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Journal of Animal Science - Animal Nutrition

Bacillus cereus var. toyoi promotes growth, affects the histological organization and microbiota of the intestinal mucosa in rainbow trout fingerlings1

 

This article in JAS

  1. Vol. 91 No. 6, p. 2766-2774
     
    Received: Apr 26, 2012
    Accepted: Mar 08, 2013
    Published: November 25, 2014


    2 Corresponding author(s): enric.gisbert@irta.cat
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doi:10.2527/jas.2012-5414
  1. E. Gisbert 2,
  2. M. Castillo,
  3. A. Skalli*,
  4. K. B. Andree* and
  5. I. Badiola
  1. Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Centre de Sant Carles de la Ràpita, Unitat de Cultius Aqüícoles, E-43540 Sant Carles de la Rápita, Spain
    RUBINUM SA, E-08191 Rubí, Spain
    Centre de Recerca en Sanitat Animal (CReSA), Campus Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain

Abstract

In this preliminary study, we evaluated the effects of a gram-positive soil bacteria Bacillus cereus var. toyoi on the growth performance, digestive enzyme activities, intestinal morphology, and microbiota in rainbow trout Oncorhynchus mykiss fingerlings. Trout were maintained in a recirculation system and fed 2 diets: 1) a commercial trout feed deprived of the probiotic and 2) the same diet but with the spores of the probiotic bacteria dissolved in fish oil during the manufacturing of the feed (final concentration = 2 × 104 cfu/g). Each diet was tested in three 400-L cylindroconical tanks (125 fish per tank; initial density = 1.3 kg/m3; 13.2°C) for a period of 93 d. The probiotic-supplemented diet promoted growth, and the final mean BW and standard length in fish fed the probiotic were 3.4% and 2.1%, respectively, which was greater than the control group (P < 0.05). Fish fed the probiotic showed a more homogeneous distribution in the final BW, with a greater frequency of individuals around the modal of the normal distribution of the population. This result is of practical importance because homogenous production lots can improve rearing practices, reducing hierarchical dominance situations arising from individuals of larger sizes. In addition, the probiotic-supplemented diet increased the level of leukocyte infiltration in the lamina propria of the intestinal mucosa, the number of goblet cells (P < 0.010), and villi height (P < 0.001) but did not affect villi width. The administration of the probiotic changed the intestinal microbiota as indicated by 16S rDNA PCR-restriction fragment length polymorphism. In this sense, fish fed the probiotic formed a well-defined cluster composed of 1 super clade, whereas compared control fish had a greater degree of diversity in their gut microbiota. These changes in gut microbiota did not affect the specific activity of selected pancreatic and intestinal digestive enzymes. These results indicate that the inclusion of the probiotic bacteria in trout feeds could be beneficial for the host by enhancing its intestinal innate immune function and promoting growth.



INTRODUCTION

There have been numerous investigations evaluating the feasibility of supplementing diets with different probiotic bacteria in salmonid species (Merrifield et al., 2010a). Merrifield et al. (2010a) concluded that despite a lack of clarity of their precise mechanisms of action, the use of probiotics in salmonids has resulted in increased health status, growth performance, feed use, carcass composition, gastric morphology, and digestive enzyme activities, as well as reduced skeletal deformities and changes in intestinal microbiota.

Bacillus cereus var. toyoi is not an autochthonous bacterium in the intestinal microbiota of fish (Merrifield et al., 2009) but is a common soil inhabitant (Williams et al., 2009). Several studies demonstrated probiotic characteristics of this Bacillus strain (NCIMB 40112), and its use as a feed additive has been authorized in the European Union and other countries (Nakano, 2007; Williams et al., 2009). In livestock animals, this probiotic has been reported to increase BW gain, improve feed efficiency, and promote changes in gut microbiota and immune status (Jadamus et al., 2002; Scharek et al., 2007). However, few studies evaluate the inclusion of B. cereus var. toyoi in fish diets. The only published data with this probiotic bacterium were conducted with Anguilla japonica, where the dietary inclusion of spores of this bacterium reduced mortality rates, whereas its use in Seriola quinqueradiata juveniles promoted growth performance (Gatesoupe, 2010).

