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Latin american journal of aquatic research

versión On-line ISSN 0718-560X

Lat. Am. J. Aquat. Res. vol.43 no.5 Valparaíso nov. 2015

http://dx.doi.org/10.3856/vol43-issue5-fulltext-16 

Research Article

 

Effects of the probiotic Bacillus amyloliquefaciens on growth performance, hematology and intestinal morphometry in cage-reared Nile tilapia

Efectos del probiótico Bacillus amyloliquefaciens en el crecimiento, hematología y morfometría intestinal en tilapias del Nilo criadas en balsa jaula

 

Thiago Fernandes A. Silva1, Thalita R. Petrillo2, Jefferson Yunis-Aguinaga1, Paulo Fernandes Marcusso2, Gustavo da Silva Claudiano2, Flávio Ruas de Moraes2 & Julieta R. Engrácia de Moraes1,2

1Aquaculture Center of Unesp, São Paulo State University (Unesp), Jaboticabal, Brazil Via Prof. Paulo Donato Castellane, km 05, Jaboticabal, SP. 14.884-900 
2
Department of Veterinary Pathology, São Paulo State University (Unesp), Jaboticabal, Brazil

Corresponding author: Julieta Engrácia de Moraes (julietaengracia@gmail.com)
Corresponding editor: Mauricio Laterça


ABSTRACT. The aim of this study was to evaluate the effect of probiotic Bacillus amyloliquefaciens on the growth performance, blood profile and intestinal morphometry in Nile tilapia (Oreochromis niloticus) reared in cages. 936 Nile tilapias were distributed in 12 cages (1.5 m3). Fish were fed for 90 days on basal diets containing 0 (control); 1x106 CFU g-1; 5x106 CFU g-1; and 1x107 CFU g-1 of the probiotic. The results showed no significant difference on performance and proximal composition of fish. Blood glucose and hemoglobin were lower in 1x107 CFU g-1 group, suggesting improves of the homeostatic state of the fish. Other hematimetric indices did not differ between groups. It was observed significant increase of villi height and in the number of goblet cells of the intestine in fish supplemented 5x106 CFU g-1 and 1x107 CFU g-1 of food suggesting that fish improved the digestion and absorption of nutrients. However, more studies are needed to determine the efficacy of this probiotic in field conditions in Nile tilapia.

Keywords: Oreochromis niloticus, homeostatic state, intestinal morphology, blood glucose, aquaculture.


RESUMEN. El objetivo de este estudio fue evaluar el efecto del probiótico Bacillus amyloliquefaciens en el desempeño del crecimiento, hemograma y morfometría intestinal en tilapia del Nilo (Oreochromis niloticus) criadas en balsas jaulas. 936 tilapias del Nilo se distribuyeron en 12 balsas jaulas (1,5 m3). Los peces fueron alimentados durante 90 días con dietas que contenían 0 (control); 1x 106 UFC g-1; 5x106 UFC g-1 y 1x107 UFC g-1 del probiótico. Los resultados no mostraron diferencias significativas en el rendimiento y composición proximal de los peces. La glucosa en sangre y hemoglobina fue menor en el grupo 1x107 UFC g-1, lo que sugiere un mejor estado homeostático de los peces. Otros parámetros hematimétricos no difirieron entre los grupos. Se observó aumento significativo de la altura de las vellosidades y número de células caliciformes del intestino en los peces suplementados con 5x106 UFC g-1 y 1x 107 UFC g-1 de alimento que sugieren que los peces mejoraron la digestión y absorción de nutrientes. Sin embargo, se necesitan más estudios para determinar la eficacia de este probiótico en condiciones de campo en tilapia del Nilo.

Palabras clave: Oreochromis niloticus, homeostasis, morfología intestinal, glucosa sanguínea, acuicultura.


 

INTRODUCTION

Nile tilapia reared in cages presents advantages over traditional pond and tank systems due to the easier handling, high volume of production, and use of areas unsuitable for other purposes, and the biological characteristics of this species (García et al., 2014). Although the productivity advantages (Kim et al., 2014), fish at high stocking densities may present stress and depress the immune system, increasing susceptibility to bacterial and parasitic diseases (Telli et al., 2014).

The use of antibiotics as prophylactic is questioned for being a source of environmental pollution (Ayandiran et al., 2014), accumulating in the fillet, and promoting resistance to drugs with risks to human and animal health (Kersarcodi-Watson et al, 2008).

