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A Probiotic Cocktail as a Growth Promoter in Nile Tilapia (Oreochromis niloticus) a
b
M. S. Ayyat , Howaida M. Labib & Hemat K. Mahmoud
a
a
Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig, Egypt b
Department of Microbiology, Faculty of Agriculture, Zagazig University, Zagazig, Egypt Published online: 21 Aug 2014.
To cite this article: M. S. Ayyat, Howaida M. Labib & Hemat K. Mahmoud (2014) A Probiotic Cocktail as a Growth Promoter in Nile Tilapia (Oreochromis niloticus), Journal of Applied Aquaculture, 26:3, 208-215, DOI: 10.1080/10454438.2014.934164 To link to this article: http://dx.doi.org/10.1080/10454438.2014.934164
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Journal of Applied Aquaculture, 26:208–215, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1045-4438 print/1545-0805 online DOI: 10.1080/10454438.2014.934164
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A Probiotic Cocktail as a Growth Promoter in Nile Tilapia (Oreochromis niloticus) M. S. AYYAT1 , HOWAIDA M. LABIB2 , and HEMAT K. MAHMOUD1 1
Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig, Egypt 2 Department of Microbiology, Faculty of Agriculture, Zagazig University, Zagazig, Egypt
The aim of this study was to evaluate Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium bifidum, and yeast Saccharomyces cerevisiae singly and in combination as probiotic supplements in Nile tilapia (Oreochromis niloticus) fingerling diets. All the probiotic-supplemented diets resulted in growth higher than the control. Best growth rate, food consumption, and food conversion (P < 0.05) were in the group fed a cocktail of the three bacteria. Feed cost, return on weight gain, and profit margin increased in fish groups fed diets inoculated with probiotics. Supplementation with Lactobacillus acidophilus, Bifidobacterium bifiduim, and the three-bacteria cocktail were most effective in eliminating mortality in an A. hydrophila challenge. KEYWORDS Probiotic; profitability of feed additives, disease management, Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium bifidum, yeast
INTRODUCTION Bacterial diseases are responsible for severe economic losses in aquaculture (Wang et al. 2008). The indiscriminate use of antibiotics to control pathogenic microorganisms brings important changes in the micro biota of the aquaculture systems and surrounding environment, creating bacterial resistance (Resende et al. 2012) and affecting natural beneficial bacteria (He et al. 2010, 2011, 2012). A promising alternative approach for controlling fish diseases is the use of probiotics. Address correspondence to M. S. Ayyat, Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt. E-mail:
[email protected] 208
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Lactic acid bacteria (LAB) are the most commonly applied probiotics in terrestrial animal nutrition, and their use has been proposed for aquatic species (Gatesoupe 1991, 2002; Ringo & Gatesoupe 1998). LABs potentially have several probiotic properties: They may stimulate the growth of preferred microorganisms, crowd out potentially harmful bacteria, and reinforce organisms’ immune systems (Salminen et al., 1998; Villamil et al. 2002). Studies showing such effects on aquatic species have assessed strains of Lactobacillus acidophilus, L. sporogenes, L. rhamnosus, L. plantarum, Lactococcus lactis, and Pediococcus acidilactici (Strom & Ringo 1993; Gatesoupe 1991, 2002; Gatesoupe et al. 1989; Gildberg et al. 1995, 1997; Nikoskelainen et al. 2001; Planas et al. 2004). We evaluated the efficiency of using three of these bacteria (Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium bifidum) and yeast (Saccharomyces cerevisiae), separately and in combination, as a probiotic for Nile tilapia (Oreochromis niloticus).