However, the information regarding the effects of this probiotic on fish performance and digestive histological organization and physiology is not available. Thus, the aim of this study was to evaluate the effects of supplementation of feed with the probiotic B. cereus var. toyoi on rainbow trout fingerlings by assessing its impact on fish growth performance, the organization and functionality of the digestive system, and possible changes in the gut microbiota.


MATERIAL AND METHODS

All animal experimental procedures were conducted in compliance with the experimental research protocol (reference number 4978-T9900002) approved by the Committee of Ethic and Animal Experimentation of the Institut de Recerca i Tecnologia Agroalimentàries (IRTA) and in accordance with the Guidelines of the European Union Council (86/609/EU) for the use of laboratory animals and the recommendations of the Association for the Study of Animal Behavior (ASAB, 2003).

Diets

The standard commercial diet (2.0-mm pellets, Aller Futura; Aller Aqua, Christiansfeld, Denmark) was used as the control diet. The diet containing B. cereus var. toyoi (CNCM I-1012/NCIMB 40112, Toyocerin; Rubinum, SA, Rubi, Spain) was prepared by using the same commercial diet (Aller Aqua). The spores of the probiotic bacteria were dissolved in 12% fish oil (999 Fish Oil; TripleNine Fish Protein, Esbjerg, Denmark) at 60°C, transferred with standard nozzles to the vacuum mixer (−2 atmospheric pressure), and sprayed on top of feed pellets. The final concentration of the probiotic in the experimental feed was 2 × 104 cfu/g, which was within the nontoxic and nonpathogenic levels of the inclusion for this probiotic species (Williams et al., 2009). The proximate composition of both tested diets was similar (Table 1).


View Full Table | Close Full ViewTable 1.

Composition of both diets used in this study

 
Item Control diet1 Probiotic diet
B. cereus var. toyoi, cfu/g 2 × 104
CP, % 64.2 ± 0.4 64.3 ± 0.3
Fat, % 11.8 ± 0.3 11.9 ± 0.2
Ash, % 11.2 ± 0.2 11.1 ± 0.2
Fiber, % 0.50 ± 0.01 0.50 ± 0.01
GE,2 KJ/kg 2,166 2,172
1Control diet: commercial diet (Aller Futura; Aller Aqua, Christiansfeld, Denmark).
2Gross energy content was estimated by using the following: total carbohydrate × 17.2 J/kg, fat × 39.5 J/kg, and protein × 23.5 J/kg.

Animals, Experimental Conditions, and General Procedures

Rainbow trout (O. mykiss) fingerlings (average, 2.8 g) were purchased from a commercial hatchery (Alevines del Moncayo SA, Vozmediano, Spain), transported by road to the IRTA-Sant Carles de la Ràpita (SCR) facilities (Sant Carles de la Rapita, Spain), and acclimated for 3 wk in a 3-m3 rectangular fiberglass tank. During this period, fish were fed twice a day (Microbaq 8; Dibaq SA, Fuentepelayo, Spain) at 2% of the stocked biomass. Before the onset of the trial, all fish were individually weighed (BWi) and measured for standard length (SL) to the nearest 0.1 g and 1 mm, respectively, and then were distributed into six 400-L fiberglass cylindroconical tanks (3 tanks/diet and125 fish/tank; initial density = 1.3 kg/m3). During the acclimation and experimental periods, water temperature, conductivity, pH (pH meter 507; Crison Instruments, Barcelona, Spain), and dissolved oxygen (OXI330; Crison Instruments) were 13.2°C ± 0.2°C, 1800 ± 200 µS/cm, 7.5 ± 0.01, and 8.0 ± 0.3 mg/L, respectively. Water flow rate in experimental tanks was maintained at approximately 9.0 L/min via a recirculation system that maintained adequate water quality (total ammonia and nitrite were ≤0.15 and 0.5 mg/L, respectively) through UV, biological, and mechanical filtration (Carbó et al., 2002). Photoperiod followed natural changes according to the season of the year (December to March; latitude 40°37′41″N). Each diet was tested in triplicate for a period of 93 d. Diets were distributed 4 times/d by automatic feeders (ARVO-TEC T Drum 2000; Arvotec, Huutokosk, Finland) at a rate of 3.3% of the stocked biomass, which approached apparent satiation.

