Effect of different doses of coated butyric acid on ...

Effect of different doses of coated butyric acid on growth performance and energy utilization in broilers S. A. Kaczmarek,∗,1 A. Barri,† M. Hejdysz,∗ and A. Rutkowski∗ ∗

Department of Animal Nutrition and Feed Management, Poznan University of Life Sciences, Woly´ nska 33, 60–637 Pozna´ n, Poland; and † Animal Nutrition and Health EMEA Toekomstlaan 42, Herentals, Belgium corrected for nitrogen (AMEN ) were improved after BP supplementation (P < 0.05). In experiment II, A or AB diets improved (P < 0.05) body weight gain compared to the control treatment. The diets Av, BP, and AvB improved (P < 0.05) feed conversion ratio compared to the control treatment. Birds from the treatment diet were characterized by having the thickest mucosa (P < 0.05). On days 14, 35, and 42, the use of AB diets improved AMEN content compared to the control treatment (P < 0.05). The apparent ileal digestibility of amino acid data showed that Av or AvB treated birds were characterized by higher Asp, Glu, Cys, Gly, and Ala ileal digestibility than the control animals (P < 0.05). The use of Av, BP, or AvB increased ileal digestibility of Thr, Ser, and Pro (P < 0.05). There is an indication that BP, alone or in combination with avilamycin, improve the digestion and absorptive processes and consequently birds performance results.

Key words: avilamycin, broiler chicken, calcium butyrate, ileal digestibility, metabolizable energy 2016 Poultry Science 00:1–9 http://dx.doi.org/10.3382/ps/pev382

INTRODUCTION

growth in villi height increases the absorptive surface of the small intestine leading to better nutrient utilization, which is why recent studies have shown that when B is orally ingested it can have beneficial effects on animal performance (Bergman, 1990; Smulikowska et al., 2006; Biagi et al., 2007; Hu and Guo, 2007; Mazzoni et al., 2008; Czerwi´ nski et al., 2012). Short chain fatty acids are quickly and quantitatively absorbed and metabolized by mucosa cells, so when given orally, its absorption and metabolization starts in the crop’s mucosa, in case of birds or in the oral cavity in case of mammals, and may continue throughout the whole gastrointestinal tract lining, limiting the amount of B that reaches the small intestine, restricting its practical use in animal production. Consequently, protection of B through microencapsulation, such as MicroPEARL technology (Kemin, Herentals, Belgium), improves B efficacy. MicroPEARL technology helps prevent the rapid absorption of B in the upper gastrointestinal tract (GIT), and thus its utilization far away from the action site of interest, thereby increasing the region exposed to the molecule (Smith et al.,

Butyric acid, a short-chain fatty acid produced by microbial fermentation, primarily in the large intestine, is known to be involved in the mucosal immune response, and to have an anti-inflammatory effect in animals. It is also known to have a direct effect on mucosal cell proliferation, maturation and differentiation, because it can influence gene expression and protein synthesis (Sengupta et al., 2006). It is certain that butyric acid, while it is a very simple molecule, it can have very complex and diverse modes of action. Because of its very pungent smell, butyric acid is commonly used in its butyrate (B) form (calcium or sodium salt). After butyrate supplementation, an increase in villi height and crypt depth has been observed in pigs and poultry (Galfi and Bokori, 1990; Leeson et al., 2005). The

 C 2016 Poultry Science Association Inc. Received July 1, 2015. Accepted October 27, 2015. 1 Corresponding author: [email protected]

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ABSTRACT We recently applied four dietary treatments in experiments I and II to determine the effect of protected calcium butyrate (BP) on growth performance and nutrient digestibility in broiler chickens. A group of one-day-old male Ross 308 broiler chicks (total 960, 480 per trial) were used in the study. In experiment I, the basal diets were fed with protected BP inclusion (0.2, 0.3, or 0.4 g/kg of finished feed) (BP) or without (C). In experiment II, 4 different diets were tested: 1) basal diet with no supplementation (C), 2) basal diet supplemented with protected BP (0.3 g/kg) (BP), 3) basal diet supplemented with avilamycin (6 mg/kg, active substance) a common antibiotic growth promoter (AGP) (Av), and 4) basal diet supplemented with the combination of both avilaymicin and BP. In experiment I, considering the entire study period, the use of BP improved feed conversion ratio (P < 0.05) irrespective of the dose. Apparent total tract crude fat digestibility and apparent metabolizable energy

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KACZMAREK ET AL.

MATERIALS AND METHODS All animal procedures were conducted in accordance with the guidelines of the Polish Council of Animal Care. The protocol for this study was approved by the Local Animal Care Committee of Poznan University of Life Sciences.

