Use of probiotic as growth promoter in african catfish Clarias gariepinus (Burchell, 1822) Juveniles

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1 AMERICAN JOURNAL OF BIOTECHNOLOGY AND MOLECULAR SCIENCES ISSN Print: , ISSN Online: , doi: /ajbms , ScienceHuβ, Use of probiotic as growth promoter in african catfish Clarias gariepinus (Burchell, 1822) Juveniles 1 Nwanna, L. C., Tope-Jegede, O. H. and 2 Jegede T. 1 Department of Fisheries & Aquaculture Technology, Federal University of Technology, Akure, Ondo State. 2 Department of Fisheries and Aquaculture Management, Ekiti State University, Ado Ekiti, Ekiti State, Correspondence:temitopejegede@yahoo.com ABSTRACT This study was conducted to evaluate the effect of Lactobacillus plantarum on the growth and feed utilization of Clarias gariepinus juveniles. Two hundred and twenty five (225) Clarias gariepinus juveniles, average body weight of 9.2g , were used for the investigation. The Fish were randomly distributed into five groups each at a rate of forty five (45) fish per treatment. The test diets were fed to the fish in 15 aquaria with three replicates per treatment. Diet 1 was the control diet (basal diet). Diets 2, 3, 4 and 5 were the basal diet plus 10 3 cfu/g, 10 5 cfu/g, 10 7 cfu/g and 10 9 cfu/g of (Lactobacillus plantarum) respectively. The fish were fed to apparent satiation twice daily for seventy (70) days. At the end of the experiment, the results revealed that the four groups fed Lactobacillus plantarum supplemented diets showed significant increase (P > 0.05) in body weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio. It may be concluded that Lactobacillus plantarum can be used as a growth promoter in Clarias gariepinus juveniles. Key words: Probiotics, Lactobacillus plantarum, Clarias gariepinus, growth promoter, nutrient utilization, MRS. INTRODUCTION Proper nutrition has long been recognized as a critical factor in promoting normal growth and sustaining fish health. Prepared diets not only provide the essential nutrients that are required for normal physiological functioning but also may serve as the medium by which fish receive other components that may affect their health (Gatlin 2002). But most of the intensive aquaculture farms have been facing major hindrance because of disease outbreak, poor growth and low survival of fish. The aquaculture industry has experienced widespread use of antimicrobial drugs in their practices while the use of such products have an obvious benefit to treat animals infected by bacterial disease, these drugs have been either prophylactic (preventative), or for growth enhancement (Van den Bogaard and Stobberingh, 2000). There is an urgent need in aquaculture to develop microbial control strategies to improve growth and one of the alternatives is probiotics. The use of probiotics in aquaculture has not only resulted in the reduction of use of harmful antimicrobial compounds, particularly the antibiotics but also improved the appetite and bio-growth performance of the farmed species. (Gatesoupe 1999 and Naik et al., 1999). Several reports suggest that probiotics supplementation can reduce the cost of culture by improving the growth and feed utilization efficiency of fish (Swain et al., 1996; Bogut et al., 1998; Ghosh et al., 2003; Wang & Xu 2006; Marzuurkiewicz et al., 2007). Therefore in order to make aquaculture products safe for human consumption, it is important to develop an alternative to antibiotics by using living microbial cells as additives in fish feed to control pathogens and improve growth in fishes. Though extensive research had been conducted on the nutrient requirements and feeding regimes of Clarias gariepinus (Jambo & Alfred-Ockiya 2009) but the effects of probiotics as growth promoter in Clarias gariepinus are yet to be fully investigated. Therefore, the objective of this study is to investigate the effects of Lactobacillus plantarum as a growth promoter in Clarias gariepinus juveniles. The results of this research will undoubtedly help to reduce antibiotics

