract, valeric acid was supplied as a glyceride
(Small 1991; Roy et al., 2006). In the in vivo studies
a composition of esters derived from glycerol and va-
leric acid (GVA) was used as feed additive (Perstorp
Effects of Valeric Acid Glyceride Esters on
Gut Health Parameters
Experimental Design and Diet A completely ran-
domized design was used with 2 dietary treatment
groups. One treatment group received GVA (5 g/kg)
as feed additive, and the other treatment group
received no feed additive. Both treatment groups were
 VALERIC ACID GLYCERIDE AS FEED ADDITIVE
Table 2. Nutrient composition of the diet of 2 feeding trials,
evaluating the effects of valeric acid glyceride esters on gut health
parameters.
Nutrient (%) Starter Day 1–10 Grower Day 11–28
Crude protein 21.0 18.0
Crude fat 6.0 4.8
Crude ash 5.5 5.5
Crude fiber 3.0 3.2
Lysine 1.17 0.97
Methionine 0.52 0.45
Calcium 0.85 0.85
Phosphorus 0.58 0.53
Sodium 0.15 0.15
replicated 3 times. A commercial broiler feed formula
(Versele-Laga, Deinze, Belgium) was used, of which the
analyzed nutrient composition is given in Table 2.
Animals and Experimental Procedures In a first
experiment, 24 day-of-hatch male Ross 308 broiler
chicks were randomly assigned to one of the 2 dietary
treatment groups of 12 birds each. Each pen had a sur-
face of 1.44 m2 and had a solid floor covered with wood
shavings. At 28 d of age, birds were euthanized by an
intravenous injection of sodium pentobarbital (Natrium
Pentobarbital 20%, Kela Veterinaria, Sint-Niklaas,
Belgium). Standardized segments of jejunum were col-
lected and fixed in 4% formaldehyde. Intestinal contents
of duodenum, jejunum, ileum, colon and both ceca were
collected and stored at –20◦ C.
In a second experiment, for analysis of GLP-2-
immunoreactive cells, 24 day-of-hatch male Ross 308
broiler chicks were randomly assigned to one of the 2
dietary treatment groups of 12 birds each, and eutha-
nized at 10 d of age. All other experimental procedures
were the same as those in the first experiment. Stan-
dardized segments of jejunum and ileum were collected
and fixed in 4% formaldehyde.
Samples Processing and Analyses In the first ex-
periment, the formalin-fixed intestinal segments were
dehydrated in xylene, embedded in paraffin, and 4 μm
tissue sections were prepared. The sections were stained
with hematoxylin and eosin. Villus height and crypt
depth were measured using a light microscope with
Leica LAS software (Leica Microsystems, Diegem,
Belgium). The average of 10 measurements per segment
per animal was calculated.
The method previously described by De Weirdt et al.
(2010) was used in the first experiment to quantify the
amounts of butyric, propionic, acetic, and valeric acid in
intestinal contents of duodenum, jejunum, ileum, colon,
and cecum. In short, SCFAs were extracted from the
samples using diethyl ether. Methyl hexanoic acid was
added as an internal standard. The extracts were ana-
lyzed using a GC-2014 gas chromatograph (Shimadzu,
‘s-Hertogenbosch, the Netherlands), equipped with a
capillary fatty acid-free EC-1000 Econo-Cap column
(dimensions: 25 mm × 0.53 mm, film thickness 1.2 mM;
Alltech, Laarne, Belgium), a flame ionization detector
and a split injector. The injection volume was 1 mL,
and the temperature profile was set from 110 to 160◦ C,
2305
with a temperature increase of 6◦ C per min. The carrier
gas was nitrogen, and the temperature of the injector
and detector were 100 and 220◦ C respectively.
In the second experiment, the method previously de-
scribed by Monir et al. (2014) was used for immuno-
histochemical staining of glucagon-like peptide (GLP)-
2-immunoreactive cells in the small intestine of the
chicken. The formalin-fixed intestinal segments of je-
junum and ileum were dehydrated in xylene, embed-
ded in paraffin, and 4 μm tissue sections were pre-
pared. Paraffin was removed, and the samples were re-
hydrated. After incubation with 0.5% antigen retrieval
agent, the horseradish peroxidase-labeled streptavidin-
biotin method was used (Sigma-Aldrich, Overijse, Bel-
gium). The samples were incubated with 10% normal
goat serum (Invitrogen, Merelbeke, Belgium) for 20 min
before 24 h incubation with rabbit anti-human-GLP-
2-serum (Abcam, Cambridge, UK). Visualization was
done with 0.05% 3,3-d-aminobenzidine in Tris-HCL
buffer (pH 7.6) and Mayer’s hematoxylin. The area
of the mucosal layer was measured, and the GLP-2-
immunoreactive cells were counted within the area,
using a light microscope with Leica LAS software
(Leica Microsystems, Diegem, Belgium). The average
of 10 areas of at least 10 mm2 in both jejunum
and ileum per animal was used for calculation of
the cell number per area of the mucosal layer
(cell/mm2 ).
Protection Against Necrotic Enteritis
Challenge by Valeric Acid Glycerides
Clostridium Strain and Culture Conditions C.
perfringens strain 56 is a netB-positive toxin type A
strain isolated from the intestine of a broiler chicken
with necrotic enteritis lesions. This strain has been
shown to have the capacity to induce necrotic enteri-
tis lesions in previous in vivo trials (Gholamiandehko-
rdi et al., 2007; Timbermont et al., 2009). Bacteria
were grown in Brain Heart Infusion broth (Oxoid, Bas-
ingstoke, UK) at 37◦ C in anaerobic conditions (84% N2 ,
8% CO2 and 5% H2 ).
Vaccines The commercial IBD vaccines Poulvac
Bursa Plus (Zoetis Belgium S.A., Louvain, Belgium)
and Nobilis Gumboro D78 (MSD Animal Health, Brus-
sels, Belgium) were used. Also, 2 anticoccidial vaccines
were used, i.e., Paracox-5TM (MSD Animal Health,
Brussels, Belgium) containing oocysts from precocious
lines of Eimeria acervulina, Eimeria maxima (2 lines),
Eimeria mitis, and Eimeria tenella, and Hipracox (Lab-
oratorios Hipra, Amer, Spain) containing oocysts from
precocious lines of Eimeria acervulina, Eimeria max-
ima, Eimeria mitis, Eimeria praecox, and Eimeria
tenella.
Experimental Design and Diet In a first trial, 108
day-of-hatch male Ross 308 broiler chicks were divided
in 4 pens of 27 chickens each, assigned to 4 differ-
