CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES EFFECT OF Fructo-Oligosaccharides (FOS) ON DIGESTIVE ENZYMES OF STRIPED CATFISH (Pangasianodon hypophthalmus) By NGUYEN KHANH LINH A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Aquaculture Can Tho, December 2013 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES EFFECT OF Fructo-Oligosaccharides (FOS) ON DIGESTIVE ENZYMES OF STRIPED CATFISH (Pangasianodon hypophthalmus) By NGUYEN KHANH LINH A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Aquaculture Supervisor Assoc. Prof. Dr. DO THI THANH HUONG Can Tho, December 2013 APPROVEMENT The thesis “Effect of fructooligosaccharides (FOS) on digestive enzymes of striped catfish (Pangasianodon hypophthalmus)” defended by Nguyen Khanh Linh,which was edited and passed by the committee on 12-27-2013. Sign of Supervisor Assoc. Prof. DO THI THANH HUONG Sign of Student NGUYEN KHANH LINH ACKNOWLEDGEMENT Firstly, I want to express special thanks to my supervisor, Assoc. Prof. Dr. Do Thi Thanh Huong for her invaluable guidance, advice, and encouragement. I would also like to dedicate my great appreciation to Nguyen Thi Kim Ha for her kind help in finishing the research. Secondly, many thanks are also given to all other doctors of the college of aquaculture and fisheries, and especially to those of the department of Aquatic Nutrition and Products Processing for providing me with great working and learning conditions. Thirdly, I would love to express my sincere appreciation to many friends, especially Le Thi Mai Anh, Nguyen Minh Thuat, Dang Minh Quan, Tran Thi Be Gam, Nguyen Chi, Vo Van Dao and Tran Thi Bich Thuan for their unconditionally kind help throughout the experimental period. Finally, I want to thank my academic adviser, Dr. Duong Thuy Yen, who was guiding and encouraging me over the last four years, and my family for their great lifetime support which makes everything possible for me. Thank you very much, Nguyen Khanh Linh i ABSTRACT The aim of the experiment was conducted to assess the impact of the supplement of fructooligosaccharide on on digestive enzyme activities and total bacteria in the intestine of stripped catfish fingerling. The experiments were consists of concentrations (0%; 0.5; 1%; 1.5% and 2%/kg food). Each treatment was three replicated; the test period was 90 days. The digestive enzymes activities as pepsine, trypsine, chymotrypsine and amylase in the stomach and intestine of the fish were measured at 0, 1, 3, 7, 10, 30, 60 and 90 days and the total bacteria in these organs also counted. The results show that the fish eating with supplement FOS were improved on digestive enzyme activities and total bacteria in the intestine at the treatment of 0.5% and % of FOS. ii TABLE OF CONTENTS Acknowledgement . i Abstract ii Table of contents . iii List of figures . v List of tables vi List of abbreviations vii CHAPTER 1: Introduction 1.1 General introduction 1.2 Research Objective 1.3 Research Content . CHAPTER 2: Literature reveiw 2.1 Tra Catfish (Pangasianodon hypophthalmus) . 2.2 Prebiotic . 2.3 FOS (Frustooligosaccharides) . 2.4 Application of FOS in terrestrial animals 2.5 Application of FOS in aquatic animals . 2.6 Digestive enzymes in fish CHAPTER 3: Research methodology . 3.1 Time and place 3.2 Materials 3.3 Methods . 3.3.1 Experimental design 3.3.2 Evaluate the total microorganisms in intestine 3.3.3 Evaluate enzyme in stomach and intestine 3.4 Data collection, calculation, and analysis . CHAPTER 4: Results and disscussions . 10 4.1 Environment parameters 10 4.2 Total bacterial count 11 4.3 Enzymes activity . 12 CHAPTER 5: Conclusions and recommendations 17 iii REFFERNCES 18 APPENDICES 21 Appendix 1: Enzyme analysis . 21 Appendix 2: Chemicals preparation 24 Appendix 3: Total bacterial count . 