Journal of Research in Ecology

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1 Journal of Research in Ecology Journal of Research in Ecology ISSN No: Print: ; Online: An International Scientific Research Journal Original Research Effect of Jerusalem artichoke (Helianthus tuberosus L.) on histological parameters of small intestinal segments of broiler chickens Authors: Abbas AM Al-Abboodi and Hasan SA Jawad Institution: Department of Animal Production, Faculty of Agriculture, University of Baghdad, Iraq. Corresponding author: Hasan SA Jawad ABSTRACT: A study was undertaken to examine the broiler growth performance effectiveness by daily supplementing H. tuberosus from 1 to 6 week of age. A total of 390 one-day-old broiler chicks were allocated to five treatments, with three replicates per treatment and 26 birds per replicate. Experimental treatments including 0% (T 1 ), 0.5% (T 2 ), 1% (T 3 ), 1.5% (T 4 ) and 2% (T 5 ) H. tuberosus powder were used. Three birds per treatment at week four and six (3 male and 3 female) at the marketing age were isolated based on the mean treatment weight and euthanized to perform the necropsy. The birds were slaughtered and the duodenum, jejunum and ilium cross sections were measured by an ocular micrometer after stained by Haematoxylin and Eosin stain. In comparison with the control group, supplementing 1% H. tuberosus caused greater (P<0.05) effect on the total duodenum wall thickness at week 4 of age. Same observation was shown in jejunum wall for T 3 and T 4. At week 6 of age, partnered significant effect was obtained in all the H. tuberosus treatments. The improvement percentage in the duodenum wall parameters were 7-16% in villi length, 8-15% in crypt depth and 6-15% in the total wall thickness. While in jejunum the improvement percentages were 16-25% in villi length, 1-17% in crypt depth and 12-23% in the total wall thickness. Similarly, in ilium the improvement percentages were, 4-33% in villi length, 11-26% in crypt depth and 5-31% in the total wall thickness. This study gives evidence that there is a positive relationship between the developments in the intestinal wall layers and increase the supplementation percentage of H. tuberosus in broiler diet. Keywords: Jerusalem artichoke, Inulin, Histological parameters, Small intestinal. Article Citation: Abbas AM Al-abboodi and Hasan SA Jawad Effect of Jerusalem artichoke (Helianthus tuberosus L.) on histological parameters of small intestinal segments of broiler chickens Journal of Research in Ecology (2018) 6(2): Web Address: documents/ec0588.pdf Dates: Received: 29 April 2018 Accepted: 26 May 2018 Published: 01 Aug 2018 This article is governed by the Creative Commons Attribution License ( licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. Journal of Research in Ecology An International Scientific Research Journal JRE 2018 Vol 6 No 2

2 INTRODUCTION The Helianthus tuberosus L., (Jerusalem artichoke) is an erect, rhizomatous perennial herb, up to 3-4 m height. Though perennial, it is mainly grown as an annual and is considered as one of the prebiotic sources (Snel et al., 2002). The dominant prebiotics are fructo-oligosaccharide products (FOS, oligofructose, inulin) (Patterson and Burkholder, 2003); glucooligosaccharides, stachyose, malto-oligosaccharides, and oligochitosan, and have also been investigated in the broiler chickens (Zhan et al., 2003; Gao and Shan, 2004; Jiang et al., 2006; Huang et al., 2007). Inulin belonged to a class of fructose-based, highly soluble polysaccharides collectively called fructans. Fructans are the major non-structural carbohydrates in many plant species, particularly in the prevalent and evolutionarily advanced orders of Asterales, Liliales and Poales eg. chicory, onions, wheat (Hendry, 1993). Small