ent treatment groups. Dietary treatment groups were a
2306 ONRUST ET AL.
positive control (no feed additive, and challenged with
C. perfringens), a negative control (no feed additive
and no challenge with C. perfringens), a group receiv-
ing feed containing GVA at higher concentrations (5.0,
2.5, 2.0 g/kg in resp. week 1, 2, 3) and challenged with
C. perfringens and a group receiving GVA at lower con-
centrations (2.5, 2.0, 1.5 g/kg in resp. week 1, 2, 3) and
challenged with C. perfringens.
In a second trial, 243 day-old male Ross 308 broiler
chicks were divided in 9 pens of 27 chickens each, as-
signed to 3 different treatment groups. Each dietary
treatment was replicated 3 times. Dietary treatment
groups were a positive control (no feed additive), and a
group receiving a diet containing GVA at 1.5 g/kg and
a group receiving GVA at 0.5 g/kg.
All birds were given a wheat- and rye-based (43% +
7.5%) diet with soybean meal as main protein source for
the first 16 d. From d 17 onwards, soybean meal was
replaced with fish meal as main protein source (30%)
as described by Gholamiandehkordi et al. (2007)
Experimental Procedures Day-of-hatch Ross 308
broilers were obtained from a local hatchery and ran-
domly divided in pens of 1.44 m2 . There were 27 birds
in each treatment group. The pens had solid walls and
a solid floor covered with wood shavings. The exper-
imental procedures were as described previously by
Gholamiandehkordi et al. 2007, with some modifica-
tions. At d 4 Poulvac Bursa Plus and at d 9 Nobilis
Gumboro D78 were given by oral gavage. The birds were
orally inoculated with a 10-fold dose of 2 different coc-
cidiosis vaccines at d 14 (Hipracox) and d 16 (Paracox-
5). At d 18, 19, and 20, all birds (except for the nega-
tive control group) were challenged by oral gavage with
1 mL bacterial culture containing approximately 4 ×
108 cfu of C. perfringens strain 56 (3 times a d). At
d 21, all birds were euthanized for macroscopic lesion
scoring, and bodyweight was measured.
At necropsy lesions in the small intestine were scored
as described by Keyburn et al. 2006 as follows: 0 = no
gross lesions; 2 = small focal necroses or ulcerations
(1 to 5 foci); 3 = focal necroses or ulcerations (6 to
15 foci); 4 = focal necroses or ulcerations (16 or more
foci); 5 = patches of necrosis more than 1 cm long;
6 = diffuse necrosis. Scoring was performed for duode-
num, jejunum and ileum separately. Final lesion score
was determined by the highest of these 3 scores. The
score 1 used for congested intestinal mucosa was not
applied here because of difficulties in scoring this objec-
tively, and due to the lack of scientific documentation of
an association between “congested intestinal mucosa”
and necrotic enteritis. Final lesion scores of 2 or more
were classified as necrotic enteritis-positive.
Ethical Approval
The experiments were carried out according to the
recommendations of, and following approval from,
the Ethical Committee of the Faculty of Veterinary
Medicine, Ghent University (EC 2013/25 and EC
2015/60).
Statistical Analysis
Statistical analysis was carried out with InVivoStat
(Cambridge, UK), a statistical software package which
uses R as its statistics engine (Clark et al., 2012).
Model assumptions were checked by visual inspection
of the residuals. Differences of the mean between di-
etary treatment groups were analyzed with each pen
as experimental unit. The differences were considered
statistically significant at P ≤ 0.05.
SCFA data, villus height and crypt depth measure-
ments, and density of GLP-2-immunoreactive cells were
assessed by one-way analysis of variance (ANOVA) us-
ing the following model: Yij = μ + τi + εij , where
Yij represents the jth replicate (j is 1 – 12) fed the ith
treatment (i = control or GVA). μ is the overall mean
response, τ i is the ith treatment effect, and εij is the
random error associated with the jth replicate fed the
ith treatment.
Performance data, a 2 by 2 factorial design, were first
analyzed using a 2-way ANOVA to evaluate whether
there was an interaction present between treatment
and gender. The following model was used: Yij k =
μ + τi + βj + γij + εij , where Yijk represents the kth
replicate (k is 1 – 8) fed the ith treatment (i = control
or GVA) to the jth gender (j = male or female). μ is
the overall mean response, τ i is the ith treatment ef-
fect, βj is the jth fixed effect of gender, and γij is the
interaction effect between the treatment and gender.
For the data with interaction, a one-way ANOVA was
used with the same procedure as SCFA data. For data
that were not normally distributed, the nonparametric
Mann-Whitney U test was performed.
Differences in mean necrotic enteritis lesion score and
body weight in necrotic enteritis model were statisti-
cally analysed using one-way ANOVA using the same
model as SCFA data, where for the first trial Yij repre-
sents the jth replicate (j is 1 – 4) fed the ith treatment
(i = negative control, positive control, low regimen
GVA or high regimen GVA). For the second trial Yij
represents the jth replicate (j is 1 – 9) fed the ith treat-
ment (i = control, low GVA or high GVA).
RESULTS
Effects of Valeric Acid Glyceride Esters on
Performance
In this study, broiler performance was evaluated
with and without supplementation of GVA to the diet.
Body weight and feed intake were measured, and FCR
and growth were calculated. In the starter phase, in-
teraction between treatment and sex was observed.
For the male birds, a significant difference in body
weight, growth and FCR was observed comparing GVA
 VALERIC ACID GLYCERIDE AS FEED ADDITIVE 2307
Table 3. Effect of glyceride esters of valeric acid (GVA) on feed conversion ratio, body weight (g), feed intake
(g/d/bird) and weight gain (g/d/bird) measured at 3 different time points. The data shown are the mean and
standard deviation of these parameters of broilers fed a diet without or with GVA (1.5 g/kg). Each dietary
treatment consisted of 4 pens of 43 female birds and 4 pens of 37 male birds.