Probiotics are live microorganisms able to establish, multiply and colonize the intestine of the host in order to promote a beneficial balance of microorganisms. These benefits are explain due to these microorganisms inhibit the proliferation of harmful agents in the intestinal mucosa, improve the digestibility and absorption of nutrients (Nayak, 2010), promote the synthesis of vitamins (Lee et al., 2013), and improve the growth performance of animals (Mohapatra et al., 2014) by increasing the survival percentage (Wu et al., 2014) and water quality (Chi et al., 2014).

Bacteria from genus Bacillus are one of the main probiotics used in aquaculture. These bacteria are ease to cultivate and form spores, which facilitates its conservation (Wang et al., 2008; Nayak, 2010; Han et al., 2015). In addition, several bacteria of this genus have the capacity to secrete antimicrobial compounds and different exoenzymes that aid digestion (Ziaei-Nejad et al., 2006).

Bacillus amyloliquefaciens (ex Fukomoto, 1943) Priest et al., 1987 is a non-pathogenic bacterium rodshaped, aerobic, Gram-positive, and catalase positive (Loncar et al., 2014). This bacterium is highly resistant to environmental changes (Mahdhi et al., 2012; Das et al., 2013), presents antibacterial activity of broad spectrum (Kaewklom et al., 2013), and is an important producer of a-amylase, an enzyme that acts in the digestion of carbohydrates (Wang et al., 2008).

Recently, in studies with B. amyloliquefaciens Reda & Selim (2015) observed in O. niloticus reared in laboratory conditions; improve in body composition and intestinal morphology. Ridha & Azad (2012) reported in the same fish improvements on growth performance and immune response. Cao et al. (2011) observed in Anguilla anguilla, antagonist activity of B. amyloliquefaciens and Aeromonas hydrophila. The same effect was observed by Ran et al. (2012) in Ictalurus punctatus against A. hydrophila and Edwardsiella ictaluri. Das et al. (2013) observed an increase in disease resistance in Catla catla.

The aim of this study was to evaluate the effect of diet containing three concentrations of the probiotic B. amyloliquefaciens on performance, blood profile and intestinal morphology in Nile tilapia, O. niloticus, reared in cages.

MATERIALS AND METHODS

Fish culture, diet and feeding

Masculinized Nile tilapia (Oreochromis niloticus) (35 ± 5 g) from the same spawning were obtained from commercial breeding. The cages (1.5 m3) were distributed in four experimental groups (triplicate) in a completely randomized design (78 fish per cage). The experiment was conducted in the reservoir 3 of the Aquaculture Center of Sao Paulo State University, Jaboticabal (21°14.33'S, 48°17.55'W) in April-June 2014. This reservoir has approximately 10,000 surface m2.

The animals were acclimated for 10 days and fed with basal diet (pellets used in this experiment without addition of probiotics). It was used the program "Super Crac Premium" to formulate the diet according the suggestions of Furuya (2010). Feed was extruded. The feed rate was 3% of body weight, 3 times a day for 90 days on basal diets containing 0 (control); U106 CFU g-1; 5x106 CFU g-1; and U107 CFU g-1 of Bacillus amyloliquefaciens.

Diet composition is shown in Table 1. To achieve homogeneous final concentrations of bacteria in the diet and to facilitate the adhesion of the probiotic, the bacterial suspension was added and homogenized manually with commercial vegetable oil (4% by weight; 4.000 kcal L-1). Thus, according to the recommendations of Das et al. (2013) food was prepared every 7 days to maintain optimal probiotic levels.

 

Table 1. Ingredients and proximal composition
of the basal diet.

1Composition of vitamin-mineral supplement
(Fri-Ribe): vitamin A, 600,000 IU; Vitamin
D3 600,000 IU; Vitamin E 12,000 IU; Vitamin
K3, 1200 mg; vitamin B1, 1200 mg; vitamin
B2, 1536 mg; vitamin B6, 1287 mg; B12, 4000 mg;
folic acid, 198 mg; pantothenic acid, 3800 mg;
 Vitamin C, 48,000 mg; biotin, 20 mg; choline,
30,000 mg; niacin 19,800 mg; Fe, 25,714 mg; Cu,
1960 mg; Mn, 13,334 mg; Zn, 6000 mg; I, 948 mg;
Co, 2 mg; Se, 30.10 mg.

 

Fish were measured at 30 and 60 days for diet amount adjustment. During the experimental period, except temperature, there were no important changes in the water quality that could have interfered in the experiment results (Boyd & Massaut, 1999). The values were maintained as follows: the water temperature varied from 27.1 to 21.9°C (24.35 ± 2.43°C) and dissolved oxygen (7.31 ± 0.48 mg L-1). Both were measured using an YSI 53 device (YSI Company, USA); pH (7.50 ± 0.30) and electrical conductivity (45.54 ± 10.40 µV) were measured using a 63 YSI device (YSI Company, USA). Water transparency (26.03 ± 2.10 cm) was measured using the Secchi disk at 11:00 h on alternate days.