Bird Management and Sample Collection Four dietary treatments were used in experiments I and II to investigate the effect of protected butyrate on growth performance and nutrient digestibility in broiler chickens. One-day-old male Ross 308 broiler chicks

(total 960, 480 per trial) were obtained from a local hatchery. The birds were individually weighed and allocated to 10 weight classes. Groups of 10 birds were then randomly assigned to pens in such a way that the average initial bodyweight of birds was similar across pens. Ten replicate pens of 10 birds each were randomly assigned to four dietary treatments. All diets were fed in pelleted form throughout the 6-week experimental period. The birds were held in pens and had ad libitum access to water and feed. Body weight and feed intake (FI) were monitored weekly with the pen as the experimental unit. Before weighing, the birds were fasted for 4 h. Mean weight gain, FI, and feed-to-gain ratio were used to determine the growth performance. On day 35 (experiment I) and on days 14, 35, and 42 (experiment II), excreta samples from each pen were collected over a 3 h period and subsequently frozen, freeze-dried, and finely ground. Collection trays were installed in floor pens to allow for excreta collection; ten excreta samples per treatment were collected. On day 35 (experiments I and II), 10 birds from each treatment were randomly selected and killed via cervical dislocation (Close et al., 1997). The contents of the terminal ileum (15 cm, adjacent to the ileo-cecal junction) were collected. The digesta samples from 3 birds within each treatment were pooled to provide sufficient material for chemical analysis. The digesta samples were frozen, freeze-dried, and ground before further analyses. Additionally, samples (n = 8) of the whole intestinal tract were removed, and segments of approximately 2 cm were collected. A segment of the ileum was washed in physiological saline solution and fixed in 10% buffered formalin.

Experimental Diets The diets used in experiments I and II are presented in Table 1. Starter (1 to 14 d) and grower (14 to 42 d) diets were corn-soybean meal (SBM) and were formulated to meet or exceed Aviagen recommendations for broiler chickens (Aviagen, 2009). In experiment I, the basal diets were fed with or without protected (MicroPEARL encapsulation using hydrogenated palm oil) calcium butyrate (BP) (ButiPEARL, Kemin) inclusion (0.2, 0.3, or 0.4 g/kg). According to the manufacturer’s declaration, ButiPEARL contained 50% BP. Protected BP was added to the basal diet substituting corn. All grower diets were supplemented with titanium dioxide (3.0 g/kg) which was used as an internal marker to calculate the apparent total tract digestibility (ATTD) of crude fat, and to determine diet AMEN values. In experiment II, the basal diets were fed with (BP) or without (C) protected BP (0.3 g/kg), (ButiPEARL, Kemin) or with avilamycin (Av), (6 mg/kg, active substance), (Maxus, Elanco) or with a combination of both (AvB). Protected BP and avilamycin were added

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2012). Targeting the release of B in the small intestine results in positive effects on gut morphology and by consequence function (Jerzsele et al., 2012) and, in addition, may reduce pathogenic bacteria colonization (Van Immerseel et al., 2005). Accordingly, several experiments have shown that administering low doses of protected B to healthy animals result in enhanced animal performance (BW = Body weight; FCR = Feed conversion ratio) through the positive histomorphological changes on the intestinal mucosa as explained above. Additional to the already mentioned benefits of the MicroPEARL technology, it is of interest to some producers to find out if it would be possible to give a matrix value to B in poultry diet formulation, allowing for more economical diets. Nonetheless, there is limited information on the effect of protected B on the AME of diets or crude fat and amino acid (AA) availability in broiler chickens. The incomplete literature data available have shown that B may improve total tract digestibility, but not ileal nutrient digestibility in pigs (Le Gall et al., 2009). In contrast, Manzanilla et al. (2006) reported that B reduced ileal digestibility of starch in weaned pigs. This study will help build up the existent knowledge on B regarding its nutritional benefits. Avilamycin (Av) is a widely used antibiotic growth promoter (AGP) belonging to the ortosomycine class of antibiotics, which shows antibacterial activity against gram-positive bacteria. Available literature data report that a bird’s performance could be improved after avilamycin supplementation due to the decreased microbial burden in the GIT and reduced susceptibility to disease (Pedroso et al., 2006; Chowdhury et al., 2009; Kim et al., 2011). Despite the ban of AGP in Europe and a few countries in North Africa, many different countries across the world continue to use these as a common poultry and livestock production practice, and thus, it is commonly used as a control treatment in trials where alternatives for antibiotic growth promoters are tested. The aim of this study was to investigate the effect of a MicroPEARL protected B, alone or in combination with avilamycin, on diet digestibility, AMEN , birds’ performance and ileum histomorphology This study consists of the results of two trials.

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COATED BUTYRIC ACID IN BROILER CHICKEN NUTRITION Table 1. Composition of basal diets and their nutritional value (experiments I and II). Experiment I 1 to 14 d 1

Gross energy [kcal/kg] Crude protein Crude fat

AMEN [kcal/kg]4 Crude protein available P Ca Available Lys Available Met + cys

52.892 38.548 4.759 1 0.937 0.633 0.360 0.278 0.066 0.408 0.099 0.02 –

– – –

14 to 42 d

1 to 14 d

14 to 42 d

57.324 52.492 57.324 33.456 38.548 33.456 5.783 4.759 5.783 1 1 1 0.748 0.937 0.748 0.369 0.633 0.369 0.295 0.360 0.295 0.227 0.278 0.227 0.218 0.066 0.218 0.194 0.408 0.194 0.066 0.099 0.066 0.02 0.02 0.02 0.3 0.3 0.3 Determined nutritional value (% unless otherwise noted) 4,395 4,260 20.9 23.2 7.66 7.37 Calculated nutritional value (% unless otherwise noted)

