Valeric acid glyceride esters in feed promote broiler performance and reduce the incidence of necrotic enteritis

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 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 jejunal 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 significant reduction of the number of birds with necrotic lesions was found at d 21, using in-feed supplementation 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.

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 investigated. Valeric acid derivatives can be produced as glyceride esters and added to broiler feed. In the current study, experiments were carried out to evaluate the effect of valeric acid glycerides (GVA) on growth performance, on the morphology of the small intestinal mucosa and on protection against necrotic enteritis. In a first feeding trial, Ross-308 chicks were randomly divided into 2 dietary treatment groups and fed either a non-supplemented diet or a diet supplemented with GVA (1.5 g/kg). In the GVA supplemented group, the feed conversion ratio was significantly decreased during the entire trial period (D1–37). In a second trial, gut wall morphology was evaluated. In broilers fed a GVA-containing diet at 5 g/kg, the villus height/crypt

Key words: valeric acid, broiler, necrotic enteritis, intestinal health 2018 Poultry Science 0:1–9 http://dx.doi.org/10.3382/ps/pey085

INTRODUCTION

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

Intestinal health is a major issue in broiler production, 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: [email protected]

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gradual release of the acids along the intestinal tract (Fernandez-Rubio et al., 2009; Namkung et al., 2011; Khan and Iqbal, 2016). The MCFA include caproic (C6), caprylic (C8), capric (C10), and lauric (C12) acid. Coconut oil and palm kernel oil constitute rich sources of MCFA. MCFA also have documented beneficial effects on intestinal health (Dierick et al., 2004; Bertevello et al., 2012; Zentek et al., 2012). Valeric acid is a C5 fatty acid which is sometimes classified with the SCFA and other times with the MCFA (Høverstad et al., 1984). It is naturally present in the lower intestinal tract, albeit at an approximately 5 times lower concentration than butyric acid. It is produced by certain members of the intestinal microbiota, mostly those belonging to the Oscillibacter genus (Lino et al., 2007). As valeric acid in the intestine is of microbial origin, it may be more appropriate to classify it 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 health in general. Nevertheless, there are indirect indications from epidemiological observational studies in humans suggesting that valeric acid might beneficially support intestinal health (Mondot et al., 2011; Cai et al., 2016). Indeed, the valeric acid-producing Oscillibacter valericigenes is found to be more abundant in the stools of healthy people as compared to the stools of patients with Crohn’s disease (Mondot et al., 2011). Moreover, valeric acid is found together with propionic acid and butyric acid in significantly higher concentrations in the stools of healthy centenarians compared to elderly people (Cai et al., 2016). Conversely, Oscillibacter spp. are associated with diet-induced obesity in mice (Lam et al., 2012), suggesting that valeric acid might promote energy harvest in the intestinal tract. This can be of benefit for food-producing animals. To the best of our knowledge, no feeding trials using valeric acid or any of its derivatives have been carried out in any animal species. Therefore, the aim of the present study was to investigate the effects of valeric acid glyceride esters on intestinal health in broilers. Five different experiments, focusing on different aspects of intestinal health, were carried out with different concentrations of GVA. One study was a trial in which performance was measured over the whole rearing period, while 2 trials, using higher dosages of GVA, were carried out to study gut wall morphology and structure. Two necrotic enteritis trials were carried out with multiple dose regimens.

MATERIALS AND METHODS

Table 1. Nutrient composition of the diet of the first feeding trial, evaluating the effects of valeric acid glyceride esters on performance. Nutrient (%)

Starter, d 1–11

Grower, d 21–31

Finisher, d 32–37

ME (kcal/kg) Crude protein Lysine Methionine + Cysteine Methionine Threonine Tryptophan Calcium Phosphorus

2,800 21.0 1.17 0.85 0.57 0.72 0.21 0.80 0.38

2,925 19.5 1.06 0.80 0.54 0.69 0.18 0.65 0.33

3,000 18.0 0.98 0.74 0.50 0.65 0.16 0.50 0.30

BV, Waspik, The Netherlands). The ester composition consisted of 45% monovalerin (26% VA), 24% divalerin (18.8% VA), 3.2% trivalerin (2.9% VA) and glycerol.