Isolation and identification of the probiotic bacterial strain

The bacterium was isolated from a commercial product containing Bacillus amyloliquefaciens. To confirm the presence of the bacteria in the product, 1.0 g of the probiotic was homogenized in 99 mL of phosphate buffered saline (145 mM NaCl, 1.4 mM NaH2PO4, 8 mM Na2HPO4, pH 7.4). Thus, it was performed serial dilutions with peptone solution (4%) (Sigma-Aldrich, USA), distributed into Petri dishes containing culture medium trypticase soy Agar (TSA), and incubated for 48 h under aerobic conditions at 26°C. Colonies of Bacillus sp. were identified according their morphological, biochemical and dyeing features (Priest et al., 1987). The sequencing of 16S rDNA gene was performed from isolated colonies by the method of Sanger et al. (1977).

The quantification of the probiotic bacteria in the feed was performed by homogenizing 1.0 g of diet in 99 mL of PBS. Then, it was performed serial dilutions as described above. Finally, they were evaluated the colonies that showed morphological, biochemical, and dyeing features compatible with B. amyloliquefaciens. Samples of all treatments were subjected to molecular identification of the bacterium as described previously.

Growth performance

After 90 days, fish were measured and weighed to determinate performance indices:

• Weight gain (WG) = Final weight - initial weight

• Specific growth rate (SGR) = (Infinal weight] -In[initial weight]) x 100/number of days

• Feed efficiency (FE) = weight gain x 100/food consumed

Proximal composition

After the feeding period, 10 fish from each group were euthanized using benzocaine (0.5 g L-1) (Wedemeyer, 1970) and stored at -18°C for later analysis of proximal composition. Then the samples were ground to obtain a homogenous product and dried in an oven at 55°C for 48 h. The humidity was determined by drying in an oven at 105°C for 24 h. The ash content was obtained after incineration in a muffle furnace at 550°C and the energy by bomb calorimetry. Protein was determined by Kjeldahl method and the ether extract by Soxhlet method (AOAC, 2005).

Hematological profile and glucose

After anesthesia, blood of 10 fish per replicate were collected by puncture of the caudal vessel and distributed in tubes containing EDTA (10%) for determination of hematocrit (Goldenfarb et al, 1971), hemoglobin (Collier, 1944) and, red blood cell counts using the solution Natt & Herrick as diluent and dye. Then, they were calculated the mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and, mean corpuscular hemoglobin concentration (MCHC).

Blood smears were stained with Panotic dye (Laborclin, PR, Brazil) to count leukocytes and thrombocytes. The determination of blood glucose was performed with the rapid glucose analyzer One Touch UltraMini® (Lifescan, CA, USA).

Analysis of intestinal morphometry

The collection of the intestine was performed in the same fish used in the blood analysis. After deep anesthesia, it was performed a longitudinal incision in the abdomen and collected a fragment of the anterior portion of the intestine located two centimeters after the pylorus. The fragments were fixed in Bouin solution for 24 h. Then, they were dehydrated in 70% alcohol to histological processing (Honorato et al., 2011). The slides were stained with hematoxylin-eosin (HE) for morphometric evaluation of villi and with periodic acid-Schiff (PAS) for goblet cell count.

The measurement of the height and width of the villi, epithelial thickness, and counting of goblet cells were performed according to Mello et al. (2013). It was used a Zeiss Axio Vision image analyzer.

Statistical analysis

Data were subjected to analysis of variance (ANOVA), differences between treatments were compared by Tukey test (P < 0.05). Data were analyzed by statistic R software (version 0.98.1) and presented as mean ± standard deviation.

RESULTS

Performance and proximal composition

Under the conditions evaluated, there was no statistical difference in weight gain, feed conversion, specific growth rate, and feed efficiency (Table 2). Proximal analysis of fish carcass showed no significant difference between treatment and control groups (Table 3).

 

Table 2. Growth performance indices of Nile tilapia fed with
experimental diets. WG: weight gain, SGR: specific growth rate,
FE: feed efficiency. Mean ± SD. There were no statistical
differences (P > 0.05) between treatments.

 

Table 3. Body composition (%) and gross energy (kcal kg-1) of Nile tilapia fed
with experimental diets. CP: crude protein, EE: ether extract, M: moisture, Ash;
GE: gross energy. Mean ± SD. There were no statistical differences (P > 0.05)
between treatments. Values are in % of dry weight.

