Effect of dietary butyric acid on performance ... - Livestock Science

Livestock Science 183 (2016) 78–83

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Effect of dietary butyric acid on performance, intestinal morphology, microflora composition and intestinal recovery of heat-stressed broilers Anas Abdelqader n, Abdur-Rahman Al-Fataftah Department of Animal Production, Faculty of Agriculture, The University of Jordan, Amman 11942, Jordan

art ic l e i nf o

a b s t r a c t

Article history: Received 18 August 2015 Received in revised form 26 November 2015 Accepted 27 November 2015

The study investigated the effect of butyric acid on heat-stressed broilers performance, intestinal histological changes, beneficial intestinal bacteria counts and recovery responses. One hundred and twentyeight Hubbard male broilers were equally distributed into 4 treatment groups, with 8 replicates per treatment (4 birds each). At 21 d of age, birds were assigned into 2 dietary treatments and fed either a control diet (CONTR) or the control dietþ 0.5 g/kg butyric acid (BUT). Each dietary treatment was further divided into 2 experimental groups; thermoneutral (TN) or heat stress (HS), each of which included one group fed with CONTR and one fed with BUT. The TN-CONTR and TN-BUT birds were kept at 21 °C from d 21 to d 42. The HS-CONTR and HS-BUT birds were kept at 32 °C from d 21 to d 34 (heat stress period) and returned back to 21 °C from d 35 to d 42 (recovery period). During the heat stress period, HS-CONTR birds had reduced (P o0.05) body weight, daily gain, villus height, villus surface area and intestinal weight compared with other treatment groups, while HS-BUT birds exhibited growth performance and intestinal histological parameters similar to TN-CONTR birds (P 40.05). During the recovery period, butyric acid enhanced the recovery of body weight, villus height, villus surface area, epithelial cell area, intestinal weight and viable counts of Lactobacillus and Bifidobacterium. Butyric acid had extra positive effects in heat-stressed broilers as revealed by temperature diet interactions (Po 0.05) detected in final body weight, daily gain, feed conversion ratio, villus height, villus surface area, intestinal weight and beneficial intestinal bacteria. It is concluded that dietary inclusion of butyric acid for heat-stressed broilers can reduce intestinal epithelia damage and accelerate subsequent recovery of growth performance and intestinal histological characteristics. & 2015 Elsevier B.V. All rights reserved.

Keywords: Butyric acid Chickens Heat stress Intestinal morphology Recovery

1. Introduction The single cell layer of intestinal epithelium plays a number of key roles in nutrient digestion and absorption and it forms the most important barrier between internal and external environment. It restricts both transcellular and paracellular passage of luminal antigens and other noxious substances to circulation (Söderholm and Perdue, 2006). The ability of the intestinal epithelium to act as a barrier is essential for maintaining the health of the organism. Damaging the intestinal epithelium can cause intestinal barrier dysfunction which permits endotoxins passage from gut lumen to blood circulation leading to multi-organ

Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; BW, body weight n Corresponding author. E-mail address: [email protected] (A. Abdelqader). http://dx.doi.org/10.1016/j.livsci.2015.11.026 1871-1413/& 2015 Elsevier B.V. All rights reserved.

dysfunction (Bouchama and Knochel, 2002). It has become increasingly recognized that heat stress can negatively affect intestinal barrier integrity and functions through damaging intestinal epithelium (Liu et al., 2009; Pearce et al., 2013), villouscrypt histological injuries (Song et al., 2013, 2014), alterations in intestinal permeability (Lambert et al., 2002; Lambert, 2009) and changing the conditions for resident bacteria–enterocyte contact which increase intestinal susceptibility to pathogenic bacteria colonization (Quinteiro-Filho et al., 2010, 2012). Heat stress has many profound effects on poultry health and performance, particularly in broilers because they are more susceptible to heat load than slower growing domestic fowl (Lin et al., 2006; Abdelqader and Al-Fataftah, 2014). Probiotics and prebiotics have been used as a dietary approach to remedy this situation (Sohail et al., 2012; Song et al., 2013, 2014; Al-Fataftah and Abdelqader, 2014). These feed additives can enhance intestinal microbial fermentation and production of short-chain fatty acids (SCFA), particularly butyrate