MATERIALS AND METHODS The study was carried out at the wet Laboratory of the Animal Production Department, Zagazig University, Egypt. The experiment lasted 98 days from June to September 2011. Healthy fingerlings of Nile tilapia (Oreochromis niloticus) were provided by the Fish Hatchery of the Central Laboratory for Aquaculture Research (CLAR) at Abbassa, Egypt. Two hundred and ten fingerlings (weighing approximately 1 g after an adaptation period of three weeks under normal laboratory conditions) were randomly distributed into 21 glass aquaria (35 × 40 × 70 cm) containing 75L of water, representing seven treatments (three replicates per treatment). The photoperiod was 12 h light:12 h darkness. The water temperature was 24–28◦ C. All fish groups were fed on a basal pelleted diet consisting of fish meal 30.0%, soybean meal 20.0%, corn 20.0%, wheat bran 15.0%, alfalfa hay 10.0%, sunflower oil 2.5%, minerals mixture 0.5%, vitamin mixture 1.0%, and carboxymethyl cellulose 1.0%. The chemical composition of the diet was: crude protein 40.12%, ether extract 6.40%, crude fiber 5.32%, and gross energy 4280.0 Kcal/Kg. The feeding rate was 7% of live body weight for the first month, 5% for the second month, and 3% for the rest of the experimental period. The experimental diets were offered three times daily at 8:00, 12:00, and 17:00 hours, with uneaten feed collected by siphoning in between. Every second day, each aquarium was partially cleaned, including the fish feces, and the water was partially changed (about 50%). Probiotic bacteria Streptococcus thermophilus TH4 , Lactobacillus acidophilus YC X11 , and Bifidobacterium bifidum Bb 12 were kindly supplied by the Agricultural Microbiology Department, Zagazig University.
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Saccharomyces cerevisiae was available in the market from the Al Nuboria yeast factory. For use as a pathogenic challenge, Aeromonas hydrophila was obtained from CLAR. Probiotics were prepared by inoculation of the bacterial isolates in MRS broth for 24 h at 37◦ C for 48 h and centrifuged at 3000 rpm for 30 min (De-Man et al. 1960). One gram of commercial yeast was suspended in 9 ml of sterilized distilled water and serially diluted to 105 (Kreger-Van Rij 1984). Bacterial probiotics were incorporated into the diet and offered to fish over a 98-day period. Nile tilapia fingerlings were divided into seven groups:T1 (control) was given a diet without probiotics; T2 was fed a diet supplemented with Lactobacillus acidophilus (7 × 106 cells/100 g diet); T3 was fed a diet supplemented with Streptococcus thermophilus (8 × 107 cells/100 g diet); T4 was fed a diet supplemented with Bifidobacterium bifidum (1 × 107 cells/100 g diet); T5 was fed a diet supplemented with a threebacteria cocktail (Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium bifidum, 3.2 × 107 cells/100 g diet); T6 was fed a diet supplemented with yeast (Saccharomyces cerevisiae; 4.2 × 108 cells/100 g diet); and the last group, T7 , was fed a diet supplemented with the three-bacteria cocktail plus yeast (3.2 × 107 cells/100 g diet). All fish were individually weighed to the nearest 0.1 g at the beginning of the experiment and at biweekly intervals throughout the experimental period. Food consumption was calculated as g/fish/day by dividing the amount of food consumed each day by the number of fish in the aquarium. Food conversion ratio (FCR) was calculated according to Berger and Halver (1987). Dissolved oxygen was maintained above 7.0 mg/l with an aerator. Total ammonia nitrogen, nitrite, and pH were monitored weekly with a Hach kit model HI 83205 (multipurameter bench photometer). No water-quality issues were observed over the course of the experiment, and there were no significant differences in water quality among treatments. At the end of the study, blood samples were collected from the caudal vein of three randomly selected fish (one from each replicate) with a heparinized syringe, and the plasma was separated by centrifugation at 3000 rpm for 20 min and stored at –20◦ C for analysis of total protein; albumin (Sundeman 1964) and plasma transaminase enzymes (aspartate amino transferase [AST] and alanine amino transferase [ALT]; Reitman & Fankel 1957) were determined by using commercial kits. Economic evaluation was done according to Ayyat (1991) as the income from additional body weight gain less the additional feed cost. Other costs were assumed constant. Price of 1 kg of diet was 2.159 LE (Egyptian pound = US$0.185), and the price of selling of 1 kg live body weight of tilapia was 10.0 LE.