Sampling to monitor fish growth took place monthly from the onset of the feeding period. For that purpose, 50 fish were randomly sampled for each tank and anesthetized with 150 mg/L tricaine methanesulfonate (MS-222; Sigma-Aldrich, Madrid, Spain), and their wet BW (g) and SL (cm) were determined. At the end of the trial (93 d), all fish from each tank were measured for their final BW (BWf, g) and final SL (SLf); in addition, 60 specimens per experimental condition (20 per replicate) were sacrificed with an overdose of anesthetic for histological purposes (n = 5), assessment of the functionality of the digestive system (n = 5), proximate biochemical composition (n = 5), and assessment of possible changes to the intestinal microbiota (n = 5).

Fish growth and feed use from different experimental groups was evaluated by means of the G:F and the following indices:

The K factor is a morphometric index that estimates the body condition of the fish, which is determined by measuring the weight and length of individual fish. This approach assumes that heavier fish of a given length are in better condition (Sutton et al., 2000). Body proximate composition of fish carcass was determined at the end of the study in 15 specimens per dietary condition (n = 5 per tank). Fish were homogenized, and small aliquots were dried (120°C for 24 h) to estimate water content. The total fat content from feed and fish was quantified gravimetrically after extraction in a chloroform-methanol solution (2:1) and evaporation of the solvent under a stream of N followed by vacuum desiccation overnight (Folch et al., 1957). Protein and carbohydrate contents were determined according to Lowry et al. (1951) and Dubois et al. (1956), respectively. Ash contents were determined by keeping the sample at 500°C to 600°C for 24 h in a muffle furnace (AOAC, 1990). All chemical analyses were performed in triplicate per fish and feed samples.

Organization and Functionality of the Digestive System and Gut Microbiota

For assessing the impact of the probiotic on the digestive system organization and functionality, sacrificed fish were dissected on a glass plate maintained at 0°C to 4°C. The pyloric ceca and anterior intestine were sampled for measuring the abundance of pancreatic proteases (trypsin, chymotrypsin, and total protease activities) and intestinal brush border enzymes (alkaline phosphatase, maltase, and aminopeptidase N). Quantification of digestive enzymes was conducted as previously described in Gisbert et al. (2009). The specific activity was expressed as international units or milli-international units per milligram of protein, whereas soluble protein of crude enzyme extracts was quantified using the Bradford method (Bradford, 1976) using BSA as a standard. For histological purposes, the posterior intestine from 12 fish per dietary treatment was dissected and fixed in 4% buffered formaldehyde (pH = 7.4), dehydrated in a graded series of ethanol, cleared with xylene, embedded in paraffin, and cut in serial sections (3 to 5 µm thick). Transversal sections of the posterior intestine were photographed (Olympus DP70 Digital Camera; Olympus Imaging Europa GmbH, Hamburg, Germany) connected to a light microscope (Leica DM LB; Leica Microsystems, Wetzlar, Germany). Digital images were processed and analyzed using an image analysis software package (ANALYSIS; Soft Imaging Systems GmbH, Münster, Germany). Measurements of total goblet cell number (full and empty) and villi height were based on the analysis of 8 randomly chosen fields from the intestinal mucosa of 12 fish per dietary group. In addition, the same fields were used for qualitatively assessing the level of leukocyte infiltration in the villi. Goblet cell counts in intestinal villi were expressed over a contour length of 100 μm, whereas villi height was calculated according to Escaffre et al. (2007).

Changes to the intestinal microbiota by means of RFLP was performed as described by Gómez-Conde et al. (2007). In brief, individual samples of the posterior intestine were processed for total DNA extraction using the kit (QIAamp DNA Stool Mini Kit; Qiagen Inc., Chatsworth, CA) according to the manufacturer’s instructions. The purified DNA was maintained at −20°C until its use. The primers 5′-CTACGGGAGGCAGCAGT-3′ and 5′-CCGTCWATTCMTTTGAGTTT-3′, corresponding to regions I and II of the 16S rRNA gene (Lane, 1991), were used to amplify a 500- to 600-bp product. Amplifications were performed in a final volume of 50 μL using a PCR-Master Mix (Applied Biosystems, Foster City, CA) containing 1.25 IU of Taq polymerase, 50 ng of DNA template, 0.2 µM of each primer, and the following cycling conditions: 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min and 15 s. The last extension cycle was continued for 5 min. Aliquots of the amplified DNA fragments were digested in separate tubes with restriction endonucleases (Alu I, Rsa I, Hpa II, Sau 3A I, or Cfo I; Sigma-Aldrich). The endonuclease fragments were resolved in 2% agarose gels at 150 V for 60 min, and DNA bands were visualized using an imaging system (UV Chemigenious Image System at a 4.63-s exposure; SynGene, Cambridge, UK) using software (GeneSnap; SynGene, Frederick, MD). Dendrograms showing the percentage of similarity among PCR-RFLP band patterns were generated on the basis of the Manhattan distance (Kaufman and Rousseeuw, 1990).