A. Abdelqader, A.-R. Al-Fataftah / Livestock Science 183 (2016) 78–83

(Meimandipour et al., 2010; Rebolé et al., 2010). Butyrate can be utilized by intestinal epithelial cells as a direct source of energy to stimulate their proliferation and differentiation and improve intestinal barrier function (Kinoshita et al., 2002). Under natural growing environments of broilers, butyric acid was reported to increase villi growth and heights (Guilloteau et al., 2010; Levy et al., 2015) and control intestinal pathogenic bacteria (Namkung et al., 2011). We hypothesized that butyric acid could protect intestinal epithelium from heat stress-induced histological damage during heat exposure and accelerate the post-heat exposure intestinal recovery. The purpose of this study was to evaluate the effect of dietary butyric acid inclusion on broiler performance, intestinal histological changes and beneficial bacteria counts under thermoneutral and heat stress conditions, and to evaluate the potential of butyric acid to influence intestinal recovery following heat exposure.

2. Materials and methods 2.1. Birds and experimental design Bird care and handling was in compliance with the regulations of the European Parliament and the European Council Directive on the protection of animals used for scientific purposes (2010/63/ EU). This experiment was conducted at the Environment and Animal Physiology Lab, Faculty of Agriculture, the University of Jordan. One-day old Hubbard male broiler chicks (initial body weight: 46 g) were obtained from a commercial hatchery, kept in cages (dimensions: 188 82 68 cm3; 15 chicks/cage) with wire mesh floor and reared under routine management practice. Birds were provided with optimum rearing temperature according to the strain guidelines and given ad libitum access to control diet Table 1 Ingredient and nutrient composition of the control diet (g/kg diet as fed basis). Ingredient

Maize Soybean meal (480 g/kg CP) Dicalcium phosphate Soybean oil Fish meal DL-Methionine Vitamin-mineral premixa Sodium chloride Calculated chemical compositionb (g/kg diet as fed basis) MEn (MJ/kg) Analyzed chemical compositionc (g/kg diet as fed basis) Crude protein Calcium Total Phosphorus Lysine Methionine

Composition Starter (d 1–21)

Grower (d 22–35)

Finisher (d 36–42)

538.0 360.0 20.0 13.0 60.0 2.5 3.5 3.0

649 290.0 20.0 13.0 20.0 1.5 3.5 3.0

708.0 250.0 20.0 14.0 0 1.5 3.5 3.0

12.85

230.0 12.3 6.5 14.0 8.0

13.22

202.0 9.8 5.8 13.0 7.0

13.61

181.0 9.0 5.2 12.0 6.0

a Provided per kilogram of diet: vitamin A (trans-retinyl acetate), 8800 IU; vitamin D3 (cholecalciferol), 2695 IU; vitamin E (all-rac-tocopherol acetate), 15 mg; vitamin K (bisulfate menadione complex), 1.2 mg; riboflavin, 4.4 mg; pantothenic acid (D-calcium pantothenate), 6.6 mg; niacin 21 mg; choline (choline chloride), 358 mg; vitamin B12 (cyanocobalamin), 0.006 mg; manganese (MnSO4 H2O), 83 mg; zinc (ZnO), 55 mg; iron (FeSO4 7H2O), 32 mg; copper (CuSO4 5H2O), 3.9 mg; iodine (KI), 1.1 mg; selenium (Na2SeO3), 0.256 mg. b Calculated according to ingredients composition provided by National Research Council (1994). c Analyzed according to the AOAC International methods(2002).