26 Appendix 4: Enzyme activity 26 iv LIST OF TABLES Table 3.1: Composition of diets . Table 4.1: Temperature, DO, pH measured in the morning and afternoon 10 Table 4.2: TAN and NO2- 10 Table 7.1: Total bacterial counted in fish’s intestine . 26 Table 7.2: α–amylase in fish’s stomach . 26 Table 7.3: α–Amylase in fish’s intestine 27 Table 7.4: Pepsine in fish’s stomach 27 Table 7.5: Trypsine in fish’s intestine 28 Table 7.6: Chymotrypsine in fish’s intestine 28 v LIST OF FIGURES Figure 2.1: Chemical structure for FOS . Figure 3.1: Total bacterial count in the intestine . Figure 4.1: Total bacterial counted in intestine of the fish . 12 Figure 4.2: α – Amylase in stomach of the fish . 13 Figure 4.3: α – Amylase in intestine of the fish . 13 Figure 4.4: Pepsine in stomach of the fish . 14 Figure 4.5: Trypsine in intestine of the fish . 15 Figure 4.6: Chymotrypsine in intestine of the fish . 15 vi LIST OF ABBREVIATIONS FOS Fructooligosaccharide XOS Xylooligosaccharide GOS Galactooligosaccharides MOS Manooligosaccharide TOS Trans-galacto-oligosaccharides ScFOS Short chain Fructooligosaccharides DO Dissolved oxygen FCR Feed conversion ratio vii mU/min/mg protein control 0.50% 1% 1.50% 2% 10 Days 30 60 90 Figure 4.2: α – Amylase activity in stomach of the fish mU/min/mg protein According Huong and Tu, 2010, amylase present mostly in the intestine of omnivorous fish. Therefore, Amylase concentration in fish’s intestine of this research is higher than in fish’s stomach. And similarly amylase in fish’s stomach, the amylase in fish’s intestine was increased with fish age. From day to day 7, there were no significantly different among treatments. And after 10 days fed with FOS administration, 0.5 and 1% FOS treatment were significantly higher than control, 1.5 and 2% (4.13 and 4.35 mU/min/mg protein). In the other hand, after 90 days administrated, amylase level is reached highest at 0.5 and 1% of FOS diet (14.2 and 14.3 mU/min/mg protein). Besides, applied 1.5 and 2% of FOS-diet, the amylase activity were no significant different compared to the control group. The results in accordance with that of Xu et al (2002, 2009) and Renjie et al (2010) 16 14 12 10 control 0.50% 1% 1.50% 2% 10 Days 30 60 90 Figure 4.3: α – Amylase activity in intestine of the fish 13 mU/mL/mg protein 4.3.2 Pepsine activity in stomach of the fish Pepsine actives in acid environment only, suitable pH for pepsine activity is 1.45 -3 (Huong and Tu, 2010). Striped catfish is omnivorous fish and they have a real stomach also. Therefore, they have pepsine enzyme. Pepsine activity in stomach of the fish was presented in Fig. 4.4. Generally, the pepsine levels were augmented with fish age and after days applied 0.5 and 1% FOS-diet, pepsine activities were significantly higher than those of control, 1.5, 2% treatment (0.131±0.02 and 0.132±0.02 mU/mL/mg protein), at that time, the lowest level of pepsine was 0.092±0.004 mU/mL/mg protein at 1.5% FOS-diet. 10 days post supplied 1% FOS-diet; the pepsine levels reached higher level 0.191±0.008 mU/mL/mg protein compared to other treatment. At the end of experiment, the highest levels of pepsine activity were 0.337±0.0340 mU/mL/mg protein and 0.356±0.015 mU/mL/mg protein for 0.5 and 1% treatments, respectively. 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 control 0.50% 1% 1.50% 2% 10 Days 30 60 90 Figure 4.4: Pepsine activity in stomach of the fish 4.3.3 Trypsine activity in intestine of the fish No significant in trypsine activities was found within 10 days feeding of FOS. However, after 30 days fish fed 0.5 and 1% FOS showed trypsine significantly increased. The research of Xu et al (2003) also concluded that application with and 6kg FOS/kg significantly improved the trysine activity in pig. 14 U/mg protein 16 14 12 10 control 0.50% 1% 1.50% 2% 10 Days 30 60 90 Figure 4.5: Trypsine activity in intestine of the fish 4.3.4 Chymotrypsine activity in intestine of the fish Basically, chymotrypsine level showed the increasing trend with the increase of fish age. However, after one day, the chemontrypsine level was varying among treatments. But after that, the chymotrypsine concentration was stable. And after 10 days of experiment 0.5 and 1% of FOS administrated, chymotrypsine activity were significantly went up (325±21.1 and 330±3.30 mU/mL/min). In addition, at the end of experiment, chymotrypsine activity got highest at 0.5 and 1% FOS diet (606±3.43 and 609±8.12 mU/mL/min). 