1 Introduction

The use of native species in the aquaculture of Northeastern Argentina had shown an increased productivity, allowing their predominance in the National fish production chart. Piaractus mesopotamicus is the most cultivated specie in this region and the most farmed fish in the country since 2012 (Dirección de Acuicultura, 2013), with 2 017.45 tons produced in 2013, representing a 52.22% of the Argentinian aquaculture production (Dirección de Acuicultura 2014). The inclusion of indigenous fishes indicates an advantage supported by their resistance to the environmental conditions. However, they were not deeply studied, leaving gaps in some critical issues such as animal development, behavior, nutritional requirements and susceptible diseases. These facts and the high demand of larvae, juveniles and sexually mature adults, generated a deficit in the number of animals available. Thus, the requirement of techniques aimed to increase the production in aquaculture facilities becomes a gap in the area. Antibiotics are one of the applied strategies, used as growing factors, anti-infectious agents and also for tranquilization (FDA, 1998; Serrano, 2005). However, the European Union (EU), under the European Food Safety Authority (EFSA), have regulated the use of additives in animal feeding and prohibited antibiotics as additives in foods (EFSA, 2008). These regulations are now applied in most of the continents, supported mainly by various items: resistance transference (FAO/NACA/WHO, 1997), toxicity of residues (SOU, 1997), allergies and effects on human intestinal microbiota (Serrano, 2005) and environmental risks (EC, 1996). Although expected, these regulations have generated an urgent problem with many difficulties to solve: the search of more extensive production systems without the requirement of using additives, and/or the application of natural, novel and safe products to get similar results to those previously obtained with antibiotics.

The use of probiotics is widely applied in order to replace the use of chemotherapeutic agents in animal production, and to restore the indigenous microbiota. In aquaculture systems, probiotics were described as “a live microbial adjunct which has a beneficial effect on the host by modifying the host associated or ambient microbial community, by ensuring improved use of feed or enhancing its nutritional value, by enhancing the host response toward disease or by improving the quality of its ambient environment” (Verschuere et al., 2000), and later defined for a wide variety of hosts (ISAPP, 2011). Several beneficial effects were reported for different aquatic species: inhibitory activity against pathogens, nonspecific immune response stimulation, immunomodulatory effect, decrease of mortality and increase of growth rate and production (Gullian et al., 2004; Vieira et al., 2010; Nayak and Mukherjee, 2011; Rahman et al., 2011; Lakshmi et al., 2013).

Different bacterial genus, mainly those classified as lactic acid bacteria (LAB), including lactobacilli, enterococci and related (Bifidobacteria) have been potentially evaluated as probiotics (Mego et al., 2005; Marcinakova et al., 2006). Among enterococci, only Enterococcus faecium is included in most of the major probiotic products commercially available for aquatic animal nutrition in the EU (Bogut et al., 2000; Chang and Liu, 2002; Vahjen et al., 2007; Gatesoupe, 2008; Abumourad et al., 2014). Pediococcus acidilactici is a Food Grade Microorganism (FGM) frequently isolated from dairy products. As they are included in the Qualified Presumption of Safety (QPS) and found in a variety of ecosystems, are commonly used as probiotic for humans (Balgeri et al., 2013), terrestrial (Giancamillo et al., 2008; Mikulski et al., 2012), and aquatic animals (Castex et al., 2010; Neissi et al., 2013 and 2015; Giannenas et al., 2015; Ramos et al., 2015).

In previous studies E. faecium strains CRL 1937, CRL 1938, CRL 1940 and CRL 1941 and P. acidilactici strain CRL 1939 were isolated from healthy specimens of P. mesopotamicus, selected as potentially probiotic based on their “in vitro”  beneficial properties and grouped into two formulae: LAB containing strains isolated in summer (P. acidilactici CRL 1939 and E. faecium CRL 1940 and CRL 1941) and ENT containing enterococci isolated in winter (E. faecium CRL 1937, CRL 1938) (Guidoli et al., 2015). As there are no previous descriptions of autochthonous strains as probiotics in this fish species, the aim of this work was to evaluate the effect of these two formulas on the survival, mean weight, biomass and histological parameters of P. mesopotamicus larvae. Another purpose was to select the optimum doses and stages of administration of each formula for the design of a multi-strain probiotic product.

2 Materials and Methods

2.1 Strains and formulae

Autochthonous E. faecium CRL1937, CRL1938, CRL1940 and CRL1941 and P. acidilactici CRL1939 strains were isolated from diverse body areas of healthy P. mesopotamicus specimens during different seasons and selected as potentially probiotic microorganisms based on their ability to express beneficial properties in in vitro tests (Table 1) (Guidoli et al., 2015). Strains were grouped into two formulae (LAB and ENT) according to their characteristics and the season in which were isolated (Table 1). All the microorganisms were included in the CERELA strain collection and in the Laboratorio de Sanidad Animal of the Estación Experimental Agropecuaria Rafaela belonging to the Instituto Nacional de Tecnología Agropecuaria (INTA) under Budapest treaty for patent aims (Conicet and Unne, 2013).