Statistical Analyses

The mean values of BWf, SLf, and K were expressed as mean ± SD. The calculation was based on the values of the individual BWf, SLf, and K of all the fish belonging to the same treatment (fish from the 3 replicate tanks per treatment analyzed together). The mean values of survival, SGR, G:F, intestinal goblet cell number, and villi width and height were expressed as mean ± SEM. In contrast to BWf and K values, these measurements were calculated using the values of the replicates (n = 5 for each treatment), as they cannot be calculated for all individual fish. The Kolmogorov-Smirnov (K-S) sample test was employed to evaluate BWf distribution at the end of the trial (Sokal and Rohlf, 1995), and differences in the relative frequency of size classes between experimental groups were compared by means of a paired t test. All statistical analyses were performed using IBM SPSS software (Statistics Version 20; IBM, Madrid, Spain).


RESULTS

At the end of the trial, survival rates between trout fed the control and the probiotic-supplemented diets were similar (Table 2). Fish fed the diet containing the probiotic were slightly heavier and longer (44.2 ± 7.6 g, 14.4 ± 1.1 cm) than those fed the control diet (42.7 ± 7.2 g, 14.1 ± 1.1 cm). The mean BWf and SLf in fish fed the probiotic were 3.4% and 2.1% greater, respectively, than the control group. No statistically significant differences were detected between groups considering the SGR values. Fish size distribution in BWf was also affected by the diet (Fig. 1). Fish fed the control and probiotic diets showed a unimodal and symmetric distribution of BWf (K-S distance = 0.256 and 0.275, respectively), although fish fed the control diet showed a right-skewed distribution (skewness values = 1.200 and 1.522, respectively). The group fed with the probiotic had 53.6% ± 2.1% of the individuals within the mode of the normal distribution of the population (41 to 50 g), whereas the control group had only 47.1% ± 1.5% (P < 0.01). In addition, the proportion of fish with 31 to 40 g was greater in the group of fish fed the diet containing the probiotic than the control group (P < 0.01). In contrast, the group fed the probiotic showed a lower frequency of fish weighing 21 to 30 g (3.6% ± 1.2%) compared with the group fed the control diet (6.9% ± 0.9%; P < 0.01).


View Full Table | Close Full ViewTable 2.

Growth performance [specific growth rate (SGR), Fulton’s condition factor (K), and G:F], survival rate, and chemical composition of rainbow trout, O. mykiss, fingerlings1

 
Item Control diet Probiotic diet
Growth performance
    SGR0to93d,2 %/d 2.50 ± 0.20 2.54 ± 0.11
    K3 1.43 ± 0.21 1.45 ± 0.15
    G:F2 0.90 ± 0.02 0.84 ± 0.01
    Survival rate,2 % 99.20 ± 0.80 98.90 ± 1.20
Composition,4 %
    Protein 41.40 ± 3.81 40.51 ± 1.51
    Lipid 17.62 ± 1.74 20.14 ± 0.81
    Carbohydrate 1.21 ± 0.01 1.11 ± 0.01
    Ash 2.21 ± 0.05 2.33 ± 0.06
1Control diet = commercial diet (Aller Futura; Aller Aqua, Christiansfeld, Denmark); probiotic diet = commercial diet containing B. cereus var. toyoi (2 × 104 cfu/g).
2Based on 3 replicate tanks per diet with 120 to 125 fish per tank.
3Mean K factor values were calculated using values of the individual final BW and final standard length of all the fish in the same dietary treatment (3 replicate tanks per diet with 120 to 125 fish per rank).
4Based on 3 replicate tanks per diet with 5 fish per tank.
Figure 1.
Figure 1.