700 mU/mL/min 600 500 control 400 0.50% 300 1% 200 1.50% 100 2% 0 10 Days 30 60 90 Figure 4.6: Chymotrypsine activity in intestine of the fish 15 Protease refers to a group of enzymes such as fungal protease, pepsin, trypsin, chymotrypsin, papain, bromelain, and subtilisin. (Enzyme essentials, 2013). Therefore some researches, they mention in protease in spite of pepsine, trypsine, chymotrysine. Xu et al (2003), Renjie et al (2010) and Soleimani et al (2012) they concluded that supplementation FOS can improve the activities of protease. According to Huong and Tu, 2010, digestion ability increased with fish age. When the fish get older, their digestive enzyme fully made of quantity and quality. Besides, the quality of feed intake can affect digestive enzyme also. The result of total bacterial counted (Figure 4.1) was supported for the increased of enzymes activity supplemented at 0.5 and 1% FOS because healthy bacteria can produce certain enzymes including lactase, protease, and amylase to assist fish digestion (Family Health News and Nutrition, 2006). 16 CHAPTER CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions The results showed that enzyme activity in stomach of fish (such as amylase and pepsine) and intestine of fish (such as amylase, trypsine, and chymotrypsine) were significantly increased after 30 days supplementation 0.5 and 1% of FOS-diet. In addition, the number of total bacterial count was significantly raised after 30 days applied 0.5 and 1% of FOS administrated. These results supported that the supplementation 0.5 and 1% FOS-diet have the positive effect on the digestive system of striped catfish. If applied more FOS such as 1.5 and 2%, there were no significant effect. The farmers are recommended to apply 0.5 and 1% of FOS diet to increase the digestibility of striped catfish fingerling. 5.2 Recommendations Conducting the experiment with the concentration of FOS lower than 0.5%. Further research should be carried out the effect of FOS on grow-out stage Performing the experiments about effects of different kinds of prebiotic (such as XOS, MOS and GOS) on growth rate parameters, haematological parameters, and enzyme activity also. Investigating the influences of FOS on the beneficial and unbeneficial bacteria in fish. 17 REFERENCES Blaut, M., 2002. Relationship of prebiotics and food to intestinal microflora. European Journal of Nutrition 41, l11-6pp Huong. D. T. T., Tu. N. V., 2010. Fish and crustacean physiology, 70-73pp. (in Vietnamese) FAO., 2011. Culture Aquatic Species Information Programme Pangasius hypophthalmus (Sauvage, 1878). http://www.fao.org/fishery/culturedspecies/Pangasius_hypophthalm us/en Farnworth, E., 1997. Fructooligosaccharides (FOS). Medicinal Food News magazine. http://www.medicinalfoodnews.com/vol01/issue6/fructo.htm Gibson, G.R., Roberfroid MB., 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics.J Nutr 125:1401–1412 Hui-yuan, L.V., Zhi-gang. Z., Rudeaux. F., Respondek. F., 2007. Effect of dietary short chain FOS on intestine microflora, mortality and growth performance of male Oreochromis aureus x female O.niloticus. Chinese Journal of Animal Nutrition. Mahious, A.S., F.J.Gatesoupe., M.Herv., R.Metailler and F.Ollevier.,2006. Effect of dietary inulin and oligosaccharides as prebiotics for weaning turbot, Psetta maxima (Linnaeus, C.1758). Aquaculture International. Thuong. N. V., 2008. Classification of the Pangasianodon hypophthalmus in