intestine consists of duodenum, jejunum and ileum with no demarcation on gross observation between duodenum and jejunum. While, Mickel diverticulum is often used as a landmark to separate the jejunum and ileum (Dorman and Deans, 2000; Cervantes, 2006; Jawad et al., 2015). Furthermore, intestinal wall histologically consist of four layers: mucosa, submucosa, muscularis and serosa. Mucosal layer of small intestine forms villi which project into the lumen and greatly increase the overall absorption surface area of the organ. The surface epithelium of the villi is small columnar epithelium with numerous goblet cells. Intestinal absorptive cells have extensive microvilli on its apical surface. Goblet cells are scattered between the absorptive cells and produce mucous. Intestinal glands (Crypts of lieberkuhn) extend from the base of the villi into the underlying lamina properia. Undifferentiated epithelial cells located in the glands divide and migrate up to renew the glandular and surface epithelium every h. Acidophilic granular cells (paneth cells) are present in the epithelium at the base of the gland, these cells produce peptidase and lysozyme and may be phagocytic. Enteroendocrine cells are also present in the epithelium of the intestinal gland. Tunica sub mucosa is very thin in chicken with the absence of bronner gland, tunica muscularis is characterized by two layers of smooth muscle, the inner layer of circular muscle fibers are surrounded by an outer of longitudinal folds. Myentrice plexus are often present between muscle layers. A typical tunica serosa lies outside the tunica muscularis as the outermost layer of the organ (Elizabeth and Fredric, 2001; Jawad et al., 2016; Naji et al., 2016; Naji et al., 2017; Hasan et al., 2018). The current study was aimed to elucidate the effect of H. tuberosus on broiler production performance. In a previous reports, it was hypothesized that the mechanism resulted in an improvement in the body performance of the chicken fed on diet supplemented by H. tuberosus (Jawad et al., 2017a, b) due to an increase in the level of benefit bacteria in the gastro-intestinal trunk. MATERIALS AND METHODS Chicken husbandry and experimental design This research was conducted in the poultry farm of Agricultural Faculty in the University of Baghdad from 26 th November 2017 to 6 th January This study consists of 390 Day-Old broiler (Ross 308), floor bred. They were randomly assigned to five treatment groups by 78 birds/ treatment, and each treatment consisted of three replicates of 26 birds/ replicate. Feed was prepared and offered ad libitum the same diets (1 10 days: starter; days: grower; 24 day-slaughter: finisher) (Table 1) with continuous water. Furthermore, constant lighting and continuous ventilation were provided. All birds were kept under uniform management conditions throughout the experimental period of six weeks. About 60 kg of H. tuberosus fruits were purchased from the market and processed by slicing, drying 1815 Journal of Research in Ecology (2018) 6(2):

3 S. No Items (%) 1 Corn 2 Wheat 3 Soybean meal (44%) 4 Protein concentrated 5 Sunflower oil 6 Limestone 7 Dicalcium phosphate 8 Salt 9 Mineral and vitamin premix Total Crude protein (%) Metabolism energy (kilo calorie per kg. diet) Lysine (%) Methionine (%) Cysteine (%) Methionine + Cysteine (%) Arginine (%) Calcium (%) Phosphorus (%) * according to NRC (1977) Table 1. Composition of the basal diet Basal diet 1 to 10 d 11 to 24 d Calculated analysis to 42 d and grinding. H. tuberosus powder was supplemented at levels 0.5%, 1%, 1.5% and 2% to the chicken diet of T 2, T 3, T 4 and T 5 respectively. Its worthy to mention that chicken in T 1 fed on dry food without supplementation. Histometrical assay Three randomly selected birds per treatment (one bird/replicate) at