Experimental Procedures The procedures for temperature, light, and feed schedule were the same for each conducted experiment. Temperature for broilers was maintained during the trials according to the recommendations in the breeder’s manual, and light schedule was set to 18 h light and 6 h darkness from d 7 onwards. Until d 6, 1 h darkness per d was applied. Birds were allowed ad libitum access to feed and water.

Effects of Valeric Acid Glyceride Esters on Performance Experimental Design and Diet A completely randomized design was used with 2 dietary treatment groups. GVA were included as feed additive for one group, and the other group was a control group without feed additives. Both treatment groups were replicated 8 times. Inclusion level of the GVA was 1.5 g/kg in a commercial broiler feed formula (Quartes, Deinze, Belgium). Analyzed nutrient composition is given in Table 1. Animals and Experimental Procedures A total of 640 day-of-hatch Ross 308 broiler chickens were randomly divided in 16 pens, 8 pens of 43 female birds and 8 pens of 37 male birds. Each pen had a surface of 2.45 m2 with a solid floor covered with wood shavings. On d 1, 11, 32, and 39, all broilers and feed were weighed per pen to calculate the weight gain and feed conversion ratio (FCR).

Feed Additive

Effects of Valeric Acid Glyceride Esters on Gut Health Parameters

Because of the rapid absorption of SCFAs in the intestinal 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 valeric acid (GVA) was used as feed additive (Perstorp

Experimental Design and Diet A completely randomized 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

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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 Crude fat Crude ash Crude fiber Lysine Methionine Calcium Phosphorus Sodium

21.0 6.0 5.5 3.0 1.17 0.52 0.85 0.58 0.15

18.0 4.8 5.5 3.2 0.97 0.45 0.85 0.53 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 surface 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 collected 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-2immunoreactive 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 euthanized at 10 d of age. All other experimental procedures were the same as those in the first experiment. Standardized segments of jejunum and ileum were collected and fixed in 4% formaldehyde. Samples Processing and Analyses In the first experiment, 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 analyzed 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,

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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 described by Monir et al. (2014) was used for immunohistochemical staining of glucagon-like peptide (GLP)2-immunoreactive cells in the small intestine of the chicken. The formalin-fixed intestinal segments of jejunum and ileum were dehydrated in xylene, embedded in paraffin, and 4 μm tissue sections were prepared. Paraffin was removed, and the samples were rehydrated. After incubation with 0.5% antigen retrieval agent, the horseradish peroxidase-labeled streptavidinbiotin method was used (Sigma-Aldrich, Overijse, Belgium). The samples were incubated with 10% normal goat serum (Invitrogen, Merelbeke, Belgium) for 20 min before 24 h incubation with rabbit anti-human-GLP2-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-2immunoreactive 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 enteritis lesions in previous in vivo trials (Gholamiandehkordi et al., 2007; Timbermont et al., 2009). Bacteria were grown in Brain Heart Infusion broth (Oxoid, Basingstoke, 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, Brussels, 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 (Laboratorios Hipra, Amer, Spain) containing oocysts from precocious lines of Eimeria acervulina, Eimeria maxima, 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 different treatment groups. Dietary treatment groups were a

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positive control (no feed additive, and challenged with C. perfringens), a negative control (no feed additive and no challenge with C. perfringens), a group receiving 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 concentrations (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, assigned 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 randomly 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 experimental procedures were as described previously by Gholamiandehkordi et al. 2007, with some modifications. 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 coccidiosis vaccines at d 14 (Hipracox) and d 16 (Paracox5). At d 18, 19, and 20, all birds (except for the negative 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 duodenum, 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 objectively, 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 dietary 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 measurements, and density of GLP-2-immunoreactive cells were assessed by one-way analysis of variance (ANOVA) using 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 effect, 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 statistically analysed using one-way ANOVA using the same model as SCFA data, where for the first trial Yij represents 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 treatment (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, interaction between treatment and sex was observed. For the male birds, a significant difference in body weight, growth and FCR was observed comparing GVA

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VALERIC ACID GLYCERIDE AS FEED ADDITIVE 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 Parameters D1–11∗ FCR M+F FCR M FCR F BW (g) M+F BW (g) M BW (g) F FI (g/d) M+F FI (g/d) M FI (g/d) F WG (g/d) M+F WG (g/d) M WG (g/d) F d 12–31 FCR BW (g) FI (g/d) WG (g/d) d 32–37 FCR BW (g) FI (g/d) WG (g/d) d 1–37 FCR BW (g) FI (g/d) WG (g/d)