the Mekong River. Scientific magazine 1, 84-89pp. Can Tho University (In Vietnamese) Renjie, L., Shidi. S., and Bangjie. Z., 2010. The effect of FOS on blood RBC count and digestive enzyme activities of oxyeleotris lineolatus. African Journal of Microbiology Research. Soleimani. N., Hoseinifar. S. H., Merrifield. D L., Barati. MAbadi. Z. H., 2012. Dietary supplementation of fructooligosaccharide (FOS) improves the innate immune response, stress resistance, digestive enzyme activities and growth performance of Caspian roach (Rutilus rutilus) fry. Fish and shell fish immunology 32, issue 2, 316-321 pp. 18 Directorate of fisheries., 2013. Conference to review the production and consumption of fish in 2012 and 2013 deployment tasks. http://www.fistenet.gov.vn/b-tin-tuc-su-kien/a-tin-van/hoi-nghitong-ket-san-xuat-tieu-thu-ca-tra-nam-2012-va-trien-khai-nhiem-vunam-2013 Xu. Z. R., C. H. Hu., M. S. Xia., X. A. Zhan and M. Q. Wang., 2003. Effect of dietary Fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of male broilers. Poultry Science. Vol 82, 1030-1036 pp. Xu. Z. R., X. T. Zou., C. H. Hu., M. S. Xia., X. A. Zhan and M. Q. Wang., 2003. Effect of dietary Fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of growing pigs. Chinese Journal of Veterinary Science. Vol 82, 1784-1789 pp. Xu.B., Wang.Y., Li.J., Lin.Q., 2009. Effect of prebiotic xylooligosaccharides on growth performances and digestive enzyme activities of allogynogenetic crucian carp (Carassius auratus gibelio). Fish Physiology Biochemistry Journal. Vol 35, 351-357 pp. Yun. J.W., Song. S. K., 1999. Enzymatic production of fructooligosaccharides from sucrose. Carbohydrate biotechnology protocols. Vol 10, 141-151 pp. Science and technology network Ho Chi Minh., 2010. It should be correctly the prebiotic. www.cesti.gov.vn/su-i-ngu-n-tri-th-c/n-n-hi-u-ung-v-prebiotic.html Wang. X., Gibson.G.R., 1993. Effect of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. Journal of Applied Bacteriology. Vol 75, 375-380 pp. Raggi. T., Gatlin III. D. M., 2012. Prebiotics have limited effects on nutrient digestibility of a diet based on fish meal and soybean meal in goldfish. North American Journal of Aquaculture. Vol 74, 400407 pp. Enzyme essentials., 2013. Proteases in enzyme therapy. http://www.enzymeessentials.com/HTML/proteases.html Rivero-Urgell. M., Santamaria-Orleans. A., 2001. Oligosaccharides: application in infant food. Early Human Develop. Vol 65, 43-52 pp. 19 Boyd.C.E., 1998. Water quality for pond aquaculture. Research and Development 37pp. Family health news and nutrition., 2006. Finding bacteria friendly http://www.familyhealthnews.com/articles-bacteria-friendly.html 20 APPENDICES Appendix 1: Enzyme analysis Protein (Bradford, 1976) Bradford solution was diluted times by distilled water; BSA solution was prepared 1mg/mL H2O; standard solution was prepared as follows: Cuvete BSA (µL) H20 (µL) Protein content (µg/µL) Blank 30 28 0,066 28 0,066 25 0,16 25 0,16 10 20 0,33 10 20 0,33 15 15 0,5 15 15 0,5 20 10 0,66 10 20 10 0,66 11 25 0,83 12 25 0,83 13 30 14 30 Added 30µL mixture sample solution into the separate cuvette; 1mL of Bradford reagent in sample solution and standard solution; measured at wavelength 595nm by using the spectrometer (model Heliso epsilon); protein concentration in the sample is calculated based on the standard curve 21 α – Amylase (Bernfeld, 1951) Preparation of standard Maltose concentration (µM) Maltose 5µM (µL) 40 80 100 160 200 H2O (µL) 200 160 120 100 40 - Assay (duplicate) Sample Blank Supernatant 10 µL - H2O 90 µL 100 µL Incubate at 25oC in minutes 100 µL Starch 1% 100 µL Incubate at 25oC in minutes 200 µL Substrate 200 µL Incubate at 100oC in 10 minutes 2mL H2O 2mL Read at 540nm Maltose concentrate hydrolyzed is calculating base on the regression y=ax+b where y is Abs of the sample. Amylase activity = x/50/P*1000 (mU/min/mg protein) where P