Distribution of final BW of rainbow trout, O. mykiss, fingerlings fed the control diet (commercial diet, Aller Futura; Aller Aqua, Christiansfeld, Denmark) or probiotic diet (commercial diet containing 2 × 104 cfu/g of B. cereus var. toyoi). Mean frequency values for each size category are based on 3 replicate tanks with 120 to 125 fish per tank. Within a BW range, means for control and probiotic diets with asterisks differ (P < 0.01).

 

No statistically significant differences in Fulton’s condition factor were detected between the treatments (Table 2); however, differences existed in the frequency of individuals within a discrete category of K values (Fig. 2). Trout with K values ranging between 1.5 and 1.7 were more abundant in the group fed the probiotic diet (61.5% ± 1.4%) compared with that fed the control diet (56.3% ± 0.6%; P < 0.01). No statistically significant differences were detected among the rest of the K value intervals, but K values >2 were more frequent numerically in fish fed the control diet than those fed the probiotic diet (4.4% ± 1.1% vs. 0.8% ± 0.5%).

Figure 2.
Figure 2.

Distribution of Fulton’s condition factor (K) of rainbow trout, O. mykiss, fingerlings fed the control diet (commercial diet, Aller Futura; Aller Aqua, Christiansfeld, Denmark) or probiotic diet (commercial diet containing 2 × 104 cfu/g of B. cereus var. toyoi) diet. The K factor estimates the body condition of the fish and assumes that heavier fish of a given length are in better condition. Mean frequency values for each size category are based on 3 replicate tanks with 120 to 125 fish per tank. Within a BW range, means for control and probiotic diets with asterisks differ (P < 0.01).

 

The G:F values were similar between the experimental groups, and no statistically significant differences were detected in the composition (i.e., protein, fat, carbohydrate, or ash content of fish; Table 2). Similarly, the inclusion of the probiotic in the control diet did not affect the functionality of the digestive system, as indicated by the absence of statistically significant differences in the specific activity of selected pancreatic (trypsin, chymotrypsin, and total proteases) and intestinal brush border enzymes (alkaline phosphatase, aminopeptidase N, and maltase; Table 3).


View Full Table | Close Full ViewTable 3.

Specific activity of pancreatic (trypsin, chymotrypsin, and total proteases) and intestinal (alkaline phosphatase, aminopeptidase N, and maltase) enzymes of rainbow trout, O. mykiss, fingerlings1

 
Enzyme activity2 Control diet Probiotic diet
Trypsin, IU/mg protein 0.022 ± 0.003 0.024 ± 0.004
Chymotrypsin, mIU/mg protein 0.172 ± 0.060 0.124 ± 0.021
Total proteases, IU/mg protein 0.107 ± 0.011 0.112 ± 0.011
Alkaline phosphatase, IU/mg protein 0.651 ± 0.101 1.002 ± 0.141
Aminopeptidase N, IU/mg protein 0.010 ± 0.002 0.011 ± 0.001
Maltase, IU/mg protein 49.921 ± 8.561 51.762 ± 9.774
1Control diet = commercial diet (Aller Futura; Aller Aqua, Christiansfeld, Denmark); probiotic diet = commercial diet containing B. cereus var. toyoi (2 × 104 cfu/g).
2Based on 3 replicate tanks per diet with 5 fish per tank.

The inclusion of the probiotic in the control diet did not affect the width of intestinal villi (225.9 ± 8.9 vs. 224.6 ± 9.5 μm, respectively; Fig. 3, top). However, intestinal villi from the posterior intestine of fish fed the diet containing the probiotic were 43.2% greater (P < 0.01) than those from the fish fed the control diet (928.5 ± 16.0 vs. 527.3 ± 17.3 μm, respectively; Fig. 3, middle). Fish fed the diet containing the probiotic had a greater number of goblet cells counted over a contour length of 100 μm of intestinal epithelium (1.63 ± 0.03) than those fed the control diet (1.22 ± 0.05; P < 0.01; Fig. 3, bottom). Histological slides revealed that fish fed the probiotic showed a greater level of leukocyte infiltration of the lamina propria compared to those fed the control diet.

Figure 3.
Figure 3.