Glyceride Ester of Valeric Acid

Pooled SEM

P-Value

Mean

SD

Mean

SD

1.20 1.23 1.18 319.0 326.5 311.5 30.8 32.3 29.2 25.6

0.05 0.07 0.01 9.9 3.3 8.5 2.1 1.9 0.8 0.9

1.16 1.15 1.19 323.0 336.0 310.0 30.2 31.0 29.3 25.9

0.03 0.02 0.03 14.8 4.3 3.7 1.0 0.5 0.4 1.3

0.03 0.04 0.02 8.90 2.71 4.64 1.16 0.98 0.45 0.79

0.0286 0.8340 0.0102 0.6559 0.3065 0.8857 -

26.2 24.9

0.3 0.7

27.1 24.8

0.3 0.3

0.21 0.38

0.0286 0.3429

1.52 1886.0 119.8 78.4

0.04 168.8 11.2 8.01

1.48 1942.0 119.4 81.0

0.05 203.4 10.5 9.5

0.02 93.45 5.42 4.39

0.0052 0.0138 0.7888 0.3282$

1.65 2574.0 187.3 113.5

0.04 268.5 21.9 16.05

1.63 2652.0 191.9 117.9

0.07 297.2 21.0 16.8

0.06 283.21 21.45 16.43

0.1089 0.0212 0.1815 0.2786$

1.53 2574.0 104.7 68.6

0.04 286.5 10.6 7.25

1.48 2652.0 104.2 70.7

0.05 297.2 8.8 8.0

0.02 145.95 4.87 3.82

0.0211 0.0212 0.7147 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 increased 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 supplementation 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, propionate, butyrate, and valerate could be detected in the ceca of the 28-day-old broilers. In Table 5, mean values of these SCFAs are shown, indicating no differences between the chickens fed with and without GVA supplement. 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.

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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 Acid

Control Parameters Jejunum Villus height Crypt depth Villus height/crypt depth

Mean (μ m)

SD

Mean (μ m)

SD

911 176 5.58

369 82 2.01

977 145 6.76

354 39 1.83

Pooled SEM

P-value

147.61 26.21 0.78

0.0964 0.0013 < 0.0001

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 SCFA

Glyceride Ester of Valeric Acid

Mean (mM)

SD

Mean (mM)

SD

39.08 7.38 8.49 1.29

8.38 4.66 1.60 0.40

41.57 5.27 8.64 1.13

8.29 3.46 1.24 0.46

Acetate Propionate Butyrate Valerate

Pooled SEM

P Value

3.40 1.68 0.58 0.18

0.4555 0.3068 0.9151 0.6441

Densit y of G LP -2 imm unor ea ctive cells 2 (cells/m m )

SCFA, Short Chain Fatty Acid.

400

Control

(low GVA regimen: P = 0.0103, and high GVA regimen: P = 0.0011) (Table 6). In trial 2, lower concentrations of GVA were tested, which resulted in a decrease in the number of chickens with lesions of NE, but the mean lesion score was not reduced significantly. However, body weight increased significantly (P < 0.0001) in the second trial for the groups that received feed supplemented with lower concentrations of GVA (691.1 g and 71.04 g vs 622.2 g; Table 6).

GVA

300

200

100

0

J ejunum villi

J ejunum cr ypts Ileum villi

DISCUSSION

Ileum cr ypts

Figure 1. Density of GLP-2 immunoreactive cells in the small intestine of 10-day-old chickens fed a diet either or not supplemented with glyceride ester of valeric acid GVA (5 g/kg). Each dietary treatment consisted of 3 pens of 4 broilers. The bar charts showing mean values of GLP-2-immunoreactive cells per 1 mm2 of mucosa, divided in villi and crypts per segment. Error bars indicate standard deviation. The differences were considered statistically significant at ∗ P ≤ 0.05.