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(Table 1) and water. At 21 d of age, 128 birds of uniform body weight (average: 851 g 71.86 SE) were chosen and randomly assigned to 2 treatments; thermoneutral (TN) or heat stress (HS), each of which included one group fed with control diet (CONTR) and one fed with control diet þ0.5 g butyric acid /kg (BUT). The experimental design was a 2 2 factorial arrangement; the main factors were environmental temperatures (thermoneutral or heat stress) and diets (control diet or control diet þbutyric acid). The four experimental groups were: TN-CONTR; birds kept at thermoneutral conditions and fed the control diet, TN-BUT; birds kept at thermoneutral conditions and fed the control diet þ butyric acid, HS-CONTR; birds exposed to heat stress and fed the control diet and HS-BUT; birds exposed to heat stress and fed the control diet þ butyric acid. Each treatment group was replicated 8 times (8 pens) with 4 birds per pen. The pens were separate, and each had its own feeder. The experiment was divided into 2 periods: period of heat stress exposure (d 21–d 34 of age) and recovery period (d 35–d 42 of age). The TN-CONTR and TN-BUT groups were kept at thermoneutral conditions (21 71 °C; 62 72% RH) during the whole experiment; from d 21 to d 42 of age. The HS-CONTR and HS-BUT groups were exposed to chronic heat stress (32 71 °C; 64 72% RH) in climatecontrolled rooms from d 21 to d 34 of age and returned back to the thermoneutral conditions from d 35 to d 42 of age. This procedure was conducted to evaluate the recovery responses after the end of heat exposure. The self-recovery process was evaluated through comparing HS-CONTR birds to TN-CONTR birds, where a self-repair mechanism assumed to occur in HS-CONTR birds after a potential intestinal damage by heat stress. The butyric acid-promoted recovery process was evaluated through comparing HS-BUT birds to TN-CONTR birds. 2.2. Feed analyses and mixing procedures The control diet (Table 1) was a standard commercial broiler mash diet formulated to meet or exceed the nutrient requirements (National Research Council, 1994). The butyric acid was provided in an encapsulated form as micro-granules which consisted of 50% butyrate (ButiPEARL; provided by Kemin Industries, Herentals, Belgium). The micro-granules were included in the diets to provide 0.5 g butyrate/kg of diet. The crude protein of the control diet was determined using the Kjeldahl method. Lysine and methionine were analyzed according to the AOAC International Methods (2002; Method 994.12). Metabolizable energy corrected for nitrogen (MEn) was calculated based on data provided by National Research Council (1994). On a weekly basis, ButiPEARL granules were carefully incorporated in the control diet at the expense of maize. To ensure that the diet was well mixed, the ButiPEARL granules were accurately weighted and then thoroughly mixed and homogenized with all other minor ingredients (vitamin-mineral premix, methionine and sodium chloride). Prior to their addition to the mixer, the homogenized ingredients were then divided into 4 portions and blended in a small mixer with soybean meal using the quartering technique (Abdelqader et al., 2013). The resulting mixture was then added to the control diet and mixed in a vertical mixer for 5 min. 2.3. Performance parameters Total daily feed intake/replicate was measured every morning at 07:00 from d 21 to d 42 of age. Average daily feed intake was calculated at the end of each experimental period; heat exposure period (d 21–d 34) and recovery period (d 35–d 42). Body weight (BW) was measured at d 0, 21, 35, and 42 of age to calculate average daily gain (ADG). Feed conversion ratio (FCR) was

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corrected for number of birds per pen and calculated on the basis of kg of feed consumed per kg of live BW gain.

2.5. Beneficial bacterial counts At 35 and 42 d of age, the small intestinal contents from all euthanized birds above (Section 2.4) were collected and then aseptically emptied in sterile bags for bacterial enumeration. The contents of duodenum, jejunum and ileum were collected and homogenized as one sample per bird. Samples were used to assay the beneficial bacteria; Lactobacillus and Bifidobacterium. Bacterial counts were performed using the appropriate dilution and plate culture techniques according to Hu et al. (2012). Fresh intestinal contents were taken for enumeration within 1 h after collection, immediately diluted tenfold (i.e. 10% w/v) with sterile 0.9% NaCl, and homogenized by a bag mixer for 3 min. Homogenate of samples were then serially diluted from 10 1 to 10 7. One-tenth milliliter of each diluted sample was coated on the appropriate agar media, in duplicate, for enumeration of the selected microbial populations. Lactobacillus and Bifidobacterium were assayed using Rogosa agar and Beerens agar, respectively, and incubated anaerobically at 37 °C for 48 h using sealed anaerobic jars. Number of bacterial colonies was counted, and results were expressed as log10 CFU/g of fresh sample.

