Scholarly article on topic 'Effect of synbiotics between Bacillus licheniformis and yeast extract on growth, hematological and biochemical indices of the Nile tilapia (Oreochromis niloticus)'

Effect of synbiotics between Bacillus licheniformis and yeast extract on growth, hematological and biochemical indices of the Nile tilapia (Oreochromis niloticus) Academic research paper on "Animal and dairy science"

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Abstract of research paper on Animal and dairy science, author of scientific article — M.S. Hassaan, M.A. Soltan, M.M.R. Ghonemy

Abstract Twelve practical diets were formulated to contain four levels of Bacillus licheniformis (0.0, 0.24×106, 0.48×106 and 0.96×106 CFUg−1), respectively, with three yeast extract levels (0%, 0.5% and 1%), respectively. Each diet was randomly assigned to duplicate groups of 50 Nile tilapia (Oreochromis niloticus) (5.99±0.03g) in 24 concrete ponds (0.5m3 and 1.25m depth) for 12weeks. Increasing dietary B. licheniformis levels in O. niloticus and yeast extract levels significantly (P <0.01) improved growth performance and nutrient utilization. Supplementation of the experimental diets with, 0.48×106 CFU/g−1 and 1.0% yeast extract showed the best nutrient utilization compared to other treatments. All probiotic levels significantly (P <0.01) increased chemical composition (P <0.05) compared to the control group, while increasing yeast extract did not significantly alter chemical composition. Hematological indices, total protein and albumin of O. niloticus significantly increased while aspartate aminotransferase and alanine aminotransferase significantly (P <0.01) decreased with an increase in B. licheniformis level up to 0.48×106 CFUg−1 . Increasing levels of yeast extract had no effect on hematological parameters and the diets supplemented with 0.48×106 CFUg−1 and 0.5% yeast extract showed the highest hematological values.

Academic research paper on topic "Effect of synbiotics between Bacillus licheniformis and yeast extract on growth, hematological and biochemical indices of the Nile tilapia (Oreochromis niloticus)"

Egyptian Journal of Aquatic Research (2014) xxx, xxx-xxx

National Institute of Oceanography and Fisheries Egyptian Journal of Aquatic Research

Egyptian Journal of Aquatic Research

http://ees.elsevier.com/ejar www.sciencedirect.com

FULL LENGTH ARTICLE

Effect of synbiotics between Bacillus licheniformis and yeast extract on growth, hematological and biochemical indices of the Nile tilapia (Oreochromis niloticus)

M.S. Hassaan a *, M.A. Soltan b, M.M.R. Ghonemy b

a National Institute of Oceanography and Fisheries, Cairo, Egypt b Faculty of Agriculture, Benha University, Egypt

Received 7 January 2014; revised 8 April 2014; accepted 8 April 2014

KEYWORDS

Nile tilapia; Probiotics; Prebiotics; Synbiotic

Abstract Twelve practical diets were formulated to contain four levels of Bacillus licheniformis (0.0, 0.24 x 106, 0.48 x 106 and 0.96 x 106 CFU g-1), respectively, with three yeast extract levels (0%, 0.5% and 1%), respectively. Each diet was randomly assigned to duplicate groups of 50 Nile tilapia (Oreochromis niloticus) (5.99 ± 0.03 g) in 24 concrete ponds (0.5 m3 and 1.25 m depth) for 12 weeks. Increasing dietary B. licheniformis levels in O. niloticus and yeast extract levels significantly (P < 0.01) improved growth performance and nutrient utilization. Supplementation of the experimental diets with, 0.48 x 106 CFU/g-1 and 1.0% yeast extract showed the best nutrient utilization compared to other treatments. All probiotic levels significantly (P < 0.01) increased chemical composition (P < 0.05) compared to the control group, while increasing yeast extract did not significantly alter chemical composition. Hematological indices, total protein and albumin of O. niloticus significantly increased while aspartate aminotransferase and alanine aminotransferase significantly (P < 0.01) decreased with an increase in B. licheniformis level up to 0.48 x 106 CFU g-1. Increasing levels of yeast extract had no effect on hematological parameters and the diets supplemented with 0.48 x 106 CFU g-1 and 0.5% yeast extract showed the highest hematological values.

© 2014 Production and hosting by Elsevier B.V. on behalf of National Institute of Oceanography and

Fisheries.

* Corresponding author. Tel.: +20 19490092; fax: +20 242185320. E-mail address: Mohamed_shaban200065@yahoo.com (M.S. Hassaan). Peer review under responsibility of National Institute of Oceanography and Fisheries.

Introduction

Probiotics are a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring an improved use of the feed or enhancing its nutritional value, by increasing the host response towards disease, or by improving the quality of its environment (Verschuere et al., 2000). Nowadays, probiotics

1687-4285 © 2014 Production and hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. http://dx.doi.org/10.1016/j.ejar.2014.04.001

are also becoming an internal part of aquaculture practices to obtain high production. Although considerably low information is available on probiotic application for fish, they offer benefits with regard to improving immune status and fish production (Cerezuela et al., 2011). Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of specific health-promoting bacteria, which can improve host's health (Gibson et al., 2003).

Based on the studies of Mahious and Ollevier (2005) and Gibson et al. (2004) foodstuff that reaches the colon (e. g. non-digestible carbohydrates, some peptides and proteins, as well as certain lipids) is a candidate prebiotic (Yousefian and Amiri, 2009). However, most of the studies have focused on non-digestible carbohydrates, mainly oligosaccharides. Synbi-otics are nutritional supplements that combine probiotics and prebiotics, enhancing their beneficial effects (Cerezuela et al., 2011).