is protein concentration in the sample, x calculated based on y. 22 Pepsin (Worthington, T.M, 1982) Assay (duplicate) Chemical Blank Blank test spectrophotometer 500 500 500 µL Substrate µL µL Incubate at 37oC 100 100 µL Sample µL Incubate exactly 10 at 37oC for each sample 1000 1000 1000 µL TCA 5% µL µL 100 Sample µL Centrifugal at 5980rpm at 4oC 10 Read at wavelength 280nm (using UV cuvet) U/ml = Test ( ) Trypsin (Tseng, Grendell et Rothman, 1982) Test Blank Buffer pH 8,2 mL mL BAPNA 10 µL - Supernatant 15 µL - Measured at wavelength 407 nm within Trypsin = (∆Abs/min x 6.2850 x 1000/P) (mU/min/mg protein) P is protein concentration in the sample Chymotrypsin (Worthington, 1982) Assay (duplicate) Buffer pH 7.8 and BTTE (benzoyl-tosyl-ethyl ester) incubate maintain at 25oC during assay U/ml/min= 23 Test Blank Buffer pH 7,8 700 µL 700 µL BTEE 700 µL 700 µL Supernatant 50 µL - H2O - 50 µL Read at 256 nm within Using UV cuvet Appendix 2: Chemicals preparation Enzyme analysis Crush solution: Buffer pH 6.9: KH2PO4 20 mM NaCl mM α - amylase (Bernfeld, 1951) - Sodium phosphate buffer pH 6.9: NaH2PO4 20 mM NaCl mM - Substrate: 3, 5-dinitrosalisylic acid (1g/50 mL H2O) 30g sodium potassium tartrate tetrahydrate 10 mL NaOH 2M Added distilled water enough 100 mL - Starch 1%: 100mg/10 mL sodium phosphate pH 6.9, incubated at 25oC in 4-5 minutes before using. - Standard solution Maltose µM: 18mg/10mL H2O Pepsine (Worthington, T.M, 1982) HCl 1N: 20ml HCl 37% added distilled water enough 240ml TCA 5%: 5g Trichloroacetic acid + 100ml distilled water 24 Substrate: 2.6 g Hemoglobin (sigma H2656)2% + 100 ml distilled water and the solution was mixed at 60oC in minutes. 80ml of solution were added into 60ml HCl 1Nand stored at 0-4oC. PCA media: 26.5g anhydrous PCA media/L distilled water Saline peptone water: 8.5g NaCl/L of distilled water Trypsin (Tseng, Grendell et Rothman, 1982) Buffer pH 8.2: Tris HCl 50 mL CaCl2 20 mM BAPNA 0.1M (Nα-Benzoyl-DL Arginine P-nitroanilide (B 4875)) for 10.87 mg/250µL DMSO solution Buffer and BAPNA were incubated at 25oC during the process Chymotrypsine (Worthington, 1982) Methanol 50%: 60mL methanol + 50mL H2O Buffer pH 7.8: Tris HCl 80mL + CaCl2 100mM BTEE – N-Benzoyl-Tyrosine ethyl ester (Sigma B6125): 16.8 mg/50 mL methanol 50% Buffer pH 7.8 and BTEE were incubated at 25oC during the process. 25 Appendix 3: Total bacterial count Table 7.1: Total bacterial count (unit: log (TVC)) Treatment Control 0.5% 1.0% 1.5% 2.0% Day 5.58±0.07a 5.49±0.41a 5.67±0.09a 5.39±0.34a 5.65±0.06a Day 5.62±0.08a 5.68±0.13a 5.65±0.11a 5.64±0.10a 5.65±0.04a Day 5.63±0.11a 5.73±0.09a 5.66±0.13a 5.70±0.05a 5.65±0.08a Day 5.66±0.14a 5.81±0.17a 5.70±0.19a 5.76±0.15a 5.67±0.07a Day 10 5.70±0.15a 5.95±0.38a 5.91±0.48a 6.19±0.49a 5.76±0.19a Day 30 6.10±0.58a 6.73±0.16b 6.66±0.06ab 6.28±0.26ab 6.29±0.28ab Day 60 6.58±0.15b 6.98±0.07c 7.02±0.07c 6.30±0.03a 6.39±0.17ab Day 90 6.60±0.16a 7.20±0.26b 7.30±0.04b 6.49±0.13a 6.52±0.27a Note: Values are presented as mean± STD. Mean within a column and followed by same alphabet are not significantly different (Ducan test, p[...]... Evaluated the total bacteria in the intestine of striped catfish fed with different doses of FOS 1 CHAPTER 2 LITERATURE REVIEW 2.1 Striped Catfish (Pangasianodon hypophthalmus) Striped catfish (Pangasianodon hypophthalmus) have compressed body with a short dorsal fin (one or two spines), a developed adipose fin, long anus, strong pectoral spines and two pairs of barbels There are six branched dorsal fin...CHAPTER 1 INTRODUCTION 1.1 General introduction In the recent years, many countries in the word including Vietnam have been developed aquaculture to contribute to the food consumption Compared to many species that cultured in the Mekong Delta, striped catfish is one of important export products Until the end of 2012, seed production of striped catfish was nearly 4.6 billion