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As a disease challenge, A. hydrophila (0.3 ml of 107 bacterial cells ml−1 ) was injected I/P to 20 fish/treatment (Aly et al. 2008). Inoculated fish were observed daily for 10 days and mortalities recorded. The relative level of protection (RLP) among the challenged fish was determined according to Ruangroupan et al. (1986): RLP % = 100 – (treatment mortality %/control mortality %) × 100. Data were statistically analyzed with SAS (2002). Significant differences between treatments were tested with Duncan’s multiple range test (Duncan 1955).
RESULTS AND DISCUSSION All the probiotic-supplemented diets improved growth relative to the control (Table 1). Best growth rate and highest food consumption (P < 0.05) were in the group fed the diet inoculated with the three-bacteria cocktail (T5 ). There was no obvious effect of probiotics on the water quality in the treatment groups. All water parameters were stable and within acceptable ranges (Boyd & Tucker 1998). Also, Wang et al. (2008) showed that the tilapia fish supplemented with the probiotic recorded better water quality than those fed the basal diet. Survival rate and FCR over 98 days were not significantly affected by probiotic supplementation. Blood albumin and AST were significantly (P < 0.01 and 0.001) affected by probiotic supplementation, while total protein, globulin, and ALT were not affected (Table 2). Highest albumin was found in the group of fish fed the three-bacteria cocktail (T5 ). Lowest values for AST and ALT were recorded in the fish fed the Bifidobacterium bifiduim (T4 ) supplemented diet. Estimated profitability of the probiotic diets was higher than the control group (Table 3) and highest in the group fed the three-bacteria cocktail. Though feed cost increased by 24.66%, 8.22%, 9.59%, 35.62%, 9.59%, and 19.18% in fish fed diets T2 –T7 respectively, weight gain was 35.53%, 14.21%, 18.27%, 61.42%, 25.89%, and 25.89% respectively. Mortality after the Aeromonas hydrophila challenge was higher in the control than in the groups fed probiotics (Table 4). Lactobacillus acidophilus (T2 ), Bifidobacterium bifidum (T4 ), and the three-bacteria cocktail (T5 ) provided 100% RLP. The same results were obtained by Balcázar et al. (2008), Nikoskelainen et al. (2001), and Robertson et al. (2000). RLP was 75%, 50%, and 25%, respectively, in groups fed Saccharomyces cerevisiae (T6 ), the three-bacteria cocktail mixture + yeast (T7 ), and Streptococcus thermophilus (T3 ). Joborn et al. (1997) and Robertson et al. (2000) reported that groups of lactic acid bacteria (LAB) exhibit in vitro, inhibitory activities against Gram-positive and Gram-negative fish pathogens.