week four of age and three males, three females at the marketing age were slaughtered and small intestinal organs were collected. It is worthy to mention that selected birds at week four were within the average treatments weight, as well as, the selected birds at week six were within the males and females average treatments weight. A 1 cm segment from the midpoint of the duodenum, jejunum and ileum were immediately fixed in formalin (10%) for 24 h. Then, dehydrated with increasing concentration (70-100%) of ethyl alcohol and passed into two content of xylol. After that the samples were embedded in paraffin and sectioned by the rotary microtome at 5 µm. The sections were fixed on the glass slides and remoistening process was done by passing the sections through decreasing concentration (100-70%) of ethylic alcohol and in xylol. The histological slides were stained by hematoxylin and eosin stain (Munro, 1971; Jawad et al., 2016). Morphological parameters were measured by a computerized image analyzer (Leica DM LB2, Germany). An ocular micrometer was used to measure the thickness of the intestinal wall layers. Variation ration was counted based on the formula which were reported by Jawad et al. (2015). Research design and data analysis This research used one way complete random sampling. The gained data which was resulted were analyzed by one way analysis of variance (ANOVA). If the treatment significantly affected the chicken, LSD and Duncan s (1955) Multiple Range would be applied (DRMT) (Vincent, 1991; Genstat, 2003). Differences between treatments subjects were considered significant level at P<0.05. RESULTS AND DISCUSSION The effects of supplementing 0.5, 1, 1.5 and 2 g of H. tuberosus powder in the chicken diet on the histo- Journal of Research in Ecology (2018) 6(2):

4 S. No Table 2. Effect of H. tuberosus on histometrical parameters of broiler small intestinal organs at four weeks of age TRT Organ VILLI LEB MM TMT SM MA S Total Al-Abboodi and Jawad, 2018 T1 T2 T3 T4 T5 I 69.6± ± ± ± ± ±0.77 a 5.4± ±2.44 Mean values with (a, b, c) common superscript in column differ significantly (P<0.05). D, J and I compared separately between TRT. TRT: treatment; D: duodenum; J: jejunum; I: ileum; Vl: villi length; DL: Depth of Crypts of Lieberkuhn; MM: Muscularis Mucosa; TMT: Total Mucosa Thickness (VL+DL+MM); T1 T2 T3 T4 T5 Mean values with (a, b, c) common superscript in column differ significantly (P<0.05). M, F and M+F compared separately between TRT. TRT: Treatment; M: Male; F: Female; D: Duodenum; J: Jejunum; I: Ileum; Vl: Villi Length; DL: Depth of Crypts of Lieberkuhn; MM: Muscularis Mucosa; TMT: Total Mucosa D ±4.24 b 25.87±1.07 ab 0.15± ±4.96 b 3.67± ± ± ±5.38 b 1 J 98.53±8.58 b 20.47±1.62 c 0.17± ±10.09 b 3.87± ± ± ±20.36 b I 72.87± ± ± ± ± ±0.28 c 5.13± ±2.11 D ±5.81 b 25.4±1.03a b 0.16± ±5.86 b 3.73± ± ± ±5.74 b 2 J 96.93±3.28 b 21.93±0.62 bc 0.17± ±3.7 b 3.67± ± ± ±3.94 b I 70.73± ± ± ± ± ±0.75 ab 5.73± ±2.62 D 183.8±6.28 a 27.4±0.94 a 0.14± ±6.38 a 3.73± ± ± ±6.49 a 3 J ±2.91 a 25.33±1.04 a 0.15± ±3.5 a 3.6± ± ± ±3.46 a I 75.33± ± ± ± ± ±0.46 bc 5.27± ±3.52 D 161.4±3.85 b 24.13±0.65 b 0.15± ±3.94 b 3.6± ± ± ±4.07 b 4 J ±2.92 a 24.8±1.23 ab 0.17± ±3.5 a 3.53± ± ± ±3.6 a I 71.67± ± ± ± ± ±0.65 abc 5.53± ±2.87 D 166±5.89 b 24.2±0.51 b 0.15± ±6.32 b 3.67± ± ± ±6.01 b 5 J ±3.73 a 23.53±0.51 abc 0.17± ±3.97 a 3.6± ± ± ±3.79 ab SM: Submucosa; MA: Muscularis; S: Serosa; TWT: Total Wall Thickness (TMT+SM+MA+S). S. No Table 3. Effect of Helianthus tuberosus on histometrical parameters of broiler duodenum at six weeks of age TRT SEX VILLI LEB MM TMT SM MA S Total M ±2.8 c 31± ± ±3.86 c 3.47± ± ± ±4.12 c 1 F ± ± ± ±8.09 b 3.53± ± ± ±8.22 b M+F 167.2±4.66 c 31.97±0.92 b 0.15± ±4.8 c 3.5± ± ± ±4.85 c M ±4.03 ab 32.33± ± ±4.04 b 3.6± ± ± ±4.05 