2 use in aquaculture and will make aquaculture products more acceptable to consumers. MATERIALS AND METHODS Experimental fish: Two hundred and twenty five (225) Clarias gariepinus juveniles (9.2g ) were obtained from fish hatchery of The Federal University of Technology, Akure, Ondo State, Nigeria and were transported life to the Fisheries laboratory of the same University. The Fish were randomly distributed into five groups each at a rate of forty five (45) fish per treatment. The treatments were five (5) altogether. Each treatment were replicated and subjected to similar conditions. Fifteen (15) juveniles were stocked into each glass aquarium with three (3) replicates per treatment. Diet 1 was the control diet (basal diet). Diets 2, 3, 4 and 5 were the basal diet plus 10 3 cfu/g, 10 5 cfu/g, 10 7 cfu/g and 10 9 cfu/g of (Lactobacillus plantarum) respectively. The fish were fed to apparent satiation twice daily for seventy (70) days. Experimental system: The feeding trial was conducted in full glass aquaria (75x45x40) cm in dimension. Two-thirds (2/3) of each tank were filled with clean oxygenated water. Fish were acclimated in the glass tanks for seven (7) days without feeding. Each treatment group of fish was fed to satiation twice daily between hours and hours for 70 days. All fish were removed from each aquarium every 14 days and batch-weighed and the amount of feed was adjusted accordingly. Siphoning of faecal samples for protein digestibility was done during the last 15 days of the experimental period. Any uneaten feed or faeces from each tank was carefully removed by siphoning about 30 minutes after the last feeding. Collected faeces were then filtered, sun dried, kept in airtight containers and later analyzed for their proximate composition. Dead fish were removed, counted and recorded on daily basis. Preparation of probiotic microorganism used for the study: One adult Clarias gariepinus 1.4kg was purchased from fishermen at Ala River in Akure, Ondo State, Nigeria. The fish was washed with sterile distilled water to reduce external surface contamination. The fish was dissected with a sterile scapel on a dissecting board overlayed with sterile alluminium foil. The gastrointestinal tract was removed and measured to be 47cm. It was then divided into three sectional parts namely: foregut, midgut and hindgut. The contents of each gut were aseptically emptied into a sterile petri dish where 1ml was obtained with a sterile pipette and serially diluted. 1ml each of 10 3, 10 5, 10 7 and 10 9 were pour plated with Mann Rogosa and Sharpe (MRS) Agar, sterilized at121 o C for 15minutes but cooled to about 45 o C after which the seeded plates were swirled and allowed to gel. The plates were incubated in an anaerobic jar at an inverted position placed in an incubator at 37 o C for 72hours. After counting, colonies on different plates were picked with sterile inoculating loop and purified by successive streaking on freshly prepared (MRS agar). All the purified isolates were tested for catalase. All the negative catalase colonies were then subjected to gram reaction. Other physiological tests and biochemical tests were conducted to identify the pure isolates to specie level using the criteria of Claus (1992) and Holt et al., (1994). A drop of hydrogen peroxide was put on a clean slide. An inoculating loop was flamed to pick a small innoculum of the test organism and smeared it with hydrogen peroxide. The ones that bubbled after reacting with hydrogen peroxide were catalase positive and were discarded. The ones that did not bubble were catalase negative and were retained for further identification. Previously sterilized innoculating loop was used to obtain innoculum and smeared on a greese free slide; this was air dried and heat fixed. The slide was flooded with crystal violet for one minute, rinsed with sterile distilled water, flooded with Lugohl s iodine for one minute, rinsed with sterile distilled water and decolorized with ethanol then counter stained with Safranin for 30 seconds, washed with sterile distilled water, air dried and a drop of oil immersion was put and then observed under the microscope using the oil immersion objective as described by Ronald (2001). 1 gram of peptone, 1 gram of sugar and a pinch of phenol red as indicator were dissolved in 100ml of water. 5ml was dispensed into test tube containing the sugars, Durham tube was inverted into the test tube, cotton plugged, sealed with alluminium foil and sterilized at C for 15 minutes after which it was uploaded from autoclave and allowed to cool to about 45 o C. The test organism was inoculated into the tubes with a sterile inoculating loop and incubated at 37 0 C for 48 hours. Reactions of the organism after incubation determined their characteristics to carbohydrate utilization. Positive reactions were indicated with change in colour from pink to pale yellow without gas accumulation in the Durham tube. Negative result was where no colour changes were observed. Feed preparation and concentration of probiotic organism for inoculating into feed: After serial 18