The use of probiotics and prebiotics has been regarded during recent years as an alternative viable therapy in fish culture, appearing as a promising biological control strategy and becoming an integral part of aquaculture practices for improving growth and disease resistance (Rombout et al., 2010). This strategy offers innumerable advantages to overcome the limitation and side effects of antibiotics and other drugs and also leads to high production (Sahu et al., 2008).

In recent years there has been a growing interest in understanding the mechanism of action of probiotics and prebiotics, especially in humans and other mammals. Probiotic activity is mediated by a variety of effects that are dependent on the pro-biotic itself, the dosage employed, treatment duration and route and frequency of delivery. Some probiotics exert their beneficial effects by elaborating antibacterial molecules such as bacterio-cins that directly inhibit other bacteria or viruses and, activity participating in the fight against infections; whereas, others inhibit bacterial movement across the gut wall (translocation), enhance the mucosal barrier function by increasing the production of innate immune molecules or modulate the inflammatory/immune response (Cerezuela et al., 2011).

On the other hand, the potential mechanism of prebiotics includes a selective increase/decrease in specific intestinal bacteria that modulate local cytokine and antibody production, an increase in the intestinal short chain fatty acid production, an enhanced binding of these fatty acids to G-coupled protein receptors on leucocytes, an interaction with carbohydrate receptors on intestinal epithelial and immune cells, and partial absorption resulting in a local and systemic contact with the immune system (Seifert and Watzl, 2007).

The alternative methods of disease prevention have been used as a means of reducing the presence of opportunistic pathogens and simultaneously stimulating the host immune responses. However, other effects related have been observed, as improved growth performance, feed utilization, digestive enzyme activity, antioxidant enzyme activity, gene expression, disease resistance, larval survival and gut morphology alter the gut microbiota, mediate stress response, improve nutrition, reduce risk of certain cancers (colon, bladder), produce lactase, alleviate symptoms of lactose intolerance and malabsorption (Rombout et al., 2010; Dimitroglou et al., 2011; Yousefian and Amiri, 2009; Ringo et al., 2010).

Synbiotic is defined as a combination of probiotic and prebiotic. It is presumed to impart the beneficial effects of both

ingredients. Few data are available regarding the application of synbiotics in aquaculture (Li et al., 2009; Rodriguez-Estrada et al., 2009; Zhang et al., 2010). Synbiotics can help to improve health status, disease resistance, growth performance, feed utilization, carcass composition, gastric morphology, and digestive enzyme activities. As such; many commercial dietary formulations now routinely include probi-otics or prebiotics.

Therefore, the aim of the present study is to investigate the effects of supplementation of a probiotic (Bacillus lichenifor-mis) and the prebiotic (yeast extract) and their synbiotic interaction on growth performance, chemical composition, hematological and biochemical blood parameters of the Nile tilapia (Oreochromis niloticus).

Materials and methods

Experimental design and culture technique

A 4 x 3 factorial experiment was designed to study the effect of probiotic (B. licheniformis) levels, prebiotic (yeast extract) levels, and their synbiotic interactions on growth performance, feed utilization, proximate chemical analysis of whole fish body, hematological and biochemical blood parameters of the Nile tilapia (O. niloticus).

Nile tilapia, were obtained from the Abbassa hatchery, Abou-Hammad, Sharkia Governorate, Egypt and were acclimated for two weeks at the El-Kanater El-Khayria Fish Research Station, National Institute of Oceanography and Fisheries (NIOF), Cairo, Egypt. During this period, fish were fed with a commercial diet (30% crude protein) twice a day to be adapted to pelleted feed according to Hassaan et al. (2013). The experiment was conducted in 24 concrete ponds (0.5 m3 and 1.25 m depth). The ponds were supplied with freshwater from the Darawa irrigation Baranch, Kalubiya, Governorate by a pump machine and a fine net was put in the inlet of each pond. Each pond was stocked with 50 fish with initial weight ranging between 5.69-6.05 g. Two replicates were randomly assigned to each treatment, prior to the start of experiment. During the experiment, fish were hand-fed their respective diets at a level of 3% of body weight, 6 days/week. The daily ratio was divided into three equal amounts and offered three times a day (09:00, 12:00 and 15:00 h). Fish for each pond were weighed biweekly and the amount of daily diet was adjusted accordingly. About one-third of water in each pond was daily renewed by the outlet at the bottom of the pond before feeding. All ponds were provided with continuous aeration to maintain the dissolved oxygen level near saturation and fish were held under natural light.

Water temperature and dissolved oxygen were measured every other day using a YSI model 58 oxygen meter (YSI Company, Yellow Springs Instrument, Yellow Springs, Ohio, USA). Total ammonia and nitrite were measured twice weekly using a DREL, 2000 spectrophotometer (Hash Company, Loveland, CO, USA). Total alkalinity and chloride were monitored twice weekly using the titration; pH was monitored twice weekly using a pH meter (Orion pH meter, Abilene, Texas, USA) (APHA, 1992). The water temperature was 26.17 ± 0.8 0C: dissolved oxygen, 5.6 ± 0.8 mg L_1: total ammonia, 0.18 ± 0.12 mg L_1: total alkalinity, 173 ± 42 mg L_1: chlorides, 570 ±151mgL~1 and pH 8.52 ± 0.3.