 

Hemoglobin and glucose levels showed differences between treatments. The group supplemented with the highest concentration of probiotic (1x107 UFC g-1) showed the lower values for these variables. Other red blood cell parameters (Table 4) and differential leukocyte count (Table 5) showed no statistical difference between treatments.

 

Table 4. Hematological parameters. Hct: hematocrit, Hb: hemoglobin, RBC: red blood
cells, MCHC: mean corpuscular hemoglobin concentration, MCH: mean corpuscular
hemoglobin, MCV mean corpuscular volume, and G: plasma glucose of the Nile
tilapia fed different probiotic concentrations in feed. Means ± SD. Values
(mean ± SD) with different letter are significantly different (P < 0.05).

 

Table 5. Differential leukocyte count in the circulating blood of Nile tilapia fed the
experimental diets. Means ± SD. There were no statistical differences (P > 0.05)
between treatments.

 

Intestinal morphometry

Histomorphometric evaluation of the anterior portion of the intestine showed significant difference in the height of the villi in groups supplemented with 5x10CFU g-1 and 1x107 CFU g-1 compared to 1x106 CFU g-1 and control groups (Fig. 1). There were observed no lesion in the intestine and no differences in the width and thickness of the villi between control and treatment groups (Table 6). Fish that received the highest concentration of probiotic also had significantly greater number of goblet cells comparing to control group (Table 6, Fig. 2).

 

Figure 1. Comparative photomicrographs of the anterior portion of the
intestine of Nile tilapia fed different probiotic concentrations in the diet.
a) Control group, b) 1x106 CFU g-1, c) 5x106 CFU g-1, and d) 1x107 CFU g-1.
Villi height enhanced proportionally to the concentration of the probiotic.
Scale bar: 200 gm. Stain: hematoxylin-eosin.

 

Figure 2. Comparative photomicrographs of the
epithelial layer of the intestinal villi of Nile tilapia
supplemented with different probiotic concentrations
in the diet. a) Control group, b) 1x106 CFU g-1,
c) 5x106 CFU g-1, and d) 1x107 CFU g-1. Goblet
cells (deep red colour -magenta-cells on the edge
of the villi) increased proportionally to the
concentration of the probiotic in the diet. Scale
bar: 100 pm. Stain: Periodic acid-Schiff (PAS).

 

Table 6. Histomorphometric parameters and goblet cells per villus in the intestine of Nile
tilapia fed the experimental diets. Mean ± SD. Values (mean ± SD) with different letter
are significantly different (P < 0.05).

 

DISCUSSION

The use of B. amyloliquefaciens as a probiotic in fish feeding is recent and information is scarce. However, it has demonstrated that dietary supplementation with this bacterium improves growth performance in fish. In tilapia fingerlings reared in glass aquaria, supplementation with this bacterium improved weight gain, specific growth rate and feed conversion after supplement with 106 CFU g-1 of B. amyloliquifaciens for 60 days (Reda & Selim, 2015). However, no differences were observed when the probiotic was administered at 104 CFU g-1 of concentration. Similarly, Telli et al. (2014) found no effect of Bacillus subtilis (5x106 CFU g-1) on performance of Nile tilapia, even after 84 days of feeding supplemented diet.

In this study, there was no effect of the probiotic B. amyloliquefaciens on growth performance. This can be explained due to the low temperatures in the last 2 months. Marcusso et al. (2015) reported that the homeostasis of Nile tilapia rearing at temperatures below 24°C could be affected, enhancing the susceptibility to bacterial infections and impairing the growth performance.

Ridha & Azad (2012) tested B. amyloliquefaciens (108 CFU g-1) in Nile tilapia reared in tanks for 99 days and did not find any difference in performance indices either. However, they observed 60 days after that fish previously supplemented with the bacterium showed better growth performance than control group, suggesting that the effects of this probiotic occur over time. Unlike this report, in the present study fish were reared in cages in a reservoir with abundant algae (low transparency of water). This availability of food could influence the growth performance of fish, explaining why there was no difference between treatments.

The efficiency of nutrient transfer from food to the organism can be measured by the proximal composition analysis. This is of great importance due to it may reflect the nutritional value of the fish and its organoleptic characteristics (Contreras, 1994). Several authors have reported improvements in body composition of Nile tilapia supplemented with probiotics (Mello et al., 2013; Hassaan et al., 2014; Reda & Selin, 2015). However, in this study, body composition analysis values showed no significant difference between treatment and control groups. Similar results were found by Telli et al., (2014) in Nile tilapia fed diet supplemented with B. subitilis.