b 2 F 179± ± ± ±6.64 b 3.87± ± ± ±6.71 b M+F ±3.77 b 32±0.53 b 0.15± ±3.82 b 3.73± ± ± ±3.86 b M ±5.04 b 35.93± ± ±5.72 a 3.6± ± ± ±5.75 a 3 F ± ± ± ±2.94 b 3.53± ± ± ±2.93 b M+F ±3.2 ab 34.5±0.8 a 0.15± ±3.56 ab 3.57± ± ± ±3.51 b M 189.2±7.06 b 35.93± ± ±6.97 ab 3.47± ± ± ±6.92 ab 4 F 198± ± ± ±4.87 a 3.6± ± ± ±4.9 a M+F 193.6±4.55 a 35.7±0.7 a 0.16± ±4.25 a 3.53± ± ± ±4.21 a M ±6.29 a 40.4± ± ±6.69 ab 3.67± ± ± ±6.79 a 5 F ± ± ± ±4.1 ab 3.6± ± ± ±4.44 ab M+F 186.5±3.73 ab 36.83±0.4 a 0.15± ±3.96 ab 3.63± ± ± ±4.11 a thickness (VL+DL+MM); SM: submucosa; MA: muscularis; S: serosa; TWT: total wall thickness (TMT+SM+MA+S) 1817 Journal of Research in Ecology (2018) 6(2):

5 S. No Table 4. Effect of Helianthus tuberosus on histometrical parameters of broiler jejunum at six weeks of age TRT Sex VILLI LEB MM TMT SUB MUSC SER Total Al-Abboodi and Jawad, 2018 T1 T2 T3 T4 T5 M 86.8±2.86 b 29.13±0.54 d 0.15± ±2.74 b 3.6± ± ± ±2.75b 1 T1 F 81.53±1.32 b 28.87±0.69 c 0.14± ±1.78 b 3.8± ± ± ±1.74b M+F 81.53±0.92 c 28.87±0.48 c 0.14± ±1.24 c 3.8± ± ± ±1.21c M 86.33±1.86 b 31.73±1.51 cd 0.17± ±1.09 b 3.93± ± ± ±1.03b 2 T2 F 83.33±3.02 b 33.27±1.1 ab 0.15± ±3.35b 3.53± ± ± ±3.45b M+F 84.83±1.76 c 32.5±0.93 b 0.15± ±1.74 c 3.73± ± ± ±1.77c T3 T4 T5 M 89.33±3.14 c 32.4±0.67 b 0.15± ±3.26 c 3.8± ± ± ±3.26 c 1 F 76.07± ±0.96 b 0.15± ±2.78 b 3.87± ± ± ±2.69 b M+F 82.7±2.42 b 30.87±0.64b 0.15± ±2.59 b 3.83± ± ± ±2.53 b M ±6.86 bc 32.27±0.53 b 0.16± ±6.56 bc 3.8± ± ± ±6.57 bc 2 F 87.8± ±0.95 b 0.17± ±4.39 a 3.53± ± ± ±4.75 ab M+F 95.83±4.44 a 31.23±0.57 b 0.17± ±4.23 a 3.67± ± ± ±4.28 a M 108±3.31 ab 33.33±0.48 b 0.18± ±3.15 b 3.53± ± ± ±3.54 b 3 F 87.8± ±1.18 a 0.16± ±4.31 a 3.67± ± ± ±4.11 a M+F 97.9±3.16 a 33.73±0.63 a 0.17± ±3.18 a 3.6± ± ± ±3.17 a M ±7.42 ab 33.73±0.89 b 0.18± ±8.02 ab 3.8± ± ± ±8.31 ab 4 F 93.33± ±0.76 a 0.14± ±6.39 a 3.8± ± ± ±6.55 a M+F 103±5.11 a 34.33±0.58 a 0.16± ±5.31 a 3.8± ± ± ±5.49 a M ±4.89 a 38.33±2.57 a 0.13± ±5.28 a 3.6± ± ± ±5.57 a 5 F 85.2± ±1.13 a 0.15± ±2.21 a 4.07± ± ± ±2.4 a M+F ±4.34 a 36.2±1.43 a 0.14± ±4.69 a 3.83± ± ± ±4.73 a Mean values with (a, b, c) common superscript in column differ significantly (P<0.05). M, F and M+F compared separately between TRT. TRT: Treatment; M: Male; F: Female; D: Duodenum; J: Jejunum; I: Ileum; VL: Villi Length; DL: Depth of Crypts of Lieberkuhn; MM: Muscularis Mucosa; TMT: Total Mucosa thickness (VL+DL+MM); SM: Submucosa; MA: Muscularis; S: serosa; TWT: Total Wall Thickness (TMT+SM+MA+S) S. No Table 5. Effect of Helianthus tuberosus on histometrical parameters of broiler ilium at six weeks of age TRT Sex VILLI LEB MM TMT SUB MUSC SER Total M 85.13±1.44 b 33±0.92 bc 0.15± ±1.68 b 3.8± ± ± ±1.63 b 3 F 82.67±1.46 b 31.33±0.56 bc 0.15± ±1.59 b 3.8± ± ± ±1.74 b M+F 83.9±1.03 c 32.17±0.55 b 0.15± ±1.2 c 3.8± ± ± ±1.27 c M ±0.69 a 35.93±1.53 ab 0.15± ±1.84 a 3.73± ± ± ±1.91 a 4 F ±6.01 a 34.8±1.85 a 0.16± ±6.23 a 3.47± ± ± ±6.46 a M+F ±3.38 a 36.37±1.22a 0.16± ±3.8 a 3.67± ± ± ±3.99 a M ±3.34 a 37.93±1.54 a 0.15± ±4.58 a 3.87± ± ± ±4.91 a 5 F 80.73±3.49 b 28.73±0.56 c 0.14± ±3.73 b 3.8± ± ± ±3.93 b M+F 94.4±3.47 b 33.33±1.17b 0.15± ±4.46b 3.83± ± ± ±4.53 b Mean values with (a, b, c) common superscript in column differ significantly (P<0.05). M, F and M+F compared separately between TRT. TRT: Treatment; M: Male; F: Female; D: Duodenum; J: Jejunum; I: Ileum; VL: Villi Length; DL: Depth of Crypts of Lieberkuhn; MM: Muscularis Mucosa; TMT: Total Mucosa thickness (VL+DL+MM); SM: Submucosa; MA: Muscularis; S: Serosa; TWT: Total Wall Thickness (TMT+SM+MA+S) Journal of Research in Ecology (2018) 6(2):