Water quality criteria were suitable within the acceptable limits for rearing the Nile tilapia O. niloticus fingerlings (El-Greirsy and El-Gamal, 2012).

Preparation inoculum of probiotics (B. licheniformis)

B. licheniformis culture was prepared by adding 15 g of dried form (Microbiological Resources Center (MIRCEN), Faculty of Agriculture, Ain Shams Univ., Egypt) to 100 ml of prepared medium containing (gl-1): (peptone 5.0; beef extract, 3.0) broth and an adjusted pH at 7.0. Incubation was done at 37 0C. After 24 h, 1 ml was inoculated into 100 ml of freshly prepared medium broth that was incubated for a further 48 h at 37 0C. After incubation, the cells were harvested by centrifugation (2000g for 15 min), washed twice with phosphate buffered saline (PBS; pH 7.3; Oxoid) and re-suspended in PBS for the addition to the basal diet. Washed cells were then added dropwise into the basal mixture prior to cold press extruding after Shelby et al. (2006) to produce the probiotic diet with three levels 0.24 x 106, 0.48 x 106 and 0.96 x 106 CFU g-1. The same volume of PBS (B. licheniformis) was added to the basal mixture for the control to maintain an equal volume of PBS.

Experimental diets

The basal diet was formulated to contain approximately 30% crude protein and 19.41 KJ/kg diet1 gross energy (Table 1). B. licheniformis was supplemented separately to the basal diet to obtain 0.0, 0.24 x 106, 0.48 x 106 and 0.96 x 106 CFU g-1 after it was pelted. Each level of B. licheniformis was supplemented with 0.0, 1 and 1.5% yeast extract (Diamond VXPC®). The ingredients were ground into fine powder through 200 im mesh. All ingredients were thoroughly mixed with soybean oil and then passed the mixed feed through a laboratory pellet mill (2-mm dye) in the National Institute of Oceanography and Fisheries, Cairo Governorate, Egypt (a California Pellet Mill, San Francisco, CA, USA), and stored at -20 0C until use.

Growth and nutrient utilization parameters

Growth performance and feed utilization parameters were measured using the following equations: Weight gain (WG) = final weight (g) - initial weight (g), Specific growth rate (SGR) = InW2-InW1 x 100 where: In = the natural log; Wi -= first fish weight; W2 = the following fish weight in gram and t = period in days, Feed conversion ratio (FCR) = Feed intake (g)/Weight gain (g), Protein efficiency ratio (PER) = -Weight gain (g)/Protein intake (g), Protein productive value (PPV)% = (protein gain (g)/protein intake (g) x 100).

Blood sample and hematological and biochemical analysis

At the end of the experiment, blood samples were collected from the caudal vein of all experimental fish treatments and were divided into two groups. The first group was collected with anticoagulant 10% ethylenediaminetetraacetate (EDTA) to measure hematocrit (Ht), hemoglobin (Hb), red blood cells (RBCs) and white blood cells (WBCs). Ht was determined as described by Reitman and Frankel (1957), hemoglobin (Hb) was determined by the hemoglobin kit which is a standardized

procedure of the cyanomet hemoglobin method and the total count of white blood cells (WBCs) was carried out by the indirect method (Martins et al., 2004). The second group of the blood samples was allowed to clot overnight at 4 0C and then centrifuged at 3000 rpm for 10 min. The non-hemolyzed serum was collected and stored at -20 0C until use. Levels of serum aspartate aminotransferase (AST), and alanine aminotransfer-ase (ALT) were determined according to the method described by Reitman and Frankel (1957). Total protein (TP) and albumin were determined by the method of (Wotton and Freeman, 1982).

At the termination of the trail, a sample of three fish was randomly sampled from each pond. Fish samples were killed, ground, stored in polyethylene bags and frozen until the chemical analysis. Dry matter, crude protein, lipid and ash contents were determined according to AOAC (1995).

Statistical analysis

All data were analyzed by SAS (1996). One-way analysis of variance (One-way ANOVA) was used to determine whether significant variation existed between treatments. When overall differences were found, they were tested by Duncan's multiple rang test as described by Duncan (1955). Two-way ANOVA was used for analyzing the individual effects of B. licheniformis and yeast extract, and the interaction between them. All differences were considered significant at (P < 0.05).

Results and discussion

Growth performance

Results in Table 2 indicated that final body weight (BW), body length (BL) weight gain (WG) and specific growth rate (SGR) of O. niloticus increased with increasing probiotic (B. licheni-formis) up to 0.48 x 106 CFU g-1 diet. The diet supplemented with 0.48 x 106 CFU g-1 showed the highest significant (P < 0.05) BW, WG and SGR when compared to other fish groups. Such an increase in the growth in aquatic animals that were fed probiotic supplemented diets may be attributed to the improved digestive activity due to enhancing the synthesis of vitamins and enzymatic activities (Ding et al., 2004); consequently, improving digestibility and growth performance. Probiotics have been shown to produce digestive enzymes such as amylase, protease, lipase which may enrich the concentration of intestinal digestive enzymes. In addition, probiotics inhibit the colonization of potential pathogens in the digestive tract by antibiosis or by the competition for nutrients and the alteration of the microbial metabolism (Gatesoupe, 1999). It also improves the nutrition by detoxifying the potentially harmful compounds in feeds by producing vitamins such as biotin and vitamin B12 (Hoshino et al., 1997), and by stimulating host immunity (Gibson et al., 1997). Soltan and El-Laithy (2008) indicated that supplementation of basal diet with Bacillus subtilis, significantly (P < 0.001) improved BW, BL, WG and SGR of O. niloticus. Similarly, the application of Entero-coccus faecium as a probiotic was found to enhance the growth performance of Nile tilapia, O. niloticus (Wang et al., 2008). Al-Dohail et al. (2009) also illustrated that African catfish Clarias gariepinus that were fed the Lactobacillus acidophilus

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Table 1 Formulation and chemical proximate analysis of the experimental diets.