3 transfer of Lactobacillus plantarum in fresh MRS broth at 37 0 C for 24 hours, serial dilution up to 10 9 was done. 10 3, 10 5, 10 7 and 10 9 were chosen to be inoculated into feed. To get this done, the selected tubes were centrifuged at 5,000 rpm (revolution/minute) for 15 minutes. The concentrated cells were washed three times with sterile 0.5% NaCl solution. The number of cells was counted by plating onto MRS agar medium and the cell number ranges from 4.8 x 10 5 cfu/g to1.5 x 10 9 cfu/g.these were introduced separately each into the diets. Five diets were formulated containing fish meal, soyabean meal, groundnut cake, maize, vegetable oil, methionine, vitamin mineral premix, lysine and starch to provide 40% crude protein (Table 1). Diet 1 was the control diet (basal diet). Diets 2, 3, 4 and 5 are the basal diet plus 10 3 cfu/g, 10 5 cfu/g, 10 7 cfu/g and 10 9 cfu/g of probiotics (Lactobacillus plantarum) respectively. Soya beans were first of all picked in other to remove dirty particles after which it was washed and then boiled in a pressure cooker at C for 25 minutes to destroy the antinutrients present, after which it was sun dried on a raised platform for 72 hours. Ingredients were then weighed on a sensitive weighing balance, followed by thorough mixing of the dietary ingredients in a Hobart A-200 pelleting and mixing machine to obtain a mixer with die of 1mm in diameter. The diets were immediately air-dried at ambient temperature for 72 hours; sieved into small pellet sizes, packed in transparent air-tight containers, labeled and stored. The five diets make up treatments 1-5. The gross composition and chemical analysis of the diets is presented in Table 1. Chemical analysis of experimental diets, faecal samples and whole body fish: The proximate composition of experimental diets, faecal samples and whole body was analysed in triplicates according to the standard methods of AOAC (1990) for moisture, protein, fat, ash, fibre and carbohydrate. Moisture content was estimated by oven drying the samples at C to constant weight and crude protein percent were calculated by estimating nitrogen content by micro-kjeldahl method and multiplying with a factor Ether extract was determined by solvent extraction with petroleum ether, boiling point C, for 10-12h. Total ash content was determined by incinerating the sample at C for 6h and crude fibre by acid digestion (1.25%). The total carbohydrate (%) of the feed and whole body fish was calculated as: 100 (crude protein + Ether extract + total ash ). Water quality parameters: Over the duration of the feeding trial (70 days), the mean water quality parameters for temperature was ( C); Dissolved oxygen ( mg/L) and ph ( ) were obtained. Water temperature and dissolved oxygen were monitored every two weeks using mercury in glass thermometer ( 0 C) for temperature and a dissolved oxygen meter (YSI model 58, Yellow Spring Instrument Co., Yellow Spring, OH, USA) for dissolved oxygen; ph was also monitored once in two weeks using ph meter (Digital Mini-pH Meter, model 55, Fisher Scientific, Denver, CO, USA). All the water quality parameters during the experiment period were found to be in the optimum range of fish rearing (Environment Protection Authority, EPA (2003). Growth indices: Growth performance and nutrient utilization of the fish used in the experiment was calculated using the following parameters according to Olivera- Novoa et al., (1990). Weight gain (%) = 100 (final body weight initial body weight) Initial body weight SGR (%/day) = (100 loge final body weight loge initial body weight) Time (day) FCR = Total dry feed consumed by fish (g) Total weight gain by fish (g) PER= Weight gain per fish (g) Protein intake (g) The value obtained for AIA was used as indicator in the calculation of digestibility coefficient. The digestibility was calculated as: Apparent protein digestibility = feed) x (% nutrient in faeces) (% AIA in faeces) x (% nutrient in feeds) (% AIA in Statistical analysis: At the end of the experiment, data were analyzed statistically using one way analysis of variance (ANOVA) and tested by Duncan s Multiple Range Test carried out at P = 0.05 to separate the differences among the means using SPSS software version