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1.020 ± 0.017 1.023 ± 0.018 1.017 ± 0.020 1.017 ± 0.009 1.010 ± 0.021 1.017 ± 0.020 1.027 ± 0.003 NS
T1 T2 T3 T4 T5 T6 T7 Significance ∗∗∗
20.756 ± 0.844c 27.670 ± 1.019b 23.583 ± 1.897bc 24.307 ± 0.985bc 32.783 ± 0.593a 25.760 ± 0.804b 25.830 ± 1.960b
Final weight (g)
∗∗∗
0.201 ± 0.009c 0.272 ± 0.010b 0.230 ± 0.020bc 0.238 ± 0.010bc 0.324 ± 0.006a 0.253 ± 0.008b 0.253 ± 0.020b
Daily gain (g/day)
∗∗∗
0.343 ± 0.008d 0.430 ± 0.009ab 0.375 ± 0.008c 0.378 ± 0.0148c 0.467 ± 0.0187a 0.376 ± 0.010c 0.411 ± 0.0159bc
Daily feed intake (g/day) 1.667 ± 0.087 1.584 ± 0.028 1.656 ± 0.167 1.592 ± 0.016 1.440 ± 0.033 1.490 ± 0.040 1.632 ± 0.064 NS
Feed conversion (g food/g gain)
93.33 ± 3.33 93.33 ± 3.33 86.67 ± 6.67 90.00 ± 5.77 86.67 ± 3.33 96.67 ± 3.33 96.67 ± 3.33 NS
Survival rate (%)
∗∗
2.997 ± 0.084 2.537 ± 0.049c 2.980 ± 0.081ab 2.320 ± 0.046c 3.180 ± 0.312a 2.327 ± 0.015c 2.720 ± 0.104bc
5.627 ± 0.113 5.000 ± 0.115 5.217 ± 0.309 4.917 ± 0.442 5.430 ± 0.064 4.737 ± 0.436 5.797 ± 0.205 NS
T1 T2 T3 T4 T5 T6 T7 Significance
ab
Albumin (g/100 ml)
Total protein (g/100 ml)
Treatments
2.630 ± 0.029 2.467 ± 0.066 2.237 ± 0.228 2.597 ± 0.395 2.250 ± 0.248 2.410 ± 0.421 3.077 ± 0.101 NS
Globulin (g/100 ml)
TABLE 2 Blood components of Nile tilapia fish as affected by various effective bacteria strains.
∗∗∗
b
30.317 ± 2.214 33.450 ± 2.858b 31.050 ± 3.285b 21.500 ± 0.497c 34.800 ± 2.090b 42.300 ± 2.240a 36.930 ± 1.542ab
AST (IU)
15.650 ± 0.664 15.500 ± 1.645 17.070 ± 0.779 12.170 ± 0.791 16.407 ± 0.026 16.727 ± 0.101 15.667 ± 1.625 NS
ALT (IU)
T1 = control, T2 = Lactobacillus acidophilus, T3 = Streptococcus thermophilus, T4 = Bifidobacterium bifiduim, T5 = bacterial mixture (Lactobacillus acidophilus, Streptococcus thermophilus and Bifidobacterium bifiduim), T6 = yeast (Saccharomyces cerevisiae) and T7 = bacterial mixture (Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium bifiduim) and yeast (Saccharomyces cerevisiae). NS = not significant; ∗∗∗ P < 0.001. Means in the same column within each classification with different letters differ significantly (P < 0.05).
Initial weight (g)
Treatment
TABLE 1 Growth performance, feed efficiency, and survival rate of Nile tilapia fish as affected by various effective bacteria strains.
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TABLE 3 Economic visibility of Nile tilapia fish as affected by various effective bacteria strains.
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Treatments T1 T2 T3 T4 T5 T6 T7
Total feed intake (g)
Feed cost (LE)
Total gain (g)
Return from gain (LE)
Final margin (LE)
33.614 42.140 36.750 37.044 45.766 36.848 40.278
0.073 0.091 0.079 0.080 0.099 0.080 0.087
19.698 26.656 22.540 23.324 31.752 24.794 24.794
0.197 0.267 0.225 0.233 0.318 0.248 0.248
0.124 0.176 0.146 0.153 0.219 0.168 0.161
TABLE 4 Relative level of protection (RLP) of Oreochromis niloticus at the end of the experimental period after probiotic treatment. Treatments T1 T2 T3 T4 T5 T6 T7
Initial number (Fish)
Dead fish (Fish)
Survival %
Mortality %
RLP %
20 20 20 20 20 20 20
4 0 3 0 0 1 2
80 100 85 100 100 95 90
20 0 15 0 0 5 10
0 100 25 100 100 75 50
It can be concluded that potential probiotics can be used to enhance immune and health status, thereby improving disease resistance in Oreochromis niloticus, and they enhanced growth performance. Application for 15 days was sufficient to improve the immune status, and a mixture of the three bacteria was superior.
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