Ingredients%

Diet NO. (B. licheniformis CFU g '/Yeast extract%)

D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12

0/0 (0/0.05) (0/1) (0.24 x 106/0) (0.24 x 106/0.5) (0.24 x 106/1) (0.48 x 106/0) (0.48 x 106/0.5) (0.48 x 106/1) (0.96 x 106/0) (0.96 x 106/0.5) (0.96 x 106/1)

Fish meal 10 10 10 10 10 10 10 10 10 10 10 10

Soybean meal 46 46 46 46 46 46 46 46 46 46 46 46

Yellow corn 29.5 29 28.5 29.4 28.9 28.4 29.35 28.85 29 29.3 28.8 28.3

Wheat bran 10 10 10 10 10 10 10 10 10 10 10 10

Soybean oil 3 3 3 3 3 3 3 3 3 3 3 3

Vit. & minerala 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5

B. licheniformisb 0 0 0 0.1 0.1 0.1 0.15 0.15 0.15 0.20 0.20 0.20

Yeast extract 0 0.5 1 0 0.5 1 0 0.5 1 0 0.5 1

Proximate analysis

Protein% 30.05 30.70 30.11 30.04 30.68 30.11 30.01 30.65 30.71 30.01 30.62 30.70

Lipids% 5.70 5.75 5.76 5.65 5.73 5.66 5.63 5.72 5.70 5.70 5.72 5.74

Ash% 5.13 5.21 5.20 5.15 5.18 5.21 5.13 5.18 5.21 5.13 5.19 5.23

T. CHOc% 59.12 58.34 58.93 59.16 58.49 59.02 59.23 59.06 58.38 59.16 58.47 58.33

GE (MJ kg~')d 19.49 19.53 19.50 19.47 19.54 19.47 19.46 19.63 19.52 19.48 19.52 19.53

a Vitamin and mineral mix (mg or g/kg diet): MnSO4, 40 mg; MgO, 10 mg; K2SO4, 40 mg; ZnCO3, 60 mg; KI, 0.4 mg; CuSO4, 12 mg; Ferric citrate, 250 mg; Na2SeO3, 0.24 mg; Co, 0.2 mg; retinol, 40000 IU; cholecalciferol, 4000 IU; a-tocopherolacetate, 400 mg; menadione, 12 mg; thiamine, 30 mg; riboflavin, 40 mg; pyridoxine, 30 mg; cyanocobalamin, 80 mcg; nicotinic acid, 300 mg; folic acid, 10 mg; biotin, 3 mg; pantothenic acid, 100 mg; inositol, 500 mg; ascorbic acid, 500 mg.

b B. licheniformis was prepared to obtain 0.24 x 106, 0.48 x 106 and 0.96 x 106 CFU approximately. c T. CHO total carbohydrate = 100-(crude protein + lipid + ash).

d Calculated using gross caloric values of 23.63, 39.52 and 17.15 kJ/g for protein, fat and carbohydrate, respectively according to Brett (1973).

Table 2 Growth performance of Nile tilapia (Oreochromis niloticus) fed the experimental diets with different levels of Bacillus

licheniformis and yeast extract levels.

Diet No (B. Licheniformis! B. Licheniformis Yeast extract Growth performance

Yeast extract) cfug-1 (1%) IBW ga FBWgb FBL cmc Kd WG ge SGRf

Diet 1 (0/0) 0 0 5.96 34.55k 12.30c 1.85ab 28.59j 195g

Diet 2 (0/0) 0 0.5 5.98 40.13j 13.40b 1.67b 34.15i 2.12f

Diet 3 (0/0) 0 1 5.98 40.44i 13.25b 1.74ab 34.46h 2.12ef

Diet 4 (0.24 x 106/0) 0.24 x 106 0 6.05 40.91h 13.45b 1.67b 34.86g 2.12ef

Diet 5 0.24 x 106/0.5) 0.24 x 106 0.5 6.02 41.15 g 13.45b 1.69ab 35.13f 2.14de

Diet 6 (0.24 x 106/1) 0.24 x 106 1 6.01 42.00e 13.40b 1.75b 35.99e 2.16cd

Diet 7 (0.48 x 106/0) 0.48 x 106 0 5.98 41.26f 13.05b 1.87a 35.28f 2.15d

Diet 8 (0.48 x 106/0.5) 0.48 x 106 0.5 5.97 44.12b 14.45a 1.47d 38.15b 2.22b

Diet 9 (0.48 x 106/1) 0.48 x 106 1 6.02 45.84a 14.15a 1.62c 39.82a 2.26a

Diet 10 (0.96 x 106/0) 0.96 x 106 0 6.01 40.11j 14.20a 1.40e 34.10i 2.11f

Diet 11 (0.96 x 106/0.5) 0.96 x 106 0.5 5.99 42.30d 14.20a 1.49d 36.31d 2.18c

Diet 12 (0.96 x 106/1) 0.96 x 106 1 5.98 43.72c 14.15a 1.45d 37.74c 2.22b

One-way ANOVA

Polled S.E.M* 0.03 0.02 0.17 0.70 0.051 0.0062

F-value (B. Licheniformis) 1.94 12841.9 24.76 9.00 5838.08 292.23

F-value (Yeast extract) 0.14 13039.9 12.92 3.33 5983.18 321.06

Tow-way ANOVA

P-value (B. Licheniformis) 0.182 0.001 0.0001 0.0027 0.0001 0.0001

P-value (Yeast extract) 0.871 0.001 0.0013 0.0742 0.0001 0.0001

P-value (interaction) 0.725 0.001 0.0132 0.0672 0.0001 0.0001

Data are means of triplicate. Means in the same column sharing a same superscript letter are not significantly different (P > 0.05).