2.4. Intestinal histologic and morphometric measurements Histological alterations in intestinal epithelia were evaluated based on degree of changes in villus height, villus basal and apical width, villus surface area, absorptive epithelial cell area and crypt depth. Histological alterations of the intestinal villi and epithelial cells were assessed at d 35 (end of heat exposure period) and at d 42 (end of recovery period). Each time, 8 birds per treatment were randomly selected, anaesthetized with diethyl ether and killed by decapitation. The entire small intestine was excised, carefully washed with physiological saline and the empty weight was recorded. A 2-cm segment was taken from the middle part of the duodenum of all euthanized birds for histological measurements. Tissue samples were washed in physiological saline solution and fixed in 10% buffered formalin overnight. The tissue samples were routinely processed, cleared with xylene, embedded in paraffin wax, sectioned using a microtome at a thickness of 5 mm (3 cross sections from each sample), placed on a glass slide and stained with hematoxylin and eosin. Histological sections were examined using an image analyzer (Leica Microsystems: Leica Imaging Systems Ltd., Cambridge, UK) to measure villus height, villus basal and apical width and crypt depth. Villus height was measured from the tip of the villus to the villus-crypt junction, and the crypt depth was defined as the depth of the invagination between adjacent villi. A total of 15 intact, well-oriented crypt-villus units were selected per bird for each intestinal cross section, and the average of these values was used to express the mean values of villus height and crypt depth for each bird. Villus surface area was calculated using the formula ¼ (2π) (VW/2) (VH) in which VW¼villus width and VH ¼ villus height (De los et al., 2005). A total of 15 villus areas were calculated for each bird. Absorptive epithelium cell area was measured according to Yamauchi et al. (2006). Briefly, the area of the epithelial cell layer at the middle part of the villus was measured and the number of cell nuclei within this layer was counted, then the area was divided by the number of cell nuclei. A total of 15 epithelial cell layers per bird were selected on intact villi to calculate the single epithelium cell area.

2.6. Statistical analysis The Statistical Analysis System (SAS Institute, 2010, Version 9.1.3) was used to conduct all statistical analysis. Data were analyzed with GLM procedure of SAS that included the effects of temperature (thermoneutral or heat stress), diets (control diet or control diet þbutyric acid), replicate and their interactions. Each replicate was considered as the experimental unit. Microbial counts were log10 transformed. Differences were accepted as representing statistically significant differences when Po 0.05. Tukey’s test was used to separate means for significant interactions.

3. Results 3.1. Growth performance Butyric acid did not change (P4 0.05) final BW and BW gain under thermoneutral conditions, while HS-BUT birds showed higher (P o0.001) final BW and BW gain compared with HSCONTR birds (Table 2). Average daily gain tended to decrease

Table 2 Effect of environmental temperature and butyric acid on broiler performance. Parameters

Number of birds/treatment Thermoneutral

Heat stress

SEM

Control diet Butyric acid Control diet Butyric acid Initial body weight (g) Final body weight (g) Average daily gain (g/day) d 21 to 34 d 35 to 42 d 21 to 42 Average daily feed intake (g/pen/day)a d 21 to 34 d 35 to 42 d 21 to 42 Feed conversion (kg/kg) d 21 to 34 d 35 to 42 d 21 to 42

Temp.