Box and whisker plots of the villus width and height and intestinal goblet cell number of rainbow trout, O. mykiss, fingerlings fed the control diet (commercial diet, Aller Futura; Aller Aqua, Christiansfeld, Denmark) or probiotic diet (commercial diet containing 2 × 104 cfu/g of B. cereus var. toyoi). Measurements of total goblet cell number (full and empty) and villi height were based on the analysis of 8 randomly chosen fields from the intestinal mucosa of 12 fish per diet (3 replicate pens/diet). The asterisk denotes that means for the control and probiotic diets differ (P < 0.01). Solid and dashed lines represent the median and mean values for each set of samples, respectively, whereas dots represent the outliers.

 

The dendrogram comparing the RFLP banding pattern of the microbiota isolated from fish fed both experimental diets showed that animals fed with the probiotic formed a well-defined cluster composed of 1 super clade (Fig. 4). Compared with control animals, in which 4 of the 5 replicates partitioned together, there was further subdivision of the clade into 2 subclades, indicating a greater degree of diversity in the gut microbiota.

Figure 4.
Figure 4.

Percentage of similarity among PCR-restriction fragment length polymorphism band patterns in the posterior intestine samples (5/diet) from rainbow trout, O. mykiss, fingerlings fed the control diet (commercial diet, Aller Futura; Aller Aqua, Christiansfeld, Denmark) or probiotic diet (T; commercial diet containing 2 × 104 cfu/g of B. cereus var. toyoi). R1 to R5 correspond to replicate numbers.

 

DISCUSSION

Considering the importance of nutrition in maintaining the health of fish, with respect to nutritional involvement on immunocompetence and disease resistance, as well as its role in stress mediation, there is a trend toward incorporating dietary components of a nonnutritional nature to provide various functional attributes (Nakagawa et al., 2007; Merrifield et al., 2010b; Ray et al., 2012). In this context, a large number of studies have demonstrated the capacity of probiotics in avoiding potential pathogens and improving the welfare of farmed aquatic species (Gatesoupe, 1999; Lara-Flores et al., 2003; Macey and Coyne, 2005; Wang and Xu, 2006; Kesarcodi-Watson et al., 2008; Gatesoupe, 2010; Merrifield et al., 2010a). In addition, probiotics have also been reported to improve growth performance (Decamp et al., 2008; Ghosh et al., 2008; Luis-Villaseñor et al., 2011), and the most common probiotics used in aquaculture belong to several genera, such as Saccharomyces, Clostridium, Lactobacillus, Bacillus, Enterococcus, Shewanella, Leuconostoc, Lactococcus, Carnobacterium, Aeromonas, and some other species (Nayak, 2010). In salmonids (Oncorhynchus mykiss, Salmo salar, and S. trutta), the most commonly used probiotic bacteria are Bacillus spp., Lactobacillus spp., Aeromonas spp., Pseudomonas spp., Carnobacterium spp., Pediococcus spp., and others (Merrifield et al., 2010b). The results from this study demonstrated the beneficial effects of the dietary inclusion of the probiotic B. cereus var. toyoi in terms of growth performance (BWf and SLf) and potentially beneficial changes in the intestinal mucosa organization in rainbow trout fingerlings.

These results are similar to those obtained in S. quinqueradiata fed the same probiotic (Gatesoupe, 2010) and those reported in rainbow trout fed diets containing a mixture of 2 probiotic bacteria belonging to the same genus, B. subtilis and B. licheniformis, for a period of 10 wk (Merrifield et al., 2010b). In channel catfish (Ictalurus punctatus) ponds inoculated with a commercial bacterial mixture of Bacillus spp., the addition of the bacteria to the water improved water quality, resulting in an increase of survival and fish production (Queiroz and Boyd, 1998). In this context, Bacillus spp. have been reported to possess adhesion abilities, provide immunostimulation, and produce bacteriocins (Ray et al., 2012). Commercial products containing such bacilli have been demonstrated to improve shrimp production to a level similar to that observed when antibiotics are used (Decamp and Moriarty 2006). The use of other probiotic bacteria has also been reported as beneficial in other fish species in terms of growth performance (Gatesoupe, 1999; Bairagi et al., 2002; Wang and Xu, 2006; Mohapatra et al., 2012), although it is not clear if their mode of action was similar (Kesarcodi-Watson et al., 2008; Merrifield et al., 2010b; Ringø et al., 2010).