Polled S.E.M standard error of mean.

a IBW: Initial body weight.

b FBW: Final body weight.

c FBL: Final body length.

d K: Condition factor.

e WG: Weight gain.

f SGR: Specific growth rate.

showed a better growth performance than the control fish group.

As described in Table 2, BW, BL, WG and SGR of O. nil-oticus significantly (P < 0.05) increased with increasing prebi-otic (yeast extract) levels. Fish that were fed the diet containing 1.0% yeast extract showed the highest BW, BL, WG and SGR when compared to the other two fish groups (control and the diet supplemented with 0.5% yeast extract). In contrary, Jarolowicz et al. (2012) indicated that, the commercial diet supplemented with yeast extract did not have an impact on the final BW of Juvenile European pikeperch, Sander lucioperca.

Referring to the effect of synbiotics on growth performance, the highest final BW, WG and SGR were recorded by fish that were fed D9 containing 0.48 x 106 CUFg_1 B. licheniformis and 1.0% yeast extract, while the lowest one was shown by fish that were fed the control diet (D1). The study of Mehrabi et al. (2011) showed that after 60 days rainbow trout (Oncorhynchus mykiss) fed diets containing different levels of symbiotic (Biomin IMBO) (0.5%, 1.0% and 1.5%) increased in body weight about 50%, 59% and 53%, respectively and improved SGR and FCR in comparison with the control group. Ye et al. (2011) reported that, Japanese flounder fed diet supplemented with fructooligosaccharides (FOS), mannan oligosaccharides (MOS) and Bacillus clausii increased WG. Also, Ai et al. (2011) indicated that at each dietary FOS

level, supplemented by 1.35 x 107 CFUg_1 B. subtilis significantly increased SGR and feed efficiency ratio (FER) when compared to the control group for juvenile large yellow croaker, Larimichthys crocea.

Feed intake and feed utilization

Results of Table 3 indicated that, increasing B. licheniformis level in O. niloticus diets was followed by a significant increase in feed intake (FI) and a significant improvement in FCR, PER and PPV up to the diet supplemented by 0.48 x 106. In practical terms, this means that the use of probiotics can decrease the amount of feed necessary for animal growth which could result in a reduction in the production cost. Several studies on probiotics have been published in recent years which suggested that, probiotics provide nutritional benefits in diets for tilapia fingerling (Khattab et al., 2004; Ferguson et al., 2010).

Increasing yeast extract levels (from 0%, 0.5% or 1.0% yeast extract) significantly (P < 0.01) increased FI, and significantly improved FCR, PER and PPV (Table 3) which was subsequently followed by an increase in the growth performance. Generally, the high demand for nucleotides occurs during periods of rapid growth (Carver, 1994). Li and Gatlin (2005) indicated that sub-adult hybrid stripped sea bass (Morone chrysops x M. saxatilis) fed commercial diet

Table 3 Feed utilization of Nile tilapia (Oreochromis niloticus) fed the experimental diets with different levels of Bacillus licheniformis

and yeas extract levels.

Diet No (B. Licheniformis/ Yeast B. Licheniformis Yeast extract Feed utilization

extract) cfug-1 (1%) Fig/fisha FCRb PERc PPV%d FR%e

Diet 1 (0/0) 0 0 52.95d 1.85a 1.830e 23.51g 33.32e

Diet 2 (0/0) 0 0.5 54.64cd 1.60bc 2.12cd 26.38ef 38.53d

Diet 3 (0/0) 0 1 55.34bc 1.61bc 2.12cd 27.27de 38.63d

Diet 4 (0.24 x 106/0) 0.24 x 106 0 56.45ab 1.62b 2.10d 25.66f 38.79d

Diet 5 0.24 x 106/0.5) 0.24 x 106 0.5 56.79a 1.62b 2.10d 28.49bc 43.27b

Diet 6 (0.24 x 106/1) 0.24 x 106 1 58.04a 1.61b 2.10d 26.87e 40.59c

Diet 7 (0.48 x 106/0) 0.48 x 106 0 56.81ab 1.61b 2.11cd 26.60e 41.63c

Diet 8 (0.48 x 106/0.5) 0.48 x 106 0.5 55.75bc 1.47e 2.32a 29.34b 43.69b

Diet 9 (0.48 x 106/1) 0.48 x 106 1 56.75ab 1.43e 2.38a 31.57a 49.22a

Diet 10 (0.96 x 106/0) 0.96 x 106 0 54.57de 1.60bc 2.12cd 28.85bc 43.31b

Diet 11 (0.96 x 106/0.5) 0.96 x 106 0.5 56.60ab 1.56cd 2.18Bc 27.97cd 40.59c

Diet 12 (0.96 x 106/1) 0.96 x 106 1 58.05a 1.54d 2.21b 29.34b 44.07b

One-way ANOVA

Polled S.E.M* 0.55 0.014 0.020 0.44 0.29

F-value (B. Licheniformis) 14.46 87.44 76.59 93.29 182.20

F-value (Yeast extract) 11.39 89.12 71.09 89.06 79.53

Tow-way ANOVA

P-value (B. Licheniformis) 0.004 0.0001 0.0001 0.0001 0.0001

P-value (Yeast extract) 0.0021 0.0001 0.0001 0.0001 0.0001

P-value (interaction) 0.1025 0.0355 0.0495 0.0001 0.0001

Data are means of triplicate. Means in the same column sharing a same superscript letter are not significantly different (P > 0.05).