3,033 22.9 0.5 1.05 1.27 0.94

3,129 21.0 0.45 0.9 1.1 0.84

3,033 22.9 0.5 1.05 1.27 0.94

4,327 20.9 8.42

3,129 21.0 0.45 0.9 1.1 0.84

1 Experiment I: ButiPEARL (0.2, 0.3 or 0.4 g/kg) was added to the basal diet substituting for corn. Experiment II: ButiPEARL (0.3 g/kg), Maxus 200 (0.03 g/kg), or both were added to the basal diet substituting for corn. 2 provides per kg diet: IU: vit. A 11250, cholecalciferol 2500; mg: vit. E 80, menadione 2.50, vit. B12 0.02, folic acid 1.17, choline 379, D-pantothenic acid 12.5, riboflavin 7.0, niacin 41.67, thiamin 2.17, D-biotin 0.18, pyridoxine 4.0, ethoxyquin 0.09, Mn 73, Zn 55, Fe 45, Cu 20, I 0.62, Se 0.3, salinomycin 60. 3 HiPhos NP. 4 Metabolizable energy was obtained by calculation.

to the basal diets substituting corn. All diets were supplemented with titanium dioxide (3.0 g/kg) which was used as an internal marker to calculate ATTD and ileal digestibility, and to determine the diet AMEN values. Diets were in pelleted form and were provided for ad libitum consumption. All diets were formulated to be iso-energetic and iso-nitrogenous.

Chemical Analyses Feed samples were analyzed in duplicate for crude protein, crude fat, crude fiber, and total P using AOAC (2005) methods 976.05, 920.39, 2002.04, and 965.17, respectively. For all chemical analyses, samples were ground to pass through a 0.5-mm sieve. Excreta and ileal digesta samples were also analyzed in duplicate. Prior to analyses, excreta samples were homogenized using a stomacher homogenizer (Interscience, SaintNom-la-Bret`eche, France), freeze-dried (Christ 1825 Medizinischer Apparatebau 326; Martin Christ GmbH, Osterode, Germany), and ground (1-mm screen). Titanium dioxide was determined according to the method of Short et al. (1996); the samples were prepared according to the procedure proposed by Myers et al. (2004). Gross energy was determined using an adiabatic

bomb calorimeter (KL 12 Mn, Precyzja-Bit PPHU, Poland) standardized with benzoic acid. Crude fat was determined according to AOAC (2005) method 920.39. Butyric acid concentrations in diets were determined by using the gas chromatography with flame ionization detection. The results were used to calculate the ButiPEARL inclusion levels in the starter and grower diets. The AA content was determined with an AAA400 Automatic Amino Acid Analyzer (Ingos Ltd., Prague, Czech Republic) using ninhydrin for postcolumn derivatization. Before analyses, the samples were hydrolyzed with 6 N HCl for 24 h at 110◦ C (procedure 994.12; AOAC (2005)). Methionine and cystine were determined as methionine sulfone and cysteic acid after cold performic acid oxidation before hydrolysis (procedure 994.12, alternative 3; AOAC, 2005).

Histomorphological Evaluation Histomorphological evaluation included determination of the villi height (VH), crypt depth (CD) and mucosa thickness (MT). Fixed segments (8 per treatment) in 10% neutral buffered formalin solution were rinsed in ethyl alcohol and embedded in paraffin wax. Ileum samples were cut using a microtome A HM 340E (Thermo Scientific, MA, USA), with 5 slices per ileum

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Corn SBM (44% CP) Soy oil Premix (Ca 38%)2 MCP Limestone DL-met L-lys NaCl NaHCO3 L-thr HiPhos NP3 TiO2

Experiment II

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KACZMAREK ET AL.

sample and stained using the haematoxylin and eosin method. Samples were analyzed (in duplicate) using a light microscope (Axioimager, Carl Zeiss, Germany). If slices were characterized by the correct structure (no damage), they were scanned using Mirax Desk (Carl Zeiss, Germany). Measurements (in duplicate) were performed using AxioVision software (Carl Zeiss, Germany) for each structure per slice. The villus length was measured from the villus tip to the bottom, excluding the intestinal crypt. Measurements of mucosa thickness were done from the tip of the villi to the muscularis mucosa. Five villi, crypts and mucosa were measured per sample, and the mean was calculated.