4 Table 1: Composition and proximate chemical analysis of experimental diets (40%cp) (g/100g dry matter) Ingredients Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Fish meal (65% CP) Soya bean meal (45% CP) Groundnut cake (48% CP) Maize (10% CP) Vegetable oil Methionine Starch Vitamin premix Lysine Lactobacillus plantarum (cfu/g) Proximate composition (gkg -1 ) Moisture Crude protein Total carbohydrate Ether extract Total ash Crude fibre Key: cfu = colony forming unit. 1 Fish pre-mix. Colborne Dawes Ltd. United Kingdod.: Vitamin A, 1600 IU; Vitamin D, 2400 IU; Vitanin E 160mg; Vitamin K, 16mg; thiamin, 36mg; riboflavin, 48mg; pyridoxine, 24mg; niacin, 288mg; biotin, 1.3mg; cyanocobalamin, 48mg; ascorbic acid, 720mg; choline chloride, 320mg; calcium, 5.2mg; cobalt, 3.2mg; iodine, 4.8mg; copper, 8mg; iron, 32mg; manganese, 76mg; zinc, 160mg; EEndox (antioxidant) 200mg. RESULTS Growth performance and nutrient utilization: Crude protein of 40% was used in the formulation of the test diets (Table 1). The summary of the growth responses and nutrient utilization parameters are shown in (Table 2). There was significant difference (P< 0.05) in the feed conversion ratio. Fish fed diets supplemented with Lactobacillus plantarum had better feed conversion ratio (FCR) when compared with those fed diet 1(control), with fish fed diet 2(10 3 cfu/g) having the best feed conversion ratio of However, the values obtained for Lactobacillus plantarum supplemented diets were not significantly different from one another.there was significant difference (P< 0.05) in the protein efficiency ratio of the fish (PER). Diet 2 (10 3 cfu/g) had the best protein efficiency ratio of but there was no significant difference in the protein efficiency ratio of fish fed diets 1 and 5. The result followed the same pattern for specific growth rate (SGR). Fish fed diet 2 had the best specific growth rate of There was also no significant difference in the values obtained for specific growth rate of fish fed diets 1 and 5. The feed were well digested in all the diets but the highest 20

5 apparent protein digestibility of 84.91% was recorded for fish supplemented with Lactobacillus plantarum at (10 3 cfu/g). Summarily, the overall result of nutrient utilization and growth parameters has shown fish fed diet supplemented with Lactobacillus plantarum at 10 3 cfu/g to be the best in terms of all the parameters. Fish fed diet containing Lactobacillus plantarum at 10 3 cfu/g grew from initial mean weight of 9.52 to having a mean weight gain of as compared with a mean weight gain of in fish fed control diet over a period of 70 days. Body composition: The whole body composition (% Dry Weight) of Clarias gariepinus juveniles fed experimental diets is shown in (Table 3). The results of chemical analysis of the fish showed significant Table 2: Growth performance and nutrient utilization by Clarias gariepinus juveniles. Diets Growth parameters DIET1 Control DIET 2 L. plantarum DIE T 3 L. plantarum Initial mean weight (g) Final mean weight(g) Mean weight gain(g) Mean feed Intake(g) FCR SGR (%/day) PER Apparent Protein Digestibility Survival 8.87 a a cfu/g 9.52 c b a b c 1.53 b 1.26 a a 2.22 b a b a b variations in their body composition. There was significant difference (P<0.05) in the crude protein content of all the fish with fish fed diet 2(10 3 cfu/g) having the highest of 68.66%. There was also significant difference (P<0.05) in the lipid values of some of the fish with those fed control diet having the highest of 6.74%. These changes in protein and lipid contents in the fish body could be linked with changes in their synthesis, deposition rate in muscle and or different growth rate. There was no significant difference (P<0.05) in the ash content of fish fed diets 2, 3 and 4 while that of fish fed diets 1, and 5 also followed the same pattern. The were slight variations in the CHO content of all the fish. Fibre was not detected in any of the fish cfu/g 9.34 bc b b a 1.26 a b b ab DIET 4 L. plantarum 10 7 cfu/g 9.05 ab b b a 1.28 a b b ab DIET 5 L. plantarum 10 9 cfu/g 9.31 abc a a b 1.45 a 1.68 a a a KEY: FCR=Food conversion ratio; SGR=Specific growth rate; PER=Protein efficiency ratio.uses Harmonic Mean Sample Size = Subset for alpha = 0.05.The values in each row having the same superscript are not significantly different (P> 0.05). 21