Polled S.E.M standard error of mean.

a FI: feed intake g/fish.

b FCR: feed conversion ratio.

c PER: protein efficiency ratio.

d PPV: protein productive value.

e FR: fat retention.

supplemented with prebiotic Grobiotic®-AE, with 1020 g kg-1 obtained a significantly improved feed efficiency.

Referring to the dietary symbiotic interaction of the experimental diets with, 0.48 x 106CFUg-1 and 1.0% the yeast extract (D9) showed the highest FI, the best FCR, PER and PPV compared to other synbiotic treatments and the control group. These results were parallel to those obtained for other growth parameters (BW, BL, WG and SGR) obtained in the present study.

Gastrointestinal bacteria take part in the decomposition of nutrients, provide the microorganisms with physiologically active materials, such as enzymes, amino acids, and vitamins (Bairagi et al., 2004; Wache0 et al., 2006; Wang, 2007; Wang and Xu, 2006), and thus facilitate feed utilization and digestion. This may account for the enhanced FCR, PER and PPV by dietary B. licheniformis supplementation in the present study and previous studies (Bairagi et al., 2004; Bagheri et al., 2008). Mehrabi et al. (2011) came to similar results. They found that the addition of synbiotic to the feed of rainbow trout, Oncorhynchus mykiss fingerlings produced a better significant (P < 0.05) FCR values than the control. Ye et al. (2011) reported that, Japanese flounder fed diet supplemented with FOS, MOS and Bacillus clausii improved FCR than other diets. Also, Ai et al. (2011) showed that juvenile large yellow croaker, Larimichthys crocea fed the diet supplemented with FOS and B. subtilis 0.96 x 106 CFU g-1 significantly improved FCR and PER values when compared to the fish group fed with the control diet.

Proximate analysis

Proximate analysis of O. niloticus which was affected by probi-otic (B. licheniformis) and prebiotic (yeast extract) is presented in Table 4. With respect to the effect of B. licheniformis supplemented to the experimental diets, it is shown that all probiotic levels significantly (P < 0.05) increased dry matter, lipid and protein content when compared to the control group, while ash content was not significantly affected. Soltan and El-laithy, 2008 indicated that, O. niloticus fed diet supplemented with B. subtilis recorded a high level of dry matter and lipid content than the control group with no effect on the ash content. Bagheri et al. (2008) reported that application of 3.8 x 109CFUg-1 of Bacillus spp. in diet of rainbow trout fry made a significant increase in fish body protein content when compared to the control group.

Results of proximate analysis (Table 4) showed that, increasing yeast extract from 0 to 1.0% did not significantly alter crude protein, dry matter, lipid or ash content. The interaction between probiotic and the prebiotic (Table 4) showed no clear trend in the proximate analyses of whole fish. Ye et al. (2011) in Japanese flounder showed an increase in the body protein content in fish fed with a FOS, MOS and/or B. clausii-containing diet when compared to the control, body lipid content demonstrated an opposite trend to body protein content. Mehrabi et al. (2011) indicated that, higher body protein content in the rainbow trout (Oncorhynchus mykiss) fingerlings implies on this fact that by the application of synbiotics,

Table 4 Body composition of whole body fish of Nile tilapia (Oreochromis niloticus) fed the experimental diets with different levels of

Bacillus licheniformis and yeast extract levels.

Diet No (B. Licheniformis/Yeast B. Licheniformis Yeast extract Chemical analysis of whole fish%

extract) cfug-1 (1%) Dry matter Lipid Protein Ash

Diet 1 (0/0) 0 0 24.16d 14.40f 53.10c 14.30ab

Diet 2 (0/0) 0 0.5 23.88d 14.80f 53.30c 14.50ab

Diet 3 (0/0) 0 1 24.36d 14.60f 53.35c 14.75ab

Diet 4 (0.24 x 106/0) 0.24 x 106 0 23.70e 15.05cd 53.10c 14.75a

Diet 5 0.24 x 106/0.5) 0.24 x 106 0.5 26.20a 15.25bc 53.50ab 14.50ab

Diet 6 (0.24 x 106/1) 0.24 x 106 1 24.58d 15.25bc 53.65ab 14.55ab

Diet 7 (0.48 x 106/0) 0.48 x 106 0 24.65c 15.80 a 53.55ab 14.35ab

Diet 8 (0.48 x 106/0.5) 0.48 x 106 0.5 24.46d 15.20bc 54.10ab 14.55ab

Diet 9 (0.48 x 106/1) 0.48 x 106 1 25.55b 15.85a 53.65a 14.70a

Diet 10 (0.96 x 106/0) 0.96 x 106 0 25.67a 15.75a 53.50ab 14.80a

Diet 11 (0.96 x 106/0.5) 0.96 x 106 0.5 24.29d 15.55ab 54.25a 14.15b

Diet 12 (0.96 x 106/1) 0.96 x 106 1 24.88c 15.50ab 54.15ab 14.45ab

One-way ANOVA

Polled S.E.M* 0.21 0.15 0.26 0.15

F-value 9.47 32.47 4.85 0.39

F-value 1.99 0.47 3.90 1.56

Tow-way ANOVA

P-Value (B. Licheniformis) 0.0022 0.0001 0.0218 0.7633

P-value (Yeast extract) 0.1836 0.6342 0.0526 0.2535

P-value (interaction) 0.0001 0.0529 0.8104 0.0987

Data are means of triplicate. Means in the same column sharing a same superscript letter are not significantly different (P > 0.05).