Ileal CP digestibility, crude fat, apparent total tract digestibility, and AMEN values of the diets were calculated relative to the ratio of TiO2 to the content of the nutrient in question in the feed or excreta according to the following formula (crude protein as an example): 

Digestibility (%) = 1 −



IM% diet /IM% digesta/ excreta



× CP% digesta/ excreta /CP% diet



 

where; IM is the internal marker (titanium oxide) Total tract AME was corrected to zero nitrogen balance using 8.22 kcal/kg N retained (Hill and Anderson, 1958) according to the following formula: AMEN (kcal/kg) = GEkcal/ kg diet 

− GEkcal/ kg excreta × (IM% diet /IM% excreta )



−8.22 × {N% diet − [N% excreta × (IM% diet /IM% excreta )]} where: GE is gross energy, N is nitrogen, IM is internal marker (titanium oxide), and 8.22 is the energy equivalent of uric acid nitrogen (i.e. 8.22 kcal/kg uric acid nitrogen). All data had earlier been explored to discard any possible outliers. The analysis was performed using the appropriate procedures (distribution analyses) within SAS Software 9.2 (SAS Institute Inc., 2009, NC, USA). Individual values were considered outliers from the whole population (the 4 groups together) when they were outside the range defined as the mean ±3 standard deviations. Individual outliers represented 0 to 5% of the whole population. Experiments I and II were of completely randomized design with the pen as the experimental unit. For histomorphological investigations, an individual randomly selected bird was the experimental unit. The results were subjected to one-factorial analysis of variance using the GLM procedure (PROC GLM) within SAS. In experiment I, means were separated using Bonferroni’s adjustments for multiple comparisons, while in

RESULTS Experiment I There were numerical differences (P > 0.05) in body weight gain (BWG) of birds given BP supplementation. The chickens fed with 0.3 g BP/kg feed had 125 g higher BWG when compared to the control birds, than the birds fed with 0.2 or 0.4 g/kg of feed, who had a BWG of 80 g and 83 g, respectively. The highest dose (0.4 g/kg) of BP reduced FI, (P < 0.05) during the starter period, but 0.4 g/kg of BP showed no effect in the course of the whole experiment. Feed conversion ratio was affected by BP supplementation. The highest and moderate doses of BP improved feed conversion compared with the control treatment (P < 0.05) during the first 2 weeks of the experiment. Considering the entire study period, the use of BP improved FCR (P < 0.05) irrespective of the dose. Apparent total tract crude fat digestibility (Table 2) was improved after BP supplementation (P < 0.05); similar differences were observed when AMEN of the diet was determined (P < 0.05). Diets with BP were characterized by higher AMEN values than the control treatment (P < 0.05). Histomorphological evaluations (Table 3) showed that microencapsulation made a difference and the addition of BP had a positive effect (P < 0.05) on VH. Birds fed diets supplemented with BP were characterized by higher VH than that of un-supplemented birds (P < 0.05). At the lowest dose (0.2 g/kg), the difference was over 150 μm compared to the control treatment (P < 0.05). The MT, CD, and VH/CD were unaffected.

Experiment II Butyric acid content in starter feed samples were as follows: BP – 0.146; AvB – 0.163 g/kg. In treatments C and A, BP content was below the detection limit. In grower feeds: BP – 0.155; AvB – 0.180 g/kg and below detection limit in C and A feeds. The BP concentration calculated in supplemented starter diets was as follows: 0.26, 0.29 g/kg – BP and AvB diets, respectively. In grower diets, the calculated ButiPEARL concentrations were: 0.27 and 0.28 g/kg in BP and AvB diets. ButiPEARL concentration in un-supplemented diets (C and Av) was below the detection limit in starter and grower feeds. The protected BP had a numerical effect on BWG (17 g; P > 0.05) in the first 2 weeks of the experiment. Protected butyrate alone or in combination with Av improved (P < 0.05) BWG compared to the control treatment. A tendency (P < 0.1) for improved BWG (14 to 42 Av) was observed after BP or Av supplementation,

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Calculations and Statistical Analyses

experiment II, Dunnett’s post-hoc test was used. Significance in both trials was based on P < 0.05. In the experiments, the standard error of the mean (SEM) was adopted as the measure of error.

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COATED BUTYRIC ACID IN BROILER CHICKEN NUTRITION Table 2. Broiler chicken performance, crude fat (CF) apparent total tract digestibility (ATTD), and diet AMEN content of diets either supplemented or not with protected butyric acid (BP) (experiment I). BWG

FI [g]

FCR [g:g]

ATTD

BP dose [g/kg]

1 to 14 d

1 to 42 d

1 to 14 d

1 to 42 d

1 to 14 d

1 to 42 d

CF [%]

AMEN [kcal/kg]

0 0.2 0.3 0.4

344 363 354 357

2,616 2,696 2,741 2,699

512 511 475 467∗

4,336 4,409 4,348 4,207

1.48 1.41 1.31∗ 1.31∗

1.66 1.64∗ 1.59∗ 1.58∗

82.8 86.9∗ 85.5∗ 86.9∗

3,136 3,358∗ 3,373∗ 3,263∗

SEM Linear response

2.50 0.051

20.1 0.165

5.97 0.0042

32.5 0.166

0.0176 0.0002

0.0085 0.0009

0.4658 0.003

18.36 < 0.0001

∗

Means were significantly different compared to the control (P < 0.05).

BP dose [g/kg]

Mucosa thickness

Villous height [μ m]

Crypt depth

VH/CD

0 0.2 0.3 0.4

1,191 1,231 1,300 1,223

903 1,054∗ 1,083∗ 1,061∗

209 212 222 217

4.38 5.14 5.20 4.98

SEM Linear response

20.88 0.334

19.57 0.001

6.31 0.909

0.165 0.285

∗

Means were significantly different compared to the control (P < 0.05).