Dietary Treatment
Control Glyceride Ester of Valeric Acid Pooled SEM P-Value
Parameters Mean SD Mean SD

D1–11∗
FCR M+F 1.20 0.05 1.16 0.03 0.03 -
FCR M 1.23 0.07 1.15 0.02 0.04 0.0286
FCR F 1.18 0.01 1.19 0.03 0.02 0.8340
BW (g) M+F 319.0 9.9 323.0 14.8 8.90 -
BW (g) M 326.5 3.3 336.0 4.3 2.71 0.0102
BW (g) F 311.5 8.5 310.0 3.7 4.64 0.6559
FI (g/d) M+F 30.8 2.1 30.2 1.0 1.16 -
FI (g/d) M 32.3 1.9 31.0 0.5 0.98 0.3065
FI (g/d) F 29.2 0.8 29.3 0.4 0.45 0.8857
WG (g/d) 25.6 0.9 25.9 1.3 0.79 -
M+F
WG (g/d) M 26.2 0.3 27.1 0.3 0.21 0.0286
WG (g/d) F 24.9 0.7 24.8 0.3 0.38 0.3429
d 12–31
FCR 1.52 0.04 1.48 0.05 0.02 0.0052
BW (g) 1886.0 168.8 1942.0 203.4 93.45 0.0138
FI (g/d) 119.8 11.2 119.4 10.5 5.42 0.7888
WG (g/d) 78.4 8.01 81.0 9.5 4.39 0.3282$
d 32–37
FCR 1.65 0.04 1.63 0.07 0.06 0.1089
BW (g) 2574.0 268.5 2652.0 297.2 283.21 0.0212
FI (g/d) 187.3 21.9 191.9 21.0 21.45 0.1815
WG (g/d) 113.5 16.05 117.9 16.8 16.43 0.2786$
d 1–37
FCR 1.53 0.04 1.48 0.05 0.02 0.0211
BW (g) 2574.0 286.5 2652.0 297.2 145.95 0.0212
FI (g/d) 104.7 10.6 104.2 8.8 4.87 0.7147
WG (g/d) 68.6 7.25 70.7 8.0 3.82 0.3823$
FCR, food conversion ratio; BW, body weight; FI, feed intake; WG, weight gain.
P-values are given between the control and GVA dietary treatment within a given characteristic.
$ data was not normally distributed, analyzed with non -parametric with Mann-Whitney-U-test.
∗
In period d 1–11, there was an interaction for the analyzed measurements between treatment and sex (M = male and
F = Female), the values for male and female were analyzed with single-factor one-way ANOVA.