Diet

Temp. Diet

32 24

850.0 2800.0xy

852.5 2850.5x

851.0 2580.0z

850.1 2787.4y

1.86 18.65

0.702 0.651 0.350 o0.001 o0.001 o 0.001

32 24 24

86.6x 92.1x 88.6xy

87.5x 96.8x 91.1x

81.5z 73.6y 78.6z

83.9y 95.3x 88.1y

0.72 2.30 0.88

o0.001 0.032 0.308 o0.001 o0.001 0.001 o0.001 o0.001 o 0.001

32 24 24 32 24 24

635.4 610.7 525.4 1.82y 2.23y 1.97y

629.5 594.4 516.8 1.80y 2.05y 1.90y

639.0 601.9 523.9 1.96x 2.79x 2.23x

620.0 596.3 512.8 1.85xy 2.10y 1.94y

13.8 9.7 7.8

0.845 0.767 0.728

0.375 0.283 0.221

0.646 0.591 0.878

0.041 0.033 0.114 0.251 0.095 0.003 o0.001 0.012 0.036 o0.001 o0.001 0.006

x,y,z – means with different letters in the same row are significantly different at Po 0.05. SEM, pooled standard error of the mean; Temp., temperature effect. d 21–34, heat exposure period of the HS groups; d 35–42, recovery period (no heat exposure); d 21–42, overall experimental period. a

P values

Pen is the experimental unit. Total daily feed intake was measured per replicate based on 8 pens (replicates) in each treatment.


(P o0.05) in heat-stressed birds compared with birds kept at thermoneutral conditions. Butyric acid enhanced (Po0.05) the recovery of growth performance in heat-stressed birds during the recovery period. Heat-stressed birds provided with butyric acid showed growth performance similar (P 40.05) to TN-CONTR birds at the end of the study. Feed conversion ratio increased (P o0.05) for HS-CONTR birds, during both the heat exposure period and the recovery period, compared with other treatments. No temperature diet interaction (P 40.05) was observed for ADG, ADFI and FCR during the heat exposure period. There was a temperature diet interaction (P o0.05) for ADG and FCR during the recovery period. At the end of the study, there was a temperature diet interaction (Po 0.05) for final BW, overall ADG and overall FCR. Feed intake was not affected by any treatment during the whole experiment (P 40.05). 3.2. Intestinal histologic and morphometric measurements Under thermoneutral conditions, TN-BUT birds showed an increased (P o0.05) duodenal villus surface area and relative intestinal weight while villus height and absorptive epithelial cell area were not affected (P 40.05) compared with TN-CONTR birds. The lowest (P o0.05) villus height, villus surface area, epithelium cell area and relative intestinal weight were exhibited by HSCONTR birds compared with other treatments, during both the heat exposure period and the recovery period (Table 3). In contrast, the HS-BUT birds showed villus height, villus surface area, epithelium cell area and relative intestinal weight similar (P 40.05) to TN-CONTR birds during the period of heat exposure. There was a temperature diet interaction (P o0.05) for villus height and villus surface area at 35 and 42 d of age. Butyric acid enhanced (P o0.05) the recovery of villus height and epithelium cell area, and it had more pronounced positive effects (P o0.05) on villus surface area and relative intestinal weight in HS-BUT birds than in TN-CONTR birds during the recovery period. Crypt depth was not affected (P 40.05) by any treatment during the whole experiment. 3.3. Beneficial bacterial counts Dietary inclusion of butyric acid had no effect (P 40.05) on intestinal Lactobacillus and Bifidobacterium counts under both environmental conditions at 35 d of age (Table 4). Lactobacillus and

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Bifidobacterium populations increased (P o0.001) in HS-BUT birds compared with HS-CONTR birds, and it was similar to that exhibited by TN-CONTR birds and TN-BUT birds at the end of the study. There was a temperature diet interaction (Po 0.05) for the viable counts of Lactobacillus and Bifidobacterium during the recovery period.