Table 4. Broiler chicken performance (BWG, FI, and FCR) (1 to 42 day) (experiment II).

BWG (g) 1 to 14 d 15 to 42 d 1 to 42 d FI (g) 1 to 14 d 15 to 42 d 1 to 42 d FCR (g:g) 1 to 14 d 15 to 42 d 1 to 42 d

C

BP

Av

AvB

SEM

P

306b 1,817 2,123b

323a,b 1,999 2,323a

332a 2,024 2,356a

332a 2,029 2,361a

3.86 33.3 35.3

0.043 0.070 0.046

452 2,903 3,355

460 3,000 3,460

461 3,054 3,516

472 3,025 3,496

5.64 41.3 43.8

0.070 0.615 0.590

1.48a 1.60a 1.58a

1.42a,b 1.50b 1.49b

1.39b 1.51b 1.49b

1.42a,b 1.49b 1.48b

0.011 0.012 0.011

0.035 0.001 0.001

C, Control treatment; BP, ButiPEARL; Av, avilamycin; AvB, ButiPEARL + avilamycin. a,b Means with different superscripts within a row were significantly (P < 0.05) different.

either separately or in combination (Table 4). Similar differences (P < 0.05) between Av and BP were observed through the whole trial period (0 to 42d). Feed intake was not affected by BP or Av during the whole experiment. A tendency (P < 0.1) for higher FI was observed after the use of combination of BP and Av, being less in BP. Birds fed control diets or diets supplemented with BP or AvB were characterized by similar FCR during the first 2 weeks of the experiment. However, the use of BP improved (P < 0.05) FCR over the entire grow-out period, compared to the control treatment. To a similar extent, Av, BP and the combination of both improved (P < 0.05) FCR compared to the control treatment (Table 4). Histomorphological evaluations (Table 5) showed that MT, CD, and VH/CD were affected by the supple-

mentation with BP or Av. Birds from the control treatment were characterized by having the thickest mucosa (P < 0.05). The thinnest mucosa was observed when birds were fed the diet supplemented with the combination of BP and Av (P < 0.05). Villi height were not affected when BP or Av were used separately; however, animals fed BP showed numerically higher villi than the controls. Birds given the mixture of BP and Av consistently showed lower villi compared to the control treatment (P < 0.05). Crypt depth decreased in the ileum for all treated animals compared to the control birds (P < 0.05). The ileal digestibility of crude protein increased after BP, Av, or the combination of both (P < 0.05). Similar differences were observed in ileal crude fat digestibility, but significant differences (P < 0.05) were noted

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Table 3. Ileum mucosa thickness, villi height (VH), crypt depth (CD), and VH to CD ratio of broiler chickens fed diets either supplemented or not with protected butyric acid (BP) (wxperiment I).

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KACZMAREK ET AL. Table 5. Mucosa thickness, villous height, crypt depth, and villous height: crypt depth [μm] of ileum in broiler chickens (experiment II).

Mucosa thickness Villous height Crypt depth VH/CD

C

BP

1210a 835a 204a Ratio 4.21b

981b 918a 156b

Av

AvB

SEM

P

895b 813a 176b

837b 721b 154b

30.07 17.02 5.36

< 0.0001 0.0013 0.0014

5.06a,b

4.85a,b

0.171

0.0027

[μ m]

5.91a

C, Control treatment; BP, ButiPEARL; Av, avilamycin; AvB, ButiPEARL + avilamycin; VH/CD, villous height to crypt depth ratio. a,b Means with different superscripts within a row were significantly (P < 0.05) different.

Table 6. Ileal apparent digestibility of crude protein, total tract crude fat digestibility, and AMEN of diets on days 14, 35, and 42 (experiment II). BP

Av

AvB

SEM

P

Ileal crude protein digestibility at day: 14 69.6b 78.1a

78.8a

78.2a

1.08

0.0024

AMEN of diet at day: 14 3088b 35 3105b 42 3241b

3148b 3320a 3348a

3243a 3247a 3333a

10.8 15.5 11.9

0.0002 0.0001 0.0005

95.6a 89.2 96.0a

94.4a 85.5 95.9a

0.26 1.22 0.533

0.0019 0.1017 0.0085

3154b 3235a 3305a,b

Total tract crude fat digestibility at day: 14 92.5b 95.4a 35 82.4 84.7 42 92.0b 90.8b

C, Control treatment; BP, ButiPEARL; Av, avilamycin; AvB, ButiPEARL + avilamycin. a,b Means with different superscripts within a row were significantly (P < 0.05) different.

only between the control and avilamycin treated animals. Apparent metabolizable energy was determined on days 14, 35, and 42 of the experiment and this is presented in Table 6. On day 14, the use of BP and Av in combination improved AMEN content compared to the control treatment (P < 0.05) and the difference was over 150 kcal/kg. Considering days 35 and 42 of the experiment, determined AMEN increased after Av, BP, or AvB supplementation compared to the control treatment (P < 0.05). BP, Av, or AvB treated birds were characterized by higher total tract crude fat digestibility on days 14 and 35. On day 42, Av and AvB treated birds were different from the control (P < 0.05). The most pronounced improvement was noted after Av supplementation. Apparent ileal digestibility of AA is presented in Table 6. Avilamycin alone or AvB treated birds were characterized by higher Asp, Glu, Cys, Gly, and Ala ileal digestibility than the control animals (P < 0.05). The use of Av, BP, or AvB increased the ileal digestibility of Thr, Ser, and Pro (P < 0.05). Valine, Ile, Leu, Tyr, and His ileal digestibility was improved compared to the control after Av addition (P < 0.05) (Table 7).