Protection Against Necrotic Enteritis Challenge by Valeric Acid Glycerides Glyceride Esters of Valeric Acid Can Reduce the Number of Chickens with Lesions of NE In trial 1, the number of chickens with lesions of NE was decreased in both groups supplemented with different concentrations of glyceride derivatives compared to the positive control group. There was a reduction from 71% animals with NE lesions in the positive control group to, respectively, 26.7 and 35.5% in the supplemented groups. The mean lesion score for the GVA supplemented groups was decreased compared to the positive control group

To the best of our knowledge, this is the first study demonstrating beneficial effects of valeric acid on intestinal health parameters and performance in broiler chickens. Moreover, higher concentrations partially protected the birds from necrotic enteritis in a severe challenge model. Valeric acid is naturally produced by the microbiota in the lower gastrointestinal tract. Most of the valeric acid producing microorganisms in the intestine belong to the genus Oscillibacter (Lino et al. 2007). Valeric acid is a C5 fatty acid and, as such, its chain length is in between the short-chain fatty acids (C1 to C4) and the medium chain fatty acids (C6 to C12). Beneficial effects on broiler performance have been reported for C3 (propionate), C4 (butyrate), as well as for the MCFA (Bintvihok and Kositcharoenkul, 2006; Namkung et al., 2011; Zentek et al., 2012; Khosravinia, 2015). The current study showed an improved feed efficiency for broilers fed with GVA compared to those fed a control diet (Table 3). One of the parameters influencing feed

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VALERIC ACID GLYCERIDE AS FEED ADDITIVE

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 (%)

Group Trial 1 Negative control Positive control Low High Trial 2 Positive control Low High

Inclusion level of C. perfringens glyceride ester of valeric acid (g/kg) challenge

no yes yes yes

yes yes yes

Lesion score

Mean

SD

Mean

SD

Pooled SEM

0 0 resp. week 1,2,3 2.5, 2.0, 1.5 resp. week 1,2,3 5.0, 2.5, 2.0

10.3 71.0 26.7

– – –

0.4138 1.742 0.7000

1.240 1.365 1.317

35.5

–

0.9355

0 0.5 1.5

36.1 27.2 25.4

12.7 2.9 8.4

0.7429 0.5571 0.5211

Body weight (g) Pooled SEM

P value

Mean

SD

P value

0.50 0.50 0.50

< 0.0001

108.0 83.6 135.4

0.7101

0.0011

714.0 738.8 686.9

1.482

0.50

0.0103

766.6

104.1

0.6212

1.031 0.9268 0.908

0.31 0.31 0.31

0.2848 0.1904

622.2 710.4 691.1

91.0 83.9 83.6

< 0.0001 < 0.0001

0.1610

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 decreasing 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 absorption, 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 inflammation 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). Moreover, a study by Lam et al. (2012) describes that a valeric acid producing strain is associated with dietinduced 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 hormones playing an important role in the gastrointestinal tract, exerting diverse actions including enhancing cell differentiation and stimulation of intestinal growth (Guan et al., 2006). GLP-2 is produced by enteroendocrine 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 binding to receptors (GLP-2R) located on enteric neurons, enteroendocrine cells (Guan et al., 2006) and subepithelial myofibroblasts (de Heuvel et al., 2012). These cells subsequently produce multiple downstream mediators 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, increased expression of intestinal tight junction proteins (Cani et al., 2009) and regulation of the innate immune system by controlling the expression of antimicrobial peptides which are implicated in the maintenance 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 dietary change can influence the density of gastrointestinal 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 production, thereby suggesting that preproglucagon (precursor of GLP-1 and GLP-2) production is related to an expanded L-cell population (Aranias et al., 2015; Catry et al., 2017). It can be hypothesized that valeric acid increases the density of GLP-2 cells and thereby GLP-2 production in the intestine of chickens, which stimulates intestinal growth, and in this way, improves broilers gut health and performance. However, as the density 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

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8

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 effects might also have resulted in protection in the subclinical NE model in our study. Subclinical NE caused by C. perfringens can adversely affect growth and feed conversion rate (Lovland and Kaldhusdal, 2001). Previously 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 animals with lesions was observed with the low regimen and high regimen doses of GVA fed. At lower concentrations of GVA, the mean score of necrotic enteritis lesions was not significantly reduced, but a positive effect 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 producing L-cells, and reduce the incidence of necrotic enteritis. The effect of GVA supplementation on L-cell density may play a key role in the observed beneficial 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 performance data, and to compare treatment groups with different concentrations of GVA. Further research is thus needed to unravel the mechanism underlying the observed 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 assisted in the sampling of the in vivo trials. This work received the financial support from Perstorp 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.

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