supplemented groups with non-supplemented groups.
During the grower phase (D12–31) the FCR was 0.04
units lower (P = 0.0052), and body weight was in-
creased with an average of 56 grams (P = 0.0183) when
the chickens received GVA added to the diet compared
to chickens in the control group. Over the entire trial
period (D1–37), the FCR was also 0.05 units lower
(P = 0.0212) for the chickens that received GVA in
the diet (Table 3).
Effects of Valeric Acid Glyceride Esters on
Gut Health Parameters
Glyceride Esters of Valeric Acid Decrease Crypt
Depth and Increase the Villus Height/Crypt Depth
Ratio in Jejunum Histology showed that GVA sup-
plementation to broiler feed significantly increased
(P < 0.0001) the villus height/crypt depth ratio in
jejunum compared to controls (6.76 vs 5.58). The
crypt depth was decreased (P = 0.0013) in GVA
supplemented groups (145 μm vs 176 μm), and for the
villus height a trend in increase (P = 0.0964) could be
observed (977 μm vs 911 μm; Table 4).
Glyceride Esters of Valeric Acid Have No Effect
on SCFA Concentrations in Cecum Acetate, propi-
onate, butyrate, and valerate could be detected in the
ceca of the 28-day-old broilers. In Table 5, mean val-
ues of these SCFAs are shown, indicating no differences
between the chickens fed with and without GVA sup-
plement. In the intestinal content of the other segments
the values of SCFA were below the detection limit.
Glyceride Esters of Valeric Acid Increase the
Density of GLP-2-immunoreactive Cells in Ileum
and Jejunum Immunohistochemistry showed that the
density of GLP-2-immunoreactive cells in jejunal villi
(P = 0.001), ileal villi (P = 0.080), and jejunal crypts
(P = 0.025), obtained from broilers supplemented with
GVA, was increased compared to the control group
(Figure 1). Only in the ileal crypts no difference
(P = 0.1079) was observed between the GVA treatment
group and the control group.
2308 ONRUST ET AL.

Table 4. Effect of glyceride ester of valeric acid (GVA) on the intestinal morphological parameters of broilers.
The data shown are the mean length of the villi and mean depth of the crypts and the ratio of these parameters
in jejunal sections taken at d 28 of broilers fed a diet without or with GVA (5 g/kg). Each dietary treatment
consisted of 3 pens of 4 broilers. The average of 10 measurements per animal was calculated.

Dietary Treatment
Glyceride Ester of Valeric
Control Acid Pooled SEM P-value
Parameters Mean (μ m) SD Mean (μ m) SD

Jejunum
Villus height 911 369 977 354 147.61 0.0964
Crypt depth 176 82 145 39 26.21 0.0013
Villus 5.58 2.01 6.76 1.83 0.78 < 0.0001
height/crypt depth

Table 5. Effect of glyceride ester of valeric acid treatment (5 g/kg) on concentrations of short chain fatty
acids in cecum of broilers at the age of 28 d. Each dietary treatment consisted of 3 pens of 4 broilers. The data
shown are the mean values of 12 chickens per dietary treatment.

Dietary Treatment
Control Glyceride Ester of Valeric Acid Pooled SEM P Value
SCFA Mean (mM) SD Mean (mM) SD

Acetate 39.08 8.38 41.57 8.29 3.40 0.4555

Propionate 7.38 4.66 5.27 3.46 1.68 0.3068
Butyrate 8.49 1.60 8.64 1.24 0.58 0.9151
Valerate 1.29 0.40 1.13 0.46 0.18 0.6441
SCFA, Short Chain Fatty Acid.

(low GVA regimen: P = 0.0103, and high GVA regimen:

Densit y of G LP -2 imm unor ea ctive cells

400
Control GVA P = 0.0011) (Table 6). In trial 2, lower concentrations
of GVA were tested, which resulted in a decrease in the
300 number of chickens with lesions of NE, but the mean le-
(cells/m m )

sion score was not reduced significantly. However, body

2

weight increased significantly (P < 0.0001) in the sec-

200
ond trial for the groups that received feed supplemented
with lower concentrations of GVA (691.1 g and 71.04 g
100 vs 622.2 g; Table 6).

0 DISCUSSION
J ejunum villi J ejunum cr ypts Ileum villi Ileum cr ypts

Figure 1. Density of GLP-2 immunoreactive cells in the small in-

To the best of our knowledge, this is the first study
testine of 10-day-old chickens fed a diet either or not supplemented demonstrating beneficial effects of valeric acid on in-
with glyceride ester of valeric acid GVA (5 g/kg). Each dietary treat- testinal health parameters and performance in broiler
ment consisted of 3 pens of 4 broilers. The bar charts showing mean chickens. Moreover, higher concentrations partially pro-
values of GLP-2-immunoreactive cells per 1 mm2 of mucosa, divided in
villi and crypts per segment. Error bars indicate standard deviation.
tected the birds from necrotic enteritis in a severe chal-
The differences were considered statistically significant at ∗ P ≤ 0.05. lenge model.
Valeric acid is naturally produced by the microbiota
Protection Against Necrotic Enteritis in the lower gastrointestinal tract. Most of the valeric
Challenge by Valeric Acid Glycerides acid producing microorganisms in the intestine belong
to the genus Oscillibacter (Lino et al. 2007). Valeric
Glyceride Esters of Valeric Acid Can Reduce the acid is a C5 fatty acid and, as such, its chain length is
Number of Chickens with Lesions of NE In trial 1, in between the short-chain fatty acids (C1 to C4) and
the number of chickens with lesions of NE was decreased the medium chain fatty acids (C6 to C12). Beneficial ef-
in both groups supplemented with different concentra- fects on broiler performance have been reported for C3
tions of glyceride derivatives compared to the positive (propionate), C4 (butyrate), as well as for the MCFA
control group. There was a reduction from 71% animals (Bintvihok and Kositcharoenkul, 2006; Namkung et al.,
with NE lesions in the positive control group to, respec- 2011; Zentek et al., 2012; Khosravinia, 2015). The cur-
tively, 26.7 and 35.5% in the supplemented groups. The rent study showed an improved feed efficiency for broil-
mean lesion score for the GVA supplemented groups ers fed with GVA compared to those fed a control
was decreased compared to the positive control group diet (Table 3). One of the parameters influencing feed
 VALERIC ACID GLYCERIDE AS FEED ADDITIVE 2309
Table 6. Effect of glyceride derivatives treatment on the occurrence of necrotic enteritis in broiler chickens after a feeding period of
3 weeks. In trial 1, 4 experimental groups were included, of which each experimental group consisted of 1 pen of 27 broilers. In trial
2, 3 experimental groups were included, of which experimental groups consisted of 3 pens of 27 broilers.