DISCUSSION Previous research performed in our laboratory has shown that some organic acids may be used as growth stimulators and may affect poultry GIT microflora (Jozefiak et al., 2010). Also, other researchers have

Table 7. Ileal apparent digestibility of amino acids on day 35 (experiment II).

Asp Thr Ser Glu Pro Cys Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg

C

BP

Av

AvB

SEM

P

71.9b 61.0c 71.6b 82.8 72.8b 67.1b 66.6b 76.8b 74.4b 85.4 77.3b 75.6b 72.4b 81.0 72.8b 82.4 85.3

76.3a,b 68.9b 78.4a 84.5 77.6a 74.3a,b 72.7a,b 80.6a,b 78.4a,b 83.8 80.9a,b 79.2a,b 76.7a,b 82.5 78.4a 84.2 87.1

79.8a 74.0a,b 80.8a 87.2 79.5a 78.2a 76.2a 83.7a 82.1a 87.0 84.5a 82.7a 81.1a 85.7 81.5a 87.6 89.5

78.1a 75.3a 78.4a 85.0 79.0a 75.3a 74.6a 80.3a,b 78.7a,b 85.0 81.7a 79.3a,b 76.4a,b 83.0 78.1a 83.6 86.1

0.877 1.16 1.17 0.73 0.77 1.44 1.23 1.13 1.17 0.62 1.07 1.09 1.37 0.93 1.09 1.06 0.86

0.002 < 0.001 < 0.001 0.086 0.004 0.009 0.008 0.016 0.01 0.239 0.005 0.01 0.04 0.075 0.002 0.119 0.102

C, Control treatment; BP, ButiPEARL; Av, avilamycin; AvB, ButiPEARL + avilamycin. a–c Means with different superscripts within a row were significantly (P < 0.05) different.

shown that organic acids may influence GIT microbiota as well as birds’ performance, carcass yield, and nutrient digestibility (Leeson et al., 2005; Van Immerseel et al., 2005; Hu and Guo, 2007; Jerzsele et al., 2012). In the present studies, B affected FCR and BWG positively in both experiments. Likewise, Leeson et al. (2005) reported that 0.4 g/kg of B (in the form of a glyceride) improved FCR mostly during the first 3 weeks of the birds’

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COATED BUTYRIC ACID IN BROILER CHICKEN NUTRITION

there is still room for improvement. It is worth remembering that even small changes in starch digestibility may result in higher AMEN values for a diet and consequently in an improvement of chickens’ performance. On the other hand, Manzanilla et al. (2006) reported that sodium butyrate caused a deterioration in ileal and total tract starch digestibility in piglets. The authors speculated that sodium butyrate had an inhibiting effect on amilolytic bacteria and, consequently, depressed starch utilization. Previous studies have shown that B has a strong trophic effect on the gastrointestinal epithelium. Butyrate ingestion is known to modify the microstructure of the small and large intestines in all animals. In the small intestine, B improves enterocyte proliferation, differentiation and maturation (Guilloteau et al., 2010). This is supported by our first study, where B supplementation affected VH (P < 0.05) and mucosa thickness (P > 0.05). The fact that mucosa thickness (MT) remained unaffected when B or Av where added implies that both Av and B are able to control intestinal inflammatory responses. This supports Niewold’s theory on the mechanism of action of antibiotic growth promoters (Niewold, 2007). In experiment II, although not statistical significant, numerical changes were noted for VH after the use of protected B. The research results of the present study agree with those of Galfi and Bokori (1990), where sodium butyrate increased the length of the villi. The ratio between villi length and crypt depth is recognized as an important parameter for intestinal health. A high ratio indicates a long villus in which the epithelium is sufficiently matured and functionally active, in combination with a shallow crypt with constant cell renewal. Only numerical differences in the above ratio were recorded in experiment I and these could be attributed to the excellent experimental conditions and low GIT microbial pressure. Previously, Cook and Bird (1973) reported that germ-free chickens were characterized by shallower CD than conventional birds. In experiment II, significant differences were observed for CD after microencapsulated B addition. In combination with higher villi, this results in a higher CD/VH ratio. This may be linked with the good health status of the bird’s GIT. It can be said that the BP addition increased the intestinal absorptive area by increasing VH and, consequently, affecting crude fat and protein digestibility and improving diet AMEN . The indirect effect on VH could consist of increased pancreatic fluid secretion and, consequently, higher nutrient absorption. The use of avilamycin in poultry feed is common all over the world (except Europe and some North African countries). In our study, the application of Av either alone or in combination with B improved BWG and FCR (Kim et al., 2011; Zhang and Kim, 2014). It is suggested that avilamycin disrupts the GIT microbiota in the upper part of the GIT which, indirectly, affects birds’ performance (Knarreborg et al., 2002; Pedroso et al., 2006; La-ongkhum et al., 2011), since grampositive bacteria are the main target for avilamycin.