Polled S.E.M standard error of mean.

Table 5 Hematological parameters of Nile tilapia (Oreochromis niloticus) fed the experimental diets with different levels of Bacillus

licheniformis and yeas extract levels.

Diet No (B. Licheniformis! B. Licheniformis Yeast extract Hematological parameters

Yeast extract) cfu g^1 (1%) Hb(g/dl)a Hct%b RBCc WBC(x103 mm~3)d

Diet 1 (0/0) 0 0 10.21e 15.05de 1.80h 36.29e

Diet 2 (0/0) 0 0.5 10.51d 14.10g 1.83g 37.05c

Diet 3 (0/0) 0 1 10.83c 14.70ef 185fg 37.15c

Diet 4 (0.24 x 106/0) 0.24 x 106 0 10.49d 15.25d 1.87de 36.90d

Diet 5 0.24 x 106/0.5) 0.24 x 106 0.5 10.97bc 14.35fg 1.85ef 37.12c

Diet 6 (0.24 x 106/1) 0.24 x 106 1 10.88bc 14.70ef 185fg 37.60b

Diet 7 (0.48 x 106/0) 0.48 x 106 0 10.95bc 15.15d 1.88cd 37.20bc

Diet 8 (0.48 x 106/0.5) 0.48 x 106 0.5 11.28a 17.12a 1.97a 38.36a

Diet 9 (0.48 x 106/1) 0.48 x 106 1 11.07ab 16.15c 1.91bc 38.15a

Diet 10 (0.96 x 106/0) 0.96 x 106 0 10.97bc 16.75b 1.90bc 37.38bc

Diet 11 (0.96 x 106/0.5) 0.96 x 106 0.5 10.91bc 17.10a 1.84fg 36.94c

Diet 12 (0.96 x 106/1) 0.96 x 106 1 11.09ab 16.41bc 1.92b 37.16bc

One-way ANOVA

Polled S.E.M* 0.0070 0.11 0.0094 0.13

F-value (B. Licheniformis) 40.28 234.67 57.43 34.91

F-value (Yeast extract) 22.96 23.30 4.52 20.14

Tow-way ANOVA

P-value (B. Licheniformis) 0.0001 0.0001 0.0001 0.0001

P-value (Yeast extract) 0.0001 0.0001 0.0369 0.0002

P-value (interaction) 0.068 0.0001 0.001 0.0016

Data are means of triplicate. Means in the same column sharing a same superscript letter are not significantly different (P > 0.05).

Polled S.E.M standard error of mean.

a Hb: hemoglobin.

b Hct: hematocrit.

c RBC: red blood.

d WBCs: white blood cell.

the ingested food was converted more effectively into the structural protein and subsequently resulted in more muscle, which is a desirable aspect in fish farming. However, the application of synbiotic in trout fingerlings diet has insignificant effect on the lipid content.

Hematological indices

Hemoglobin (Hb), hematocrit (Ht), red blood cells (RBCs) and white blood cells (WBCs) of O. niloticus significantly increased with each increase in B. licheniformis level, up to 0.48 x 106CFUg_1 B. licheniformis and then decreased as the diet was supplemented with 0.96 x 106CFUg_1 B. licheniformis.

Hematology is an important factor that could be considered for the fish diet quality assessment. Ologhobo (1992) reported that the most common blood variables consistently influenced by diet are the hematocrit (Ht) and hemoglobin (Hb) levels. Probiotics and prebiotics have been used alone and together in various animals including the synbiotic, in tila-pia (Abd El-Rhman et al., 2009), which reported positive effects on hematological parameters. On the other hand, O. nil-oticus fed diet supplemented with B. subtilis (Soltan and El-Laithy, 2008) or supplemented with Pediococcus acidilactici (Ferguson et al. (2010) showed some variation (but not significant) in Hb and Ht contents among the control and fish that were fish groups fed diet enriched with probiotics.

As shown in Table 5 Hb, Ht, RBCs and WBCs were not significantly (P > 0.05) affected by the graded levels of yeast

extract used in the study. Fish fed the diet supplemented with synbiotic (0.48 x 106 CFU and 0.5% yeast extract) showed the highest values of Hb, RBCs and WBCs. Marzouk et al. (2008) reported that both fish groups fed the diet supplemented with dead Saccharomyces cerevisiae yeast and both of live B. subtilis and S. cerevisae showed significant (P < 0.05) increase in the Ht level when compared to fish fed the control diet. Also, Firouzbakhsh et al. (2012) reported that, Hb concentration, in rainbow trout (Oncorhynchus mykiss) fed different levels of synbiotic was significantly (P < 0.05) different from the control.