4. Discussion This study investigated the consequences of dietary butyric acid administration in preventing heat stress-induced changes of intestinal epithelia and on recovery process following potential damage. Extensive studies have established the positive effect of butyric acid on broilers performance and health (Levy et al., 2015; Qaisrani et al., 2015). However, studies on the role of this fatty acid in improving intestinal recovery following heat stress-induced intestinal damage are absent in broilers. The present investigation provided evidence that dietary inclusion of butyric acid had positive effects on growth performance, duodenal villi morphology, intestinal weight and intestinal content of beneficial bacteria in heat-stressed broilers. Butyric acid had a significant role in enhancing the recovery process of heat stress-damaged villi. Broilers’ fast growth rate requires high metabolic demands which present unique challenges for the digestive and absorptive functions of the intestinal epithelia (Mitchell and Moretó, 2006). Disrupting integrity of intestinal epithelia will directly influence growth performance and feed conversion efficiency (Liu et al., 2009; Song et al., 2014). In this study, the impaired intestinal integrity, as measured by the reduction in villi height, villi surface area, absorptive epithelial cell area and beneficial bacteria populations, can be directly associated with the impaired growth performance and feed conversion efficiency of heat-stressed birds. In contrast, the improved growth performance and feed conversion efficiency of birds supplemented with butyric acid was accompanied with a parallel improvement in intestinal integrity parameters. The increase in villi height, villi surface area, absorptive epithelial cell area and intestinal weight, caused by butyric acid, may indicate an increase in cell proliferation. This improvement in epithelia structure may contribute to the maintenance of intestinal epithelial integrity by reducing breaks in the mucosal barrier which will restrict passage of luminal antigens to blood circulation (Söderholm and Perdue, 2006). The significant temperature diet

Table 3 Effect of environmental temperature and butyric acid on duodenal morphology and relative intestinal weight (n¼ 8 birds/treatment at d 35 and 8 birds/treatment at d 42). Parameters

Villus height (mm) d 35 d 42 Villus surface area (mm2) d 35 d 42 Epithelial cell area (mm2) d 35 d 42 Crypt depth (mm) d 35 d 42 Relative intestinal weight (g/100 g of BW) d 35 d 42

Thermoneutral

Heat stress

SEM

Control diet

Butyric acid

Control diet

Butyric acid

1797.5w 1932.7w

1799.2w 1949.9w

1760.0x 1810.6x

1795.7w 1937.5w

0.418w 0.418y

0.441w 0.444x

0.295x 0.339z

0.440w 0.497w

5.10 14.17 0.0129 0.0093

P values Temp. Diet

Temp.

Diet

o 0.001 o 0.001

0.003 o 0.001

0.001 0.001

o 0.001 0.164

o 0.001 o 0.001

o 0.001 o 0.001

222.0w 217.3w

209.5w 217.5w

186.4x 182.6x

194.6wx 209.0w

9.46 9.00

0.012 0.023

0.823 0.150

0.282 0.158

144.6 124.7

148.4 138.4

134.8 136.6

143.4 141.1

8.74 9.87

0.625 0.547

0.214 0.346

0.358 0.702

0.14 0.05

0.014 0.004

0.021 0.007

0.143 0.005

2.44wx 1.91x

2.56w 2.11w

2.02y 1.87x

w,x,y,z – means with different letters in the same row are significantly different at Po 0.05. SEM, pooled standard error of the mean; Temp., temperature effect. d 35, end of heat exposure period; d 42, end of recovery period.

2.36x 2.08w

82


Table 4 Effect of environmental temperature and butyric acid on intestinal counts (log10 CFU/g of fresh digesta) of Lactobacillus and Bifidobacterium (n¼ 8 birds/treatment at d 35 and 8 birds/treatment at d 42). Parameters

Lactobacillus d 35 d 42 Bifidobacterium d 35 d 42

Thermoneutral

Heat stress

SEM

Control diet

Butyric acid

Control diet

Butyric acid

6.43 7.44y

6.18 7.14y

6.14 6.18z

6.77 7.43y

4.64 5.88y

5.35 6.69y

5.55 5.22z

6.14 6.28y

P values Temp.

Diet

Temp. Diet

2.11 0.02

0.487 o 0.001

0.264 o 0.001

0.344 o 0.001

0.91 0.07

0.847 o 0.001

0.447 o 0.001

0.687 o 0.001

y,z– means with different letters in the same row are significantly different at Po 0.05. SEM, pooled standard error of the mean; Temp., temperature effect. d 35, end of heat exposure period; d 42, end of recovery period.