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life. In the same paper, the authors reported contradictory results for a 0.2 g/kg dose. In this experiment (I), the supplementation of 0.2 g/kg of B exerted a positive effect on FCR only when the entire trial was considered. The dose of 0.3 g/kg had a positive effect on FCR irrespective of the birds’ age. This can be attributed to the fact that in our experiment B was protected. The microencapsulation of B allowed for the targeted release of this compound at the ileum level and directly affected intestinal morphology, potentially the microbiota, and digestive processes in this section of the intestinal tract. As reported by Bolton and Dewar (1965), unprotected/ un-encapsulated butyric acid salts (butyrates) are also rapidly absorbed in the upper parts of the GIT, thus protection of the active ingredient is crucial for these to have a positive effect in the animal’s intestinal digestive and absorptive capacity. The results of experiment I revealed that 0.4 g/kg of microencapsulated B reduced feed intake had numerical effects on body weight gain, and had a significantly positive effects on FCR at 1 to 14 d and through the whole grow-out period. Namkung et al. (2011) and Pinchasov and Jensen (1989) reported that, in contrast to propionates and acetates, butyric acid and its glyceride forms could cause feed intake depression. In the second trial, there were no differences in FI after B supplementation (0.3 g/kg) either alone or in combination with Av. Improvements in performance traits could be partially explained by the nutrient utilization results that were determined in these studies. The use of B enhanced fat digestibility and, consequently, the AMEN in the diet of the first trial. In the second trial, the addition of B in the diets was found to lead to an increase in protein and ileal AA digestibility as well as crude fat total tract digestibility and, consequently, diet AMEN on days 14, 35, and 42. Biggs and Parsons (2008) reported that the use of gluconic acid yielded inconsistent Metabolizable energy (ME) results, while the application of citric acid improved ME of the diet and enhanced AA availability. On the other hand, Hu and Guo (2007) reported that sodium butyrate had no effect on the absorptive function of chicken jejunum determined in an in vitro system. However, studies performed on some other animals have shown that B administration increased pancreatic fluid, amylase, and dose-dependent secretion of trypsin (Katoh and Tsuda, 1987; Ohbo et al., 1996; Sileikiene et al., 2005). It was suggested by Katoh and Tsuda (1987) that short-chain fatty acids, including butyrate, may increase the cellular concentration of Ca2+ ions in the pancreatic acinar cells and, consequently, activate the processes causing fluid and amylase secretions. It can be speculated that an increase in pancreatic fluid secretion may improve fat, starch, and other nutrient digestibility and that this in turn, may increase diet AMEN . In the above studies, fat and protein digestibility were improved after B supplementation, but it seems appropriate to also study starch digestibility in future trials. Starch digestibility in corn-SBM diets is almost always high (Kaczmarek et al., 2014a,b), but

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ACKNOWLEDGMENTS This work was carried out thanks to the financial support provided by the Animal Nutrition and Health EMEA (Herentals, Belgium).

REFERENCES AOAC. 2005. Agricultural Chemicals; Contaminants; Drugs. Official Methods of Analysis Vol. 1. AOAC, International, Gaithersburg Maryland.