Chickens with NE
lesions (%) Lesion score Body weight (g)

Inclusion level of
C. perfringens glyceride ester of Pooled Pooled
Group challenge valeric acid (g/kg) Mean SD Mean SD SEM P value Mean SD SEM P value

Trial 1
Negative control no 0 10.3 – 0.4138 1.240 0.50 < 0.0001 714.0 108.0 0.7101
Positive control yes 0 71.0 – 1.742 1.365 0.50 738.8 83.6
Low yes resp. week 1,2,3 26.7 – 0.7000 1.317 0.50 0.0011 686.9 135.4 0.1610
2.5, 2.0, 1.5
High yes resp. week 1,2,3 35.5 – 0.9355 1.482 0.50 0.0103 766.6 104.1 0.6212
5.0, 2.5, 2.0
Trial 2
Positive control yes 0 36.1 12.7 0.7429 1.031 0.31 622.2 91.0
Low yes 0.5 27.2 2.9 0.5571 0.9268 0.31 0.2848 710.4 83.9 < 0.0001
High yes 1.5 25.4 8.4 0.5211 0.908 0.31 0.1904 691.1 83.6 < 0.0001

P-values are given for comparison dietary treatment group with positive control group.

efficiency is gut morphology. The intestinal structure
can adapt to dietary changes by increasing or decreas-
ing the height of the villi and the depth of the crypts
(Ao and Choct, 2013). Increase in length of intestinal
villi suggests an increased surface area capable of more
absorption and digestion of nutrients, which will lead to
better performance. Conversely, shortening of the villi
indicates a loss of surface area for digestion and absorp-
tion, thereby lowering performance (Caspary, 1992).
The crypt epithelium is responsible for continuously
replacing enterocytes, and deeper crypts indicate fast
tissue turnover in response to increased requirements
for maintenance of the digestive tract, such as inflam-
mation caused by pathogens or their toxins (Yason
et al., 1987; Awad et al., 2009). In the present study,
a shortening of crypts and a higher villus height: crypt
depth ratio was measured (Table 4). This indicates a
lower turnover of enterocytes. As renewal of gut tissue
requires a lot of energy, a lower epithelial turnover can
have a significant impact on nutrient requirements for
maintenance (Taylor-Pickard and Spring, 2008). More-
over, a study by Lam et al. (2012) describes that a
valeric acid producing strain is associated with diet-
induced obesity in mice, suggesting that valeric acid
might promote energy harvest in the intestinal tract.
The gut wall morphology was, however, measured in
animals that received a higher dose as compared to the
animals in the performance trial, so a causal relation
cannot be established, but this analysis should be taken
into account in future studies.
Glucagon-like-peptide (GLP)-2 is one of the gut hor-
mones playing an important role in the gastrointesti-
nal tract, exerting diverse actions including enhancing
cell differentiation and stimulation of intestinal growth
(Guan et al., 2006). GLP-2 is produced by enteroen-
docrine cells located in the intestinal epithelial layer in
response to luminal stimuli. It is secreted at the basal
side. Following secretion in the bloodstream, GLP-2
migrates to other organs and acts via specific bind-
ing to receptors (GLP-2R) located on enteric neurons,
enteroendocrine cells (Guan et al., 2006) and subep-
ithelial myofibroblasts (de Heuvel et al., 2012). These
cells subsequently produce multiple downstream medi-
ators affecting the intestinal epithelium (Dube et al.,
2006; Sigalet et al., 2010; Drucker and Yusta, 2014).
The main biological role of GLP-2 has been associated
with intestinotrophic actions on the small intestine, in-
creased expression of intestinal tight junction proteins
(Cani et al., 2009) and regulation of the innate im-
mune system by controlling the expression of antimi-
crobial peptides which are implicated in the mainte-
nance of the gut barrier function (Lee et al., 2012). This
meal-induced gut hormone is derived from pro-glucagon
and has already been associated with positive effects
on growth performance of broilers in previous studies
(Hu et al., 2010). It has also been shown that a di-
etary change can influence the density of gastrointesti-
nal endocrine cells (El-Salhy et al., 2016). In our study,
we showed that the density of GLP-2-immunoreactive
cells was significantly increased in jejunum and ileum
villi from broilers supplemented with GVA compared
to the non-supplemented broilers (Figure 1). In mice
an increase in density of L-cells, induced by dietary
changes, correlated with an increase of GLP-1 produc-
tion, thereby suggesting that preproglucagon (precursor
of GLP-1 and GLP-2) production is related to an ex-
panded L-cell population (Aranias et al., 2015; Catry et