fingerlings (increase... diet contained oligofructose was highest and the growth of Bacillus sp than the others In 2007, the research of Hui Hui-yuan et al about Effect of dietary short chain fructo-oligosaccharides (scFOS) on hybrid tilapia was done to find out the suitable concentrations (control, 0.8, 1.2gKg-1) of scFOS And they concluded that dietary FOS had beneficial effects on growth, FCR, and intestinal microflora... breaks down long chain of starch into oligosaccharides, and maltose will degrade oligosaccharides into monosaccharides Amylase present mostly in the intestine of omnivorous fish Because of the important function of these enzymes, there are many researches were done to provide the information on digestive enzymes activity in fish that fed with different kind of prebiotics Huong and Tu also mention in some... applied 0.5 and 1% of FOS administrated These results supported that the supplementation 0.5 and 1% FOS-diet have the positive effect on the digestive system of striped catfish If applied more FOS such as 1.5 and 2%, there were no significant effect The farmers are recommended to apply 0.5 and 1% of FOS diet to increase the digestibility of striped catfish fingerling 5.2 Recommendations Conducting the experiment... objectives The aim of the study is to find out the activity levels of enzymes in stomach and intestine of striped catfish fed with various doses of FOS in order to enhance the growth and feed utilization 1.3 Research contents - Evaluated the enzymes activities in the stomach (pepsine and amylase) and in the intestine (trypsine, chymotrypsine and amylase) of striped catfish fed with different doses of FOS - Evaluated... grow-out production was about 1,255,500 tons, and export turnover was about 1.744 billion USD (decrease 3.4% compare to 2011) (Directorate of Fisheries, 2012) Since Vietnam becomes number one striped catfish export country, striped catfish become the important species Many research about nutrition, genetic, physiology, and diseases were investigated Striped catfish is intensively cultured in pond of about... rotation and incubate at 37oC in 48 or 72 hours 7 After 48 or 72 hours, the colonies were counted (acceptable rate is from 25 to 250 colonies /disk) Bacteria density is calculated by using the formula: A= Note: A: total colonies (CFU) in 1g of fish sample N: total colonies counted in disks ni: number of disks at concentration i V: volume of sample implant for one disk fi: diluted solution at concentration... Application of FOS in terrestrial animals There are many researches about applied FOS into terrestrial diet Example, the experiment of Xu et al., 2003 about the effect of different concentrations of FOS (0, 2, 4, 6 gKg-1) of growing pig, they founded that supplementation with 4 and 6g/kg FOS significantly improved average daily gain and feed conversion ratio As compared to control, supplementation with... http://www.medicinalfoodnews.com/vol01/issue6/fructo.htm Gibson, G.R., Roberfroid MB., 1995 Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics.J Nutr 125:1401–1412 Hui-yuan, L.V., Zhi-gang Z., Rudeaux F., Respondek F., 2007 Effect of dietary short chain FOS on intestine microflora, mortality and growth performance of male Oreochromis aureus x female O.niloticus Chinese Journal of Animal Nutrition Mahious, A.S., . 5 CHAPTER 3: Research methodology 6 3. 1 Time and place 6 3. 2 Materials 6 3. 3 Methods 6 3. 3.1 Experimental design 6 3. 3.2 Evaluate the total microorganisms in intestine 7 3. 3 .3 Evaluate. NO 2 - (mg/L) Control 0.40±0.22 0 .38 ±0.21 0.5% 0 .39 ±0. 23 0.42±0.22 1.0% 0. 43 0.26 0 .38 ±0.24 1.5% 0 .39 ±0.25 0 .36 ±0. 23 2.0% 0. 43 0.27 0 .37 ±0.18 Note: Values are presented as. NaCl, etc. 3. 3 Methods 3. 3.1 Experimental design There were 5 treatments in this study: + Treatment 1: without FOS + Treatment 2: add 0.5% of FOS in the diet + Treatment 3: add 1% of