6 logical parameters of small intestine segments at week 4 of age are presented in Table 2. The duodenum wall of the chicken in the third treatment was significantly (P<0.05) higher in thickness compared with other treatments. That take place because of increase the villi length (17%) and total mucosa thickness (15%) than the control group. Furthermore, 4% improvement percentage in the duodenum thickness was reported for T 2 and T 5. 17, 13 and 11% significant improvement was shown in the jejunum wall thickness for T 4, T 3 and T 5 based on the improvement of mucosal layer, especially villi length and depth of lubricant. The variation ratios of the villi length of these treatments were 21, 15 and 15% and in the depth of lubricant were 21, 24 and 15% respectively. While, non-significant effects were reported between all the treated chickens ilium wall thickness. Table 3 express the histological comparative of the duodenum wall layers of males and females at week six of age. Overall, supplementing H. tuberosus caused significant improvement (P<0.05) in the duodenal layers thickness, especially in males. Higher values were reported in T 4 and T 5 (15, 12%) followed by T 3 and T 2 (12, 6%). In males, higher duodenum wall thickness was shown in T 3, T 5, T 4 and T 2 and the variation ratios were 19, 18, 17 and 11% compared with the control group. Whereas, T 4 in females had significantly (P<0.05) higher duodenum wall thickness (10%) compared with the other H. tuberosus treatments and control group. Table 4 shows the histological measurements of the jejunum wall layers of the birds in the treatments at week six of age. All the H. tuberosus treatments reported significant increase (P<0.05) in the total jejunum wall thickness and the variation ratio was 23, 21, 16 and 12% in T 5, T 4, T 3 and T 2 respectively. Furthermore, it was 26, 17, 13, 9% in males and 12, 10, 17, 9% in females. Table 5 present the difference between the histological measurements in the ilium wall layers between H. tuberosus treatments and control group at the marketing age. Overall, birds in T 4 reported significantly (P<0.05) higher improvement in the ilium wall thickness and followed by T 5 with 31 and 16% variation ratio respectively. In the same regard, males in T 5, T 4 and T 4 in females showed significant (P<0.05) superiority in the measurements of ilium wall layers. The increasing the length of villi and depth of lubricant were behind the improvement in the total ilium wall thickness in H. tuberosus treatments. The results of present research is constant with Rehman et al. (2007) observation that supplementing 1% inulin in broiler diet notically improved the intestinal villi height and crypt depth. Whereas, Rebole et al. (2010) found that inclusion of 10, 20 g/kg inulin did not affect the jejunal histomorphology measured in broilers at 35 d of age, especially, villus height and crypt depth or microvillus length, width, density and amplification factor. However, a significant increase in the villus height: crypt depth ratio could be observed in the group receiving the diet containing 10 g of inulin/kg. Furthermore, Konosonoka et al. (2015) reported that adding 0.5 and 1% H. tuberosus in broiler diet did not significantly effect on the villi length in duodenum and ilium while increased the depth of the crypt. In the same regard, Nabizadeh (2012) found that 0.5 and 1% inulin had no impact on histological structure of broiler duodenum and ilium, but increase the height of villi in jejunum. CONCLUSION Daily supplementation of 1, 1.5 and 2% Jerusalem artichoke had significant effect on the total duodenum and jejunum wall thickness at four weeks of age. Furthermore, all the supplemented percentage of Jerusalem artichoke caused improvement impacts in most of the studied parameters at six weeks of age. Best results were reported in the fifth treatment when 2% Jerusalem 1819 Journal of Research in Ecology (2018) 6(2):

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