Hematological variables are commonly used as indicators of physiological condition in fish (Mohapatra et al., 2014). Some studies reported that after feeding with Bacillus sp., Nile tilapia presented alterations on the hematological variables (Hassaan et al., 2014; Telli et al., 2014.) or not (Kumar et al., 2006; Soltan & El-Laithy, 2008). In the present study, we did not observe differences in hematocrit, red blood cells count and hematological indices (MCHC, MCH and MCV). However, there was a higher hemoglobin levels in the fish fed with lower concentrations of probiotic. This could be related to the necessity of transport more oxygen in blood to meet the increasing energy demand of fish promoted by high levels of glucose (Nikinmaa et al., 1983).

As in current study, Reda & Selim (2015) observed increase in hemoglobin levels in blood of Nile tilapia supplemented with B. amyloliquefaciens. However, the authors also observed an increase in the number of erythrocytes and leukocytes in blood, but without changes in blood glucose. They were reporter that probiotic bacteria enhance the iron absorption due to the release of organics acids in the gut. This would increase the availability of iron to produce hemoglobin in rats (Yadav et al., 2007) and fish (Dahiya et al., 2012).

Probiotics enhance fish tolerance to stress agents of environmental origin or related to handling (Tapia-Paniagua et al., 2014). During stress, the release of catecholamines stimulates glycogenolysis (Martinez-Porchas et al., 2009). The rapid increase of glucose in blood is one indicator of stress in fish (Simoes et al., 2012). In the present study, the higher blood glucose observed in the control group compared to supplemented ones with the greater inclusion of probiotic in the diet may reflect the homeostatic state of the animals against adverse conditions. B. amyloliquefaciens seems to have positively influenced the glucose levels of fish supplemented with 5x106 CFU g-1 and 1x107 CFU g-1 of feed, enhancing the homeostatic state of fish. Similar findings have been reported by Mohapatra et al. (2014) in Labeo rohita stressed that presented lower glucose levels in fish fed with this probiotic.

Stress is one of the factors that contribute to the emergence of primary diseases and mortality of reared fish. Both food and environmental changes could promote stress modifying the gut microbiota and the establishment of pathogens in the gastrointestinal tract (Tannock & Savage, 1974). Thus, it is likely that an improvement in the homeostatic level promoted by the probiotic in this study may be related to increased stability of the gut microbiota with reduction of harmful bacteria in the epithelium (Rollo et al., 2006).

The intestinal lumen of the fish is lined by a simple columnar epithelium interspersed with goblet cells (Reifel & Travill, 1979). The integrity of the intestinal mucosa is a consequence of the intensity of epithelial cell renewal that determines the uniformity of the villi and the number of goblet cells (Mello et al., 2013).

The increased villi height observed in the supplemented groups in this study and the lower number of pathogenic bacteria in the enterocytes could be related to the increased availability of nutrients and reduced desquamation of the epithelium. Furthermore, it is known that some proteins produced by probiotic bacteria promote increased cellular survival time of the intestinal tract due to the inhibition of apoptosis signaling induced by cytokines (Sherman et al., 2009).

Mello et al. (2013) observed in Nile tilapia fed diets containing Bacillus cereus and B. subtilis an increasing of the area of absorption, nutrient retention and consequent improvement in growth performance and body composition. In this study, although the intestinal absorption area has grown substantially, there was no difference in performance. This can be explained due to the short evaluation time, which was not enough to show its effects in performance and proximal analysis. It can also relate to the low concentration of the probiotic or due to the adverse weather conditions recorded in the last month of the experiment in which the temperature decreased.

The production of mucus by goblet cells is an important mechanism of protection against the entry of pathogens through the intestinal tract (Ellis, 2001). Furthermore, the mucus has bactericidal effect, protects against toxic substances and aids the transport between the luminal contents and epithelial cells (Smirnov et al., 2005).

It is well documented that probiotics stimulate the development of goblet cells in the intestine (Nayak, 2010; Mello et al., 2013). In this study, fish that received the highest level of inclusion of probiotic showed significantly higher number of goblet cells compared to control group. This result corroborates the observations of Reda & Selim (2015) that observed growth of the villi and increased goblet cell number in fish supplemented with B. amyloliquefaciens.

From the results obtained, it is possible to suggest that in the tested conditions, the use of B. amyloliquefaciens in the diet presented good indications, increasing the size of the intestinal villi, number of goblet cells and enhancing the homeostatic state of the fish. However, it is necessary longer studies with larger concentrations of the probiotic in field conditions to asseverate that the use of this product is suitable to Nile tilapia.

ACKNOWLEDGEMENTS

We would like to thank FAPESP (2012/10090-4) for financing this study.

 

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Received: 28 July 2015;
Accepted: 25 September 2015

 

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