interactions observed in this study for final BW, ADG, FCR, villus height, villus surface area, intestinal weight and intestinal beneficial bacteria indicated that butyric acid had more pronounced positive effects in heat-stressed than in thermoneutral broilers. Generally, supplementation of different dietary acidifiers has the potential to improve broilers performance under heat stress conditions and reduce the associated economic losses (Daskiran et al., 2004). The intestinal epithelia form a single-cell barrier along the entire gastrointestinal tract. The mechanism driving repair of this barrier after a potential damage is critical for maintenance of a healthy intestine and organism. The immediate response to damage called restitution, which involves migration of surrounding epithelial cells to cover the denuded area, followed by proliferation of nearby cells to restore normal cell architecture (Frey and Polk, 2006). However, research indicates that various nutrients may be able to accelerate the restitution mechanism (Yamauchi et al., 2006; Murakami et al., 2007; Swaid et al., 2013). Butyric acid is among the molecules known to promote intestinal epithelial cell restitution by providing energy to stimulate mucosal cell proliferation and supporting epithelial repair mechanisms (Ahmad et al., 2000; Hu and Guo, 2007; Meimandipour et al., 2010). In the present study, we observed a strong increase in villi histological parameters of HS-BUT birds compared with other treatments. The increase in villi surface area was of significance during the recovery period and can be correlated with an increased proliferation rate of mucosal cells. This indicated a rapid recovery process in intestinal epithelium of HS-BUT birds compared with the selfrecovery process observed in HS-CONTR birds. This point is of significant importance for broiler industry as birds should achieve a target marketing weight in a given time period. Therefore, the rapid recovery of epithelial damage will enhance the digestive and absorptive functions of the intestinal epithelium. This explains why HS-BUT birds showed final body weight similar to that of TNCONTR birds. The butyric acid used in this study was encapsulated. Butyrate efficacy increased when it is fed in a protected form such as encapsulation (Smith et al., 2012). The aim of encapsulation is to carry the butyric acid down to the intestinal tract and bypass degradation in the foregut. Encapsulation techniques ensure that butyric acid will reach the small intestine in sufficient quantities to be effective and allows for slow release of acid in the digestive tract (Smith et al., 2012). In this way, the enterocytes could be exposed to butyric acid for a prolonged period. As butyric acid is a direct energy source for enterocytes (Ahmad et al., 2000; Kinoshita et al., 2002) it may be effective at preserving cell viability for a prolonged time which, in turn, enhances enterocyte turnover and improves intestinal recovery. One interesting finding of the present study is that heat stress-induced intestinal damage was directly associated with a significant reduction in intestinal relative weight. Garriga et al. (2006) reported that jejunum weight

reduced by 22% in heat-stressed broiler chickens compared with the control. The present study demonstrated that, intestinal damage led to intestinal hypoplasia in heat-stressed birds, apparent as decreased intestinal relative weight. The intestinal hypoplasia was partly ameliorated by the restitution mechanism and fully recovered by butyric acid administration. This was evident as increased intestinal weight of butyric acid treated birds, which is a characteristic of tissues undergoing increased cell proliferation or repair. This may suggest that butyric acid exerts its positive effect on mucosal recovery through direct stimulation of proliferation or via inhibition of enterocyte apoptosis. Histologically, villus height, villus surface area and absorptive epithelial cell area increased in response to butyric acid administration. The present study showed that heat stress had no effect on beneficial intestinal bacteria count. Although the effect of butyric acid on increasing numbers of Lactobacillus and Bifidobacterium was more obvious during the recovery period as HS-BUT birds showed higher intestinal count of these bacteria compared to HSCONTR birds at the end of the study. This can be confirmed by the significant temperature diet interaction retrieved in this study on Lactobacillus and Bifidobacterium viable counts during the recovery period. This interaction indicated that butyric acid can significantly enhance the growth of the beneficial intestinal bacteria when compared with the control diet groups and this effect can persist under high ambient temperatures. This indicated that the potential of butyric acid to reestablish the normal protective microflora may take a long time to appear, and that is why it became visible later during the recovery period.

5. Conclusion Butyric acid at a dietary level of 0.5 g butyric acid/kg diet ameliorated intestinal epithelia damage, improved intestinal integrity and accelerated intestinal recovery in heat-stressed broilers. This additive could be beneficially used to attenuate the adverse effects of heat stress on broilers performance.

Conflict of interest The authors declare that they have no conflict of interest.

Acknowledgment This research was funded by the University of Jordan under promotion of research scheme.


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