Metabolism enzymes

Alanine aminotransferase (ALT) and aspartate aminotransfer-ase (AST) enzymes are important liver enzymes. They are indicators for liver health and function through controlling the transferring amino group function of alpha-amino acids to alpha-keto acids. Large amounts of ALT and AST are released into animal blood, mostly during liver cell damage (Kumar et al., 2011).

As shown in Table 6, ALT and AST values decreased with increasing B. licheniformis level. Soltan and El-Laithy (2008) found that, ALT and AST levels significantly decreased when Nile tilapia fed diets supplemented with probiotics were compared to the control group. Similarly, Wache0 et al. (2006) observed a decrease in the activity of AST, ALT and lactate dehydrogenase in O. niloticus after being fed with diet containing Pseudomonas spp. and a mixture of Micrococcus luteus and Pseudomonas spp. Similar results were also observed in

Table 6 Biochemical blood parameters of Nile tilapia (Oreochromis niloticus) fed the experimental diets with different levels of

Bacillus licheniformis and yeas extract levels.

Diet No (B. Licheniformis/yeast B. Licheniformis Yeast extract Biochemical blood parameters

extract) CFU g-1 (1%) ALT(i/l)a AST(i/l)b TP(g/dl)c AL(g/dl)d

Diet 1 (0/0) 0 0 89.50a 17.85a 2.95d 1.250d

Diet 2 (0/0) 0 0.5 86.50b 17.25ab 3.45c 1.450c

Diet 3 (0/0) 0 1 86.00bc 17.06b 3.65abc 1.45c

Diet 4 (0.24 x 106/0) 0.24 x 106 0 85.50bc 16.25cd 3.75ab 1.55bc

Diet 5 0.24 x 106/0.5) 0.24 x 106 0.5 83.50e 16.05cd 3.70abc 1.63ab

Diet 6 (0.24 x 106/1) 0.24 x 106 1 84.50 d 16.15cd 3.80a 1.74a

Diet 7 (0.48 x 106/0) 0.48 x 106 0 86.50b 16.55bc 3.45c 1.45c

Diet 8 (0.48 x 106/0.5) 0.48 x 106 0.5 82.50f 15.00e 3.80a 1.75a

Diet 9 (0.48 x 106/1) 0.48 x 106 1 84.50d 16.06cd 3.60abc 1.25d

Diet 10 (0.96 x 106/0) 0.96 x 106 0 85.50c 15.90cd 3.50bc 1.25d

Diet 11 (0.96 x 106/0.5) 0.96 x 106 0.5 84.50d 15.65de 3.55abc 1.11d

Diet 12 (0.96 x 106/1) 0.96 x 106 1 84.00de 15.85cd 3.55abc 1.160d

One-way ANOVA

Polled S.E.M* 0.19 0.24 0.081 0.047

F-value 153.27 28.29 12.80 52.58

F-value 184.80 7.32 10.37 5.99

Tow-way ANOVA

P-value (B. Licheniformis) 0.0001 0.0001 0.0007 0.6830

P-value (Yeast extract) 0.0001 0.0095 0.0029 0.2121

P-value (interaction) 0.0001 0.1095 0.0115 0.5245

Data are means of triplicate. Means in the same column sharing a same superscript letter are not significantly different (P > 0.05).

Polled S.E.M standard error of mean.

a ALT: alanine aminotransferase.

b AST: aspartate aminotransferase.

c TP: total protein.

d AL: albumin.

Cyprinus carpio fed the extract of Cyanobacteria (Palikova et al., 2004).

Control fish group showed the highest (P < 0.05) ALT value, while fish fed 0.5% yeast extract achieved the lowest (P < 0.05) ALT and AST values indicating the positive effects of probiotic and yeast extract in enhancing and protecting liver cells.

Fish fed diet D8 (supplemented with synbiotic 0.96 x 106 and 0.5% yeast extract) recorded the lowest (P < 0.05) ALT value (82.50 i/L), while the control group showed the highest (P < 0.05) ALT values, being 89.50 i/L respectively. Recently, Jarolowicz et al. (2012) reported that juvenile pikeperch, Sander lucioperca that received yeast extract in their diets exhibited a significantly lower AST and ALT activity in comparison to the control group (P < 0.05).

Results of the present study also showed that, all levels of synbiotics significantly decreased the serum levels of ALT and AST. Marzouk et al. (2008) found that, fish groups fed on diets supplemented with dead S. cerevisae yeast and both of live B. subtilis + S. cerevisae revealed a significant (P < 0.05) decrease in ALT and AST when compared to the control group that fed on probiotic-free diet.

Total protein and albumin

Table 6 showed that total protein content significantly increased with the first level of probiotic (0.24 x 106) then decreased with increasing probiotic level. The same trend was also observed for albumin. Increasing yeast extract levels 0.5 and 1.0% increased total protein (TP) and albumin (AL) and the differences between values are significant (P < 0.05). As described in Table 6, TP and ALT recorded the highest values for fish that were fed the diet supplemented with synbiotic (0.48 x 106 and 0.5% yeast extract) than those that were fed other diets. Mehrabi et al. (2011) reported that diet supplemented with synbiotic (Biomin IMBO) increased the serum protein, albumin and globulin level of rainbow trout.

Conclusions

The results of the present study clearly indicated that the supplementation of B. licheniformis (0.48 x 106 CFU g_1) not only enhanced the growth performance and feed utilization of Nile tilapia, but also hematological and biochemical blood parameters. Moreover, the supplementation of yeast extract had significant beneficial effects and there were significant interactions between dietary B. licheniformis and yeast extract.

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