Several studies have shown that the application of probiotics can improve feed efficiency, growth rates, weight gain, and chemical composition of several fish species, including salmonids (Merrifield et al., 2010b). Under the present experimental conditions, although the diet with the probiotic promoted growth moderately in rainbow trout fingerlings (3.4% in BWf), this was not reflected in statistically significant changes in the SGR and G:F values between the 2 experimental groups.

The size heterogeneity is a common feature in salmonid farming (Stead and Laird, 2002) that affects the overall performance of the rearing process. The results of the so-called hierarchical size effect are the establishment of a group of dominant fish that do not allow smaller (subordinate) ones to feed normally. Therefore, under conditions promoting hierarchy formation, the largest fish at the beginning are expected to get the largest share of the feed, grow the fastest, and have the greatest BW at the end of the production process (Petursdottir, 2002). Under present experimental conditions, fish size distribution in BW at the end of the study was affected by the dietary treatment. Thus, rainbow trout fingerlings fed the probiotic showed a more homogeneous distribution in BW, a greater proportion of animals weighing between 51 and 70 g, and a lower proportion of fish belonging to small size classes (21 to 30 g) in comparison with the control group. These findings are of practical importance because the use of the probiotic might reduce the effort required for size selection during processing of production lots and also in a reduction of hierarchical dominance situations.

The body composition of both groups was similar. On the contrary, the promotion of feed conversion and BW gain, resulting from improvement of digestion and nutrient absorption, was observed in S. quinqueradiata fed the same probiotic (Gatesoupe, 2010) and also in poultry (Vilà et al., 2009). These results indicated that further research is needed to establish a proper dosage level of B. cereus var. toyoi and the duration of dietary supplementation in rainbow trout fingerlings to maximize growth performance and feed efficiency.

Although the exact role of gut microbiota in fish nutrition is difficult to elucidate because of the complex and variable ecology of the gastrointestinal tract of fish (Nayak 2010), several authors have reported that the dietary administration of different probiotics enhanced the maturation of the digestive tract in fish larvae (Tovar-Ramírez et al., 2004), as well as the secretion and activity of digestive pancreatic and intestinal enzymes, leading to better growth performance and feed efficiency (Wang and Xu, 2006; Nayak, 2010; Mohapatra et al., 2012; Ray et al., 2012). In the present study, no differences in specific activity of pancreatic (trypsin, chymotrypsin, and total proteases) and intestinal (maltase, alkaline phosphatase, and aminopeptidase N) enzymes were detected between groups, which seemed to indicate that the digestive physiology of the animals was not affected by the inclusion of the dietary probiotic, either by direct contribution of exogenous enzymes produced by probiotic bacteria (Ding et al., 2004; Zhang et al., 2010; Ray et al., 2012) or by dietary stimulation of endogenous enzyme production and enhanced maturation of the digestive system (Tovar-Ramírez et al., 2004; Mohapatra et al., 2012). However, considering the capacity of this probiotic strain for producing and secreting other exogenous enzymes other than those considered in this study, like chitinase or cellulase, that could enhance feed digestibility, as has been reported for other Bacillus spp. (Askarian et al., 2012; Ray et al., 2012), further research is needed to evaluate the effect of B. cereus var. toyoi on growth performance.

Although the incorporation of the probiotic did not enhance digestive enzyme activities, it had a direct effect on the intestinal microbiota. Rainbow trout fingerlings fed the diet incorporating the probiotic showed a different intestinal microbiota compared with the control group as indicated by RFLP analyses. These results are similar to those already reported for B. cereus var. toyoi administered to swine and poultry (Jadamus et al., 2002; Scharek et al., 2007; Vilà et al., 2009). Changes in the intestinal microbiota, although not clearly delineated in terms of total species number or composition, can still be an effective means for stimulating fish growth or immunity or both. Numerous studies with rodents have shown that conventionally raised animals have more and larger goblet cells than those raised germ free with a corresponding increase in the complexity of composition and amount of mucus (Forder et al., 2007). The expansion of the goblet cell population would benefit the host by providing an effective immune barrier against gut microbiota. The mucus layer produced is known to be composed of 2 structurally distinct layers in the rodent gut (Johansson et al., 2008), an inner compact layer that is adherent to the gut epithelia and an outer looser layer that exists in commensal bacteria. These commensal bacteria inhabiting the gut are capable of actively digesting the mucopolysaccharide layer, which they inhabit, surviving from nutrients acquired by the breakdown of the mucin layer itself (Deplancke and Gaskins, 2001). Assuming that the knowledge obtained from studies with rodents and the gut epithelium is also true for fish, it can be presumed that some of the indirect by-products of mucus breakdown (metabolites derived from the mucolytic bacteria) can be beneficial to the nutrition and therefore the growth of the fish directly. The observed increase in goblet cells observed in this study, which could be acting synergistically, would benefit this process as well. In addition, the larger infiltration of leukocytes in the lamina propria of villi from fish fed the diet supplemented with probiotic would support the plausibility of an enhanced immune response in fish.