Aviagen. 2009. Ross Broiler Nutrition Supplement. Aviagen Ltd, Newbridge, UK. Available at http://en.aviagen.com/ [Verified 09 December 2014]. Bergman, E. N. 1990. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol. Rev. 70:567–590. Biagi, G., A. Piva, M. Moschini, E. Vezzali, and F. X. Roth. 2007. Performance, intestinal microflora, and wall morphology of weanling pigs fed sodium butyrate. J. Anim. Sci. 85:1184–1191. Biggs, P., and C. M. Parsons. 2008. The Effects of Several Organic Acids on Growth Performance, Nutrient Digestibilities, and Cecal Microbial Populations in Young Chicks. Poult. Sci. 87:2581–2589. Bolton, W., and W. A. Dewar. 1965. The digestibility of acetic, propionic and butyric acids by the fowl. Br. Poult. Sci. 6:103–105. Chowdhury, R., K. M. S. Islam, M. J. Khan, M. R. Karim, M. N. Haque, M. Khatun, and G. M. Pesti. 2009. Effect of citric acid, avilamycin, and their combination on the performance, tibia ash, and immune status of broilers. Poult. Sci. 88:1616–1622. Close, B., K. Banister, V. Baumans, E. M. Bernoth, N. Bromage, J. Bunyan, W. Erhardt, P. Flecknell, N. Gregory, H. Hackbarth, D. Morton, and C. Warwick. 1997. Recommendations for euthanasia of experimental animals: Part 2. DGXT of the European Commission. Lab Anim. 31:1–32. Cook, R. H., and F. H. Bird. 1973. Duodenal villus area and epithelial cellular migration in conventional and germ-free chicks. Poult. Sci. 52:2276–2280. Czerwi´ nski, J., O. Højberg, S. Smulikowska, R. M. Engberg, and A. Mieczkowska. 2012. Effects of sodium butyrate and salinomycin upon intestinal microbiota, mucosal morphology and performance of broiler chickens. Arch. Anim. Nutr. 66:102–116. Elwinger, K., E. Berndtson, B. Engstrom, O. Fossum, and L. Waldenstedt. 1998. Effect of antibiotic growth promoters and anticoccidials on growth of Clostridium perfringens in the caeca and on performance of broiler chickens. Acta Vet. Scand. 39:433–441. Galfi, P., and J. Bokori. 1990. Feeding trial in pigs with a diet containing sodium n-butyrate. Acta Vet. Hung. 38:3–17. Garc´ıa, V., P. Catal´ a-Gregori, F. Hern´ andez, M. D. Meg´ıas, and J. Madrid. 2007. Effect of Formic Acid and Plant Extracts on Growth, Nutrient Digestibility, Intestine Mucosa Morphology, and Meat Yield of Broilers. J. Appl. Poult. Res. 16:555–562. Guilloteau, P., L. Martin, V. Eeckhaut, R. Ducatelle, R. Zabielski, and F. Van Immerseel. 2010. From the gut to the peripheral tissues: the multiple effects of butyrate. Nutr. Res. Rev. 23:366–384. Hill, F. W., and D. L. Anderson. 1958. Comparison of metabolizable energy and productive energy determinations with growing chicks. J. Nutr. 64:587–603. Hu, Z., and Y. Guo. 2007. Effects of dietary sodium butyrate supplementation on the intestinal morphological structure, absorptive function and gut flora in chickens. Anim. Feed Sci. Technol. 132:240–249. Jerzsele, A., K. Szeker, R. Csizinszky, E. Gere, C. Jakab, J. J. Mallo, and P. Galfi. 2012. Efficacy of protected sodium butyrate, a protected blend of essential oils, their combination, and Bacillus amyloliquefaciens spore suspension against artificially induced necrotic enteritis in broilers. Poult. Sci. 91:837–843. Jozefiak, D., S. Kaczmarek, and A. Rutkowski. 2010. The effects of benzoic acid supplementation on the performance of broiler chickens. J. Anim. Physiol. Anim. Nutr. 94:29–34. Kaczmarek, S. A., A. J. Cowieson, D. J´ozefiak, and A. Rutkowski. 2014a. Effect of maize endosperm hardness, drying temperature and microbial enzyme supplementation on the performance of broiler chickens. Anim. Prod. Sci. 54:956–965. Kaczmarek, S. A., A. Rogiewicz, M. Mogielnicka, A. Rutkowski, R. O. Jones, and B. A. Slominski. 2014b. The effect of protease, amylase, and nonstarch polysaccharide-degrading enzyme supplementation on nutrient utilization and growth performance of broiler chickens fed corn-soybean meal-based diets. Poult. Sci. 93:1745– 1753. Katoh, K., and T. Tsuda. 1987. Effects of intravenous injection of butyrate on the exocrine pancreatic secretion in guinea pigs. Comp. Biochem. Physiol. A Comp. Physiol. 87:569–572. 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.

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These bacteria potentially compete for nutrients (Elwinger et al., 1998). Some gram-positive bacteria may produce enzymes that deconjugate bile salts, resulting in depressed fat emulsification and lipid absorption (Tannock et al., 1989). The observed increase in fat digestibility in both A supplemented treatments could be the result of growth inhibition of bile salts deconjugating bacteria. It could also be stated that the improvement in digestibility observed after Av supplementation could be related to reduction in microbial use of nutrients or/and enhanced nutrients’ absorption because of thinner intestinal wall (Niewold, 2007). Changes in nutrient utilization and ME level after Av addition have previously also been reported by Kirchgessner et al. (1994). Additionally, Garc´ıa et al. (2007) found a significant improvement in dry matter and crude protein ileal digestibility after avilamycin supplementation. We also found that an application improved AA apparent ileal digestibility. Also, Zhang and Kim (2014) found a numerical or significant improvement in the apparent ileal digestibility of some amino acids after avilamycin addition. These improvements could be attributed to a better health status of GIT or larger villi surface area. Kroismayr et al. (2008) found that in piglets the use of avilamycin affected ileal morphology, overall gastrointestinal health and productivity. They hypothesized that these changes could be reflected in concomitant changes in the mRNA expression of inflammatory and apoptotic marker genes. The use of AvB combination, had no additive effect on the investigated variables with the exception of AMEN at day 14. According to the authors’ knowledge, in the literature there are no data available on the effects of AvB combination in broiler diets on performance and nutrient utilization. Future studies are warranted to investigate the AvB combination in poultry diets to explain lack of additive response. The results of our studies indicate positive effects of feeding a low dose of up to 0.3 g/kg protected butyrate throughout the 42 d broiler growth period when the compound was used either alone or in combination with avilamycin. There is an indication that butyrate helped maintain intestinal integrity and, consequently, improve the digestion and absorptive processes. Future studies are warranted to investigate the dose response to protected butyric acid on enzyme secretion and starch utilization, as well as other nutrient digestibility in birds.

COATED BUTYRIC ACID IN BROILER CHICKEN NUTRITION

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