6 Table 3: Proximate chemical analysis (% on dry matter basis) of whole body of Clarias gariepinus juveniles. Chemical analysis (%) Diets Diet 1 Control Diet 2 (10 3 cfu/g) Diet 3 (10 5 cfu/g) Diet 4 (10 7 cfu/g) Diet 5 (10 9 cfu/g) Moisture content Crude protein b a 7 a e a d a c b b Total lipid 6.74 b a a b 5.49 a Carbohydrate 0.02 ab 0.01 a 0.03 b 0.02 ab 0.02 ab Ash 5.46 b a a a b DISCUSSION The crude protein of 40% used in the formulation of the experimental diets in this study satisfied the nutrient requirements for intensively cultured Clarias gariepinus recommended by N.R.C (1993). The statistical analysis of different growth parameters of Clarias gariepinus juveniles at the end of the experimental period indicated a significant increase in the body weight gain between the four groups supplemented with Lactobacillus plantarum at 10 3 cfu/g, 10 5 cfu/g, 10 7 cfu/g and 10 9 cfu/g respectively. Fish fed diet supplemented with Lactobacillus plantarum at 10 3 cfu/g (Diet 2) were the fastest grower having the final mean weight of 45.32g from the initial mean weight of 9.52g In other words, the fish was able to gain 35.80g at the end of the feeding trial (70 days) compared to fish fed diets 1 (control) which gained 17.5g. The results of the growth parameters indicated acceptance of probiotic included in the diets. The obtained results could be attributed to the ability of Lactobacillus plantarum to adhere to the gastrointestinal tract of Clarias gariepinus juveniles producing a wide range of relevant digestive enzymes (amylase, lipase and protease) which have the ability to denaturate the indigestible components in the diets, detoxify potentially harmful components of the diets and to produce a lot of essential vitamin B. complex members particularly Biotin and vitamin B12, which enhanced feed utilization and digestibility. These results support those of Kennedy et al., (1998) who used B. subtilis in the food of common snook, Centropomus undecimalis to enhance protease level that increased feed absorption. The best feed conversion ratio (FCR) value observed with probiotic supplemented diet suggests that, addition of probotic improved feed utilization. Lara-Flores et al., (2003) similarly reported that total feed intake by fish consequently had direct effect on feed conversion ratio, protein efficiency ratio and specific growth rate. The highest result of protein efficiency ratio (PER), obtained from fish fed diet 2(10 3 cfu/g) indicated that supplementing diets with probotic significantly improved protein utilization in Clarias gariepinus juveniles, this contributes to optimizing protein used for growth which is the most expensive feed nutrient. The improvements in the biological value of the probotic supplemented diets have demonstrated its effectiveness in converting protein to flesh in fish. This agreed with the results obtained by Ringo and Gatesoupe, (1998). The better feed intake in probotic supplemented diets may have been due to the increased fish appetite, triggered by quick digestion, resulting in a higher feed intake and therefore increased growth. The results of chemical analysis of the fish showed significant variations (P < 0.05) in their body composition. Probotic supplementation significantly affected the whole fish body composition. Crude protein content of the fish is in line with the findings of Degani (1989) who reported that proteins are the major material in fish tissue and could make up to 65-75% of the total organic materials on a dry matter basis. (Abdel-Tawwab et al., 2006) added that changes in protein and lipid contents in the fish body could be linked with changes in their synthesis, 22