The intestinal microbiota can also act as a protective barrier against pathogens by depriving invading pathogens of nutrients or secreting a range of antimicrobial substances or both (Ringø et al., 2007). Thus, a probiotic-induced change in the normal microbiota in rainbow trout fingerlings might protect the host against potential pathogens, as well as promote the gut immune response, which is of special relevance as the gastrointestinal tract is the major route for the onset of any disease transmitted by the fecal-oral route or, more specifically, diseases like vibriosis, furunculosis, enteric septicemia, and aeromoniasis in fish (Nayak, 2010). In addition, the intestinal microbiota also influences the development of the gut and is a key component involved in the regulation of mucosal tolerance, development, and differentiation (Bates et al., 2006). In this study, the number of goblet cells in the intestinal mucosa of rainbow trout fed the probiotic supplemented diet was greater than in the control group. The major function of intestinal goblet cells and their main secretory products, mucins, is the formation of mucus layers, which serve as the front line for the innate host defense mechanism. These mucus layers play key roles in the establishment of the commensal intestinal microbiota and the protection from colonization and invasion by pathogenic microbiota (Kim and Ho, 2010; McGuckin et al., 2011). The larger number of mucin-producing goblet cells in rainbow trout fed the diet supplemented with the probiotic might be due to the observed change in the gut microbiota because goblet cells are modulated by the presence and abundance of microbial organisms (Kim and Ho, 2010). Thus, such an increase in the number of goblet cells might indicate an enhancement of the intestinal innate immune function in those animals (Sherman et al., 2009).

As reviewed by Merrifield et al. (2010a), there is scarce information on the effects of probiotics in the intestinal morphology of salmonids. In this sense, rainbow trout fed diets supplemented with Pediococcus acidilactici showed increased microvilli length but not density, whereas the dietary incorporation of other probiotic bacteria (B. subtilis, B. licheniformis, and Enterococcus faecium) did not affect the intestinal morphology (Merrifield et al., 2010c). In the present study, rainbow trout fed the probiotic showed larger villi than those fish fed the control diet, which seemed to indicate that B. cereus var. toyoi promoted the development of the intestinal mucosa. The larger, but not wider, size of intestinal villi might have resulted in a greater absorptive intestinal surface and an improvement in nutrient absorption, which might explain the moderate numerical improvement in growth performance in rainbow trout fed the probiotic-supplemented diet.

In conclusion, the results from this preliminary study indicated that the inclusion of B. cereus var. toyoi at the final concentration of 2 × 104 cfu/g in a commercial diet promoted growth and resulted in a more homogeneous distribution of BW in rainbow trout fingerlings. In addition, the application of the probiotic enhanced the number of goblet cells and villi height of the intestinal mucosa and changed the intestinal microbiota, even though it did not affect the specific activity of selected pancreatic and intestinal digestive enzymes. The inclusion of this probiotic in trout feeds could be beneficial and advantageous for the fish host by an enhancement of its intestinal innate immune function. In addition, these results might be of practical significance because homogenous production lots can improve rearing practices or reduce hierarchical dominance situations arising from individuals of larger sizes. However, more studies are needed to characterize the modes of action of this gram-positive soil bacteria in the gut of fish, the specifics of modulation of the intestinal microbiota, and its ability to inhibit the growth of potential pathogens, as well as its optimal dosage to administer under different rearing conditions and the minimum duration required for supplementation to achieve these perceived benefits.

 

References

Footnotes


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