2.2 Bacterial suspensions

The lactic acid bacteria (LAB) were grown daily in 400 mL LAPTg broth (Raibaud et al., 1963) and incubated at 37℃ in static conditions for 8 h. Bacterial cells were harvested by centrifugation at 3,000 g for 10 min at 4℃, washed twice with sterile distilled water and suspended to obtain the required viable cells concentration.

2.3 Live food preparation

One L freshwater containing a suspension of 1 g of brine shrimp cysts Artemia sp., 15 g of NaCl and 2 g of sodium bicarbonate was submitted to an incubation process under intense aeration and lightening for 24 h. Live hatched nauplii were harvested from the bottom of the hatching device by sedimentation, filtered to obtain those between 250 and 150 µm size and suspended in freshwater. Aliquots of the nauplii suspension previously obtained were counted in order to obtain their approximate number and concentration of the solution (Torrentera and Tacon, 1989).

2.4 Fish reproduction

P. mesopotamicus larvae were obtained by controlled reproduction from broodstock of the Instituto de Ictiologia del Nordeste (Corrientes, Argentina). Spawning was induced by injection of pituitary extract from the of Prochilodus lineatus (5 and 1.5 mg pituitary gland kg^-1 of body weight in females and males, respectively) according to Da Silva et al.(1988). The sexual gametes were obtained by the stripping technique, mixed immediately in a bowl, suspended into fresh water for 5 minutes, washed twice and suspended at the desired concentration (Gómez et al., 2014). Aliquots of 1 mL were counted in order to obtain an approximate concentration of fecundated eggs.

2.5 Experimental design and sampling

Experimental units were settled as 5 L plastic fishbowls with a constant recirculation system and approximately 300 fecundated eggs. The formulae were administered in three different concentrations, named as: 4 (6x10⁴ CFU L-1), 7 (6x10⁷ CFU L-1) and 10 (6x10¹⁰ CFU L-1) of each strain. Also at different stages of the biological cycle of larvae: E (from the time of fecundation of the eggs until the beginning of the exogenous feeding, 5 days), L (from the beginning of the exogenous feeding until the end of the assay in the laboratory, from day 5 up to day 15) and E&L (from the fecundation of the eggs until the end of the assay in laboratory, from day 0 up to day 15). Control units (CTRL) were performed with no addition of bacteria.

During stage E the bacterial suspension was added directly to the fish bowls four times a day with previous stop of water recirculation which was restarted one hour after. In stage L, bacteria were co-incubated with live food during two hours previous to administration; this procedure was performed ad libitum four times a day. Water recirculation was interrupted before alimentation and restored one hour after. Water quality determinations of pH, dissolved oxygen and temperature were performed daily in each experimental unit.

2.6 Sampling

At day 15, larvae were counted and weighed to obtain values of survival, mean weight and biomass. To evaluate the normal development, macroscopic evaluations were performed with binocular magnifier Optical Kyowa model SDZ. For histological studies, 10 larvae per treatment were collected and anaesthetized by chilling on ice, fixed in Bouin’s solution (saturated picric acid 3000 ml, formaldehyde 1000 ml, glacial acetic acid 200 ml) for 12 h, washed twice with alcohol 70° and maintained in this solution until processing. Samples were then routinely processed for histology, stained with haematoxylin and eosin and analyzed by light microscopy using a Leica DM500 microscope, a Leica ICC50 digital camera and the Leica Application Suite 3.4.1 image analysis system (Culling, 1974).

The procedures and experimental protocols applied to the animals in this work were in accordance with the ethical principles of animal experimentation, and approved according to protocol n.0019/14-2011-02204 and 14-2012-03865 by the Ethics and Biosafety Committee of the School of Veterinary Sciences of the Northeast National University (UNNE) of Argentine.

2.7 Statistical evaluation

All the assays were performed by triplicate using a completely randomized design. Each replicate corresponds to different parents, excluding the genetic factor of the experiment. Statistical analyses were carried out using Statistica 6.0 for Microsoft Windows. Comparisons were performed, first, by a one way ANOVA including the ten experimental groups followed by a control vs. treatments comparison. Later, results were compared, excluding the control, by using a factorial two-way ANOVA with subsequent post hoc test in order to evaluate the main effects of doses and stages as well as the interactions between them. When interaction and significant effects were not detected, results were evaluated by