7 deposition rate in muscle and or different growth rate. The fat content of the fish was not high which is good for human consumption because fatty fish are known to deteriorate due to oxidation of fat (lipid) content of the flesh resulting in rancidity. This is in line with the findings of El-Faer et al.,(1992) who worked on mineral and proximate composition of some commercially important fish of the Arabian Gulf. Ranges in the ash value could be due to variation in the environment and ingested metabolic rate. CONCLUSION The present study revealed that Lactobacillus plantarum improved growth performance and feed efficiency of Clarias gariepinus juveniles. Also, performance of the fish was maximized with inclusion level of Lactobacillus plantarum at 10 3 cfu/g. It may be concluded that, Lactobacillus plantarum could act as growth promoter in Clarias gariepinus juveniles. The use of Lactobacillus plantarum in aquaculture can produce healthy and safe fish for human consumption. REFERENCES Abdel Tawwab, M., Y.A.E. Khattab, M.H. Ahmed and A. M.E. Shabby (2006). Compensatory growth, feed utilization, whole body composition and haematological changes in starved juvenile Nile tilapia, Oreochromis niloticus (L). Journal of Applied Aquaculture, 18(3): Association of Official Analytical Chemists (AOAC) (1990): Official Methods of Analysis. 15 th edition, Association of Official Analytical Chemists. Arlington, Virginia, U.S.A. 1094p. Bogut, I.,Milakovic, Z., Bukvic, Z., Brkic, S. & Zimmer, R. (1998).Influence of probiotics Streptococcus faecium M74 on growth and content of intestinal micro-flora in carp Cyprinus carpio. Czech J. Anim. Sci., 43, Carnevali, O. D., Sulpizio, L., Gioacchini, R., Olivotto, I. G. & Silvi, S. (2006). Growth improvement by probiotic in European sea bass juveniles (Dicentrarchus labrax, L.), with particular attention to IGF-1, myostatin and cortisol gene expression. Aquaculture, 258, Claus, D.C. (1992). A standard Gram-staining procedure, World Journal of Microbiology and Biotechnology 8: Degani, G. (1989). The effect of different protein level and temperature and feed utilization, growth and body composition of Clarias gariepinus (Burchell 1822). Aquaculture 76: Duncan, D.B. (1955). Multiple ranges and multiple F-test. Biometrics, 11:1-42. El-Faer, M. Z., Rawdah, T. N., Khudre, M. A. and Arab, M. (1992). Mineral and proximate f some commercially important fish of the Arabian Gulf. Food Chemistry 45: El-Faer, M. Z., Rawdah, T. N., Khudre, M. A. and Arab, M. (1992). Mineral and proximate f some commercially important fish of the Arabian Gulf. Food Chemistry 45: Environment Protection Authority. (2003).Water Quality Policy an overview and a copy of the Water Quality policy with an accompanying explanatory report. Retrieved from Gatesoupe, F. J. (1999). The use of probiotics in aquaculture. Aquaculture, 180: Gatlin, D. M. III (2002). Nutrition and fish health In Halver, J. E..; Hardy, R. W. (Eds). Fish Nutrition. Academic press, San Diego, CA, USA, pp Ghosh, K., Sen, S. K. & Ray, A. K. (2003). Supplementation of an isolated fish gut bacterium, Bacillus circulans, in formulated diets for rohu, Labeo rohita, fingerlings. Israeli J. Aquacult., 55, Holt, J. G., Kneg, N. R., Sneath, P. H., Stanley, J. T., and Williams, S. T. (1994). Bergey s manual of determinative bacteriology, 9 th edition. Williams and Wilkins, Publishers, Baltimore pp 7-3. Jamabo, N. A. and Alfred-Ockiya, J. F. ( 2009). Dietary protein requirements and growth performance of Clarias gariepinus (Burchell, 1822) fingerlings. Kesarcodi-Watson, A., Kaspar, H., Lategan, M. J. & Gibson, L. (2008). Probiotics in aquaculture: the need, principles and mechanisms of action and screening process. Aquaculture, 274, Lara-Flores, M., Olivera-Novoa, B.E., Guzman-Mendez and W. Lopez-Madrid. (2003). Use of bacteria Streptococcus faecum and Lactobacillus acidophilus, and the yeast Saccharomyces cerevsiae as growth promoters in the Nile tilapia (Oreochromis niloticus). Aquaculture 216: Mazurkiewicz, J. Przybyl, A., Sip, A. & Grajek, W. (2007). Effect of Carnobacterium divergens and Enterococcus hirae as probiotics bacteria in feed for common carp, Cyprinus carpio. L. Arch. Polish Fish., 15, Mohapatra, S., Sahu, N. P., Pal, A. K., Prusty, A. K., Kumar, V. & Kumar, S. (2010). Haemato-immunology and histo-architectural changes in Labeo rohita fingerlings: Effect of dietary aflatoxin and mould inhibitor. Fish physiol. Biochem., doi: /s Naik, A. T. R., Murthy, H. S. & Ramesha, T. J. (1999). Effect of graded levels of G-probiotic on growth, survival and feed conversion of tilapia, Oreochromis mossambicus. Fish. Technol., 36,

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