al., 2017). It can be hypothesized that valeric acid in-
creases the density of GLP-2 cells and thereby GLP-2
production in the intestine of chickens, which stimu-
lates intestinal growth, and in this way, improves broil-
ers gut health and performance. However, as the den-
sity of GLP-2 cells was analyzed in birds that received
a high dosage, and at 10 d of age, no causal relation
between GLP-2 secretion and gut wall morphology and
performance could be established. To strengthen the
2310 ONRUST ET AL.
hypothesis, a further study is necessary to analyze the
density of GLP-2 cells in the intestine of broilers fed
GVA and control animals, for which good performance
data are recorded.
The intestinotrophic and gut barrier-stabilizing ef-
fects might also have resulted in protection in the sub-
clinical NE model in our study. Subclinical NE caused
by C. perfringens can adversely affect growth and feed
conversion rate (Lovland and Kaldhusdal, 2001). Pre-
viously butyric acid (C4) and MCFA (C6–C12) already
have been shown to significantly decrease the number
of birds with necrotic lesions (Timbermont et al., 2010).
In the current study, a decrease in the percentage of an-
imals with lesions was observed with the low regimen
and high regimen doses of GVA fed. At lower concen-
trations of GVA, the mean score of necrotic enteritis
lesions was not significantly reduced, but a positive ef-
fect on body weight was seen (Table 6). This is in line
with observations that valeric acid increases the villus
to crypt ratio and improves performance.
In conclusion, glycerol esters of valeric acid added
to broiler feed can increase body weight, decrease feed
conversion rate, positively affect the morphology of the
intestinal mucosa, increase the density of GLP-2 pro-
ducing L-cells, and reduce the incidence of necrotic en-
teritis. The effect of GVA supplementation on L-cell
density may play a key role in the observed benefi-
cial effects. As the effect may depend on the dose or
age of the animals, it will be interesting to analyze the
gut health parameters at starter, grower and finisher
phase in future trials, while also recording the perfor-
mance data, and to compare treatment groups with dif-
ferent concentrations of GVA. Further research is thus
needed to unravel the mechanism underlying the ob-
served effects of this feed additive, but these first data
indicate that GVA can be a valuable feed additive for
broilers.
ACKNOWLEDGMENTS
The technical assistance of C. Puttevils, D. Ameye,
and S. Loomans was greatly appreciated. The authors
acknowledge the PhD students from the department
of Pathology, Bacteriology and Avian Diseases who as-
sisted in the sampling of the in vivo trials.
This work received the financial support from Per-
storp BV, Waspik, The Netherlands. Co-author Koen
Schwarzer is employed by Perstorp BV. This does not
alter the authors’ adherence to all the Poultry Science
policies on sharing data and materials.
REFERENCES
Ao, Z., and M. Choct. 2013. Oligosaccharides affect performance and
gut development of broiler chickens. Asian-Australas. J. Anim.
Sci. 26:116–121.
Aranias, T., A. Grosfeld, C. Poitou, A. A. Omar, M. Le Gall, S.
Miquel, K. Garbin, A. Ribeiro, J. L. Bouillot, A. Bado, E. Brot-
Laroche, K. Clement, A. Leturque, S. Guilmeau, and P. Serradas.
2015. Lipid-rich diet enhances L-cell density in obese subjects
and in mice through improved L-cell differentiation. J. Nutr. Sci.
4:e22.
Awad, W. A., K. Ghareeb, S. Abdel-Raheem, and J. Bohm. 2009.
Effects of dietary inclusion of probiotic and synbiotic on growth
performance, organ weights, and intestinal histomorphology of
broiler chickens. Poult. Sci. 88:49–56.
Bertevello, P. L., L. De Nardi, R. S. Torrinhas, A. F. Logullo, and
D. L. Waitzberg. 2012. Partial replacement of omega-6 fatty acids
with medium-chain triglycerides, but not olive oil, improves colon
cytokine response and damage in experimental colitis. J. Par-
enter. Enteral. Nutr. 36:442–448.
Bintvihok, A., and S. Kositcharoenkul. 2006. Effect of dietary cal-
cium propionate on performance, hepatic enzyme activities and
aflatoxin residues in broilers fed a diet containing low levels of
aflatoxin B1. Toxicon 47:41–46.
Cai, D., S. Zhao, D. Li, F. Chang, X. Tian, G. Huang, Z. Zhu, D.
Liu, X. Dou, S. Li, M. Zhao, and Q. Li. 2016. Nutrient intake
is associated with longevity characterization by metabolites and
element profiles of healthy centenarians. Nutrients 8:564.
Cani, P. D., S. Possemiers, T. Van de Wiele, Y. Guiot, A. Everard,
O. Rottier, L. Geurts, D. Naslain, A. Neyrinck, D. M. Lambert,
G. G. Muccioli, and N. M. Delzenne. 2009. Changes in gut micro-
biota control inflammation in obese mice through a mechanism
involving GLP-2-driven improvement of gut permeability. Gut
58:1091–1103.
Caspary, W. F. 1992. Physiology and pathophysiology of intestinal
absorption. Am. J. Clin. Nutr. 55:299S–308S.
Catry, E., L. B. Bindels, A. Tailleux, S. Lestavel, A. M. Neyrinck,
J. F. Goossens, I. Lobysheva, H. Plovier, A. Essaghir, J. B. De-
moulin, C. Bouzin, B. D. Pachikian, P. D. Cani, B. Staels, C.
Dessy, and N. M. Delzenne. 2018. Targeting the gut microbiota
with inulin-type fructans: preclinical demonstration of a novel
approach in the management of endothelial dysfunction. Gut.
67:271–283.
Chan, Y. K., M. Estaki, and D. L. Gibson. 2013. Clinical conse-
quences of diet-induced dysbiosis. Ann. Nutr. Metab. 63:28–40.
Clark, R. A., M. Shoaib, K. N. Hewitt, S. C. Stanford, and S. T. Bate.
2012. A comparison of InVivoStat with other statistical software
packages for analysis of data generated from animal experiments.
J. Psychopharmacol. 26:1136–1142.
de Heuvel, E., L. Wallace, K. A. Sharkey, and D. L. Sigalet. 2012.
Glucagon-like peptide 2 induces vasoactive intestinal polypeptide
expression in enteric neurons via phosphatidylinositol 3-kinase-
gamma signaling. Am. J. Physiol. Endoc. M 303:E994–E1005.
De Weirdt, R., S. Possemiers, G. Vermeulen, T. C. Moerdijk-
Poortvliet, H. T. Boschker, W. Verstraete, and T. Van de Wiele.
2010. Human faecal microbiota display variable patterns of glyc-
erol metabolism. FEMS Microbiol. Ecol. 74:601–611.
Dierick, N., J. Michiels, and C. Van Nevel. 2004. Effect of medium
chain fatty acids and benzoic acid, as alternatives for antibiotics,
on growth and some gut parameters in piglets. Commun. Agric.
Appl. Biol. Sci. 69:187–190.
Drucker, D. J., and B. Yusta. 2014. Physiology and Pharmacology
of the Enteroendocrine Hormone Glucagon-Like Peptide-2. Annu.
Rev. Physiol. 76:561–583.
Dube, P. E., C. L. Forse, J. Bahrami, and P. L. Brubaker. 2006.
The essential role of insulin-like growth factor-1 in the intestinal
tropic effects of glucagon-like peptide-2 in mice. Gastroenterology
131:589–605.
El-Salhy, M., T. Mazzawi, T. Hausken, and J. G. Hatlebakk. 2016.
Interaction between diet and gastrointestinal endocrine cells.
Biomed. Rep. 4:651–656.
Fernandez-Rubio, C., C. Ordonez, J. Abad-Gonzalez, A. Garcia-
Gallego, M. P. Honrubia, J. J. Mallo, and R. Balana-Fouce. 2009.
Butyric acid-based feed additives help protect broiler chickens
from Salmonella Enteritidis infection. Poult. Sci. 88:943–948.
Gholamiandehkordi, A. R., L. Timbermont, A. Lanckriet, W. Van
Den Broeck, K. Pedersen, J. Dewulf, F. Pasmans, F. Haesebrouck,
R. Ducatelle, and F. Van Immerseel. 2007. Quantification of gut
lesions in a subclinical necrotic enteritis model. Avian Pathol.
36:375–382.
Guan, X., H. E. Karpen, J. Stephens, J. T. Bukowski, S. Niu, G.
Zhang, B. Stoll, M. J. Finegold, J. J. Holst, D. Hadsell, B. L.
Nichols, and D. G. Burrin. 2006. GLP-2 receptor localizes to
 VALERIC ACID GLYCERIDE AS FEED ADDITIVE
enteric neurons and endocrine cells expressing vasoactive peptides
and mediates increased blood flow. Gastroenterology 130:150–
164.
Han, W., X. L. Zhang, D. W. Wang, L. Y. Li, G. L. Liu, A. K.
Li, and Y. X. Zhao. 2013. Effects of microencapsulated Ente-
rococcus fecalis CG1.0007 on growth performance, antioxidation
activity, and intestinal microbiota in broiler chickens. J. Anim.
Sci. 91:4374–4382.
Høverstad, T., O. Fausa, A. Bjørneklett, and T. Bøhmer. 1984.
Short-chain fatty acids in the normal human feces. Scand. J. Gas-
troenterol. 19:375–81.
Hu, X. F., Y. M. Guo, B. Y. Huang, S. Bun, L. B. Zhang, J. H.
Li, D. Liu, F. Y. Long, X. Yang, and P. Jiao. 2010. The effect of
glucagon-like peptide 2 injection on performance, small intesti-
nal morphology, and nutrient transporter expression of stressed
broiler chickens. Poult. Sci. 89:1967–1974.
Huyghebaert, G., R. Ducatelle, and F. Van Immerseel. 2011. An up-
date on alternatives to antimicrobial growth promoters for broil-
ers. Vet. J. 187:182–188.
Keyburn, A. L., S. A. Sheedy, M. E. Ford, M. M. Williamson, M. M.
Awad, J. I. Rood, and R. J. Moore. 2006. Alpha-toxin of Clostrid-
ium perfringens is not an essential virulence factor in necrotic
enteritis in chickens. Infect. Immunol. 74:6496–6500.
Khan, S. H., and J. Iqbal. 2016. Recent advances in the role of or-
ganic acids in poultry nutrition. J. Appl. Anim. Res. 44:359–369.
Khosravinia, H. 2015. Effect of dietary supplementation of medium-
chain fatty acids on growth performance and prevalence of carcass
defects in broiler chickens raised in different stocking densities. J.
Appl. Poult. Res. 24:1–9.
Kim, G. B., Y. M. Seo, C. H. Kim, and I. K. Paik. 2011. Effect of
dietary prebiotic supplementation on the performance, intestinal
microflora, and immune response of broilers. Poult. Sci. 90:75–82.
Lam, Y. Y., C. W. Y. Ha, C. R. Campbell, A. J. Mitchell, A. Din-
udom, J. Oscarsson, D. I. Cook, N. H. Hunt, I. D. Caterson, A. J.
Holmes, and L. H. Storlien. 2012. Increased Gut Permeability and
Microbiota Change Associate with Mesenteric Fat Inflammation
and Metabolic Dysfunction in Diet-Induced Obese Mice. PLoS
ONE 7:e34233.
Lee, S. J., J. Lee, K. K. Li, D. Holland, H. Maughan, D. S. Guttman,
B. Yusta, and D. J. Drucker. 2012. Disruption of the murine Glp2r
impairs Paneth cell function and increases susceptibility to small
bowel enteritis. Endocrinology 153:1141–1151.
Lino, T., K. Mori, K. Tanaka, K. I. Suzuki, and S. Harayama.
2007. Oscillibacter valericigenes gen. nov., sp. nov., a valerate-
producing anaerobic bacterium isolated from the alimentary canal
of a Japanese corbicula clam. Int. J. Syst. Evol. Microbiol.
57:1840–1845.
Lovland, A., and M. Kaldhusdal. 2001. Severely impaired production
performance in broiler flocks with high incidence of Clostridium
perfringens-associated hepatitis. Avian Pathol. 30:73–81.
2311
Mondot, S., S. Kang, J. P. Furet, D. Aguirre de Carcer, C. Mc-
Sweeney, M. Morrison, P. Marteau, J. Dore, and M. Leclerc.
2011.Highlighting new phylogenetic specificities of Crohn?s dis-
ease microbiota. Inflamm. Bowel. Dis. 17:185–192.
Monir, M. M., K. Hiramatsu, K. Nishimura, C. Takemoto, and T.
Watanabe. 2014. Distribution of glucagon-like peptide (GLP)-
2-immunoreactive cells in the chicken small intestine: anti-
gen retrieval immunohistochemistry. J. Vet. Med. Sci. 76:
565–568.
Namkung, H., H. Yu, J. Gong, and S. Leeson. 2011. Antimicrobial
activity of butyrate glycerides toward Salmonella Typhimurium
and Clostridium perfringens. Poult. Sci. 90:2217–2222.
Rahimi, S., S. Kathariou, J. L. Grimes, and R. M. Siletzky. 2011. Ef-
fect of direct-fed microbials on performance and Clostridium per-
fringens colonization of turkey poults. Poult. Sci. 90:2656–2662.
Roy, C. C., C. L. Kien, L. Bouthillier, and E. Levy. 2006. Short-chain
fatty acids: ready for prime time? Nutr. Clin. Pract. 21:351–366.
Sigalet, D. L., L. Wallace, E. De Heuval, and K. A. Sharkey. 2010.
The effects of glucagon-like peptide 2 on enteric neurons in in-
testinal inflammation. Neurogastroenterol. Motil. 22:1318–e350.
Small, D. M. 1991. The Effects of Glyceride Structure on Absorption
and Metabolism. Annu. Rev. Nutr. 11:413–434.
Taylor-Pickard, J. A., and P. Spring. 2008. Gut efficiency; the key
ingredient in pig and poultry production. Pages 192 in Elevating
Animal Performance and Health. Wageningen Academic Publish-
ers, Wageningen, the Netherlands.
Teirlynck, E., M. D. Gussem, J. Dewulf, F. Haesebrouck, R.
Ducatelle, and F. Van Immerseel. 2011. Morphometric evaluation
of “dysbacteriosis” in broilers. Avian Pathol. 40:139–144.
Tellez, G., C. Pixley, R. E. Wolfenden, S. L. Layton, and B. M. Har-
gis. 2012. Probiotics/direct fed microbials for Salmonella control
in poultry. Food. Res. Int. 45:628–633.
Timbermont, L., A. Lanckriet, J. Dewulf, N. Nollet, K. Schwarzer,
F. Haesebrouck, R. Ducatelle, and F. Van Immerseel. 2010. Con-
trol of Clostridium perfringens-induced necrotic enteritis in broil-
ers by target-released butyric acid, fatty acids and essential oils.
Avian Pathol. 39:117–121.
Timbermont, L., A. Lanckriet, A. R. Gholamiandehkordi, F. Pas-
mans, A. Martel, F. Haesebrouck, R. Ducatelle, and F. Van Im-
merseel. 2009. Origin of Clostridium perfringens isolates deter-
mines the ability to induce necrotic enteritis in broilers. Comp.
Immunol. Microbiol. Infect. Dis. 32:503–512.
Yason, C. V., B. A. Summers, and K. A. Schat. 1987. Pathogenesis of
rotavirus infection in various age groups of chickens and turkeys:
pathology. Am. J. Vet. Res. 48:927–938.
Zentek, J., S. Buchheit-Renko, K. Manner, R. Pieper, and W. Vah-
jen. 2012. Intestinal concentrations of free and encapsulated di-
etary medium-chain fatty acids and effects on gastric microbial
ecology and bacterial metabolic products in the digestive tract of
piglets. Arch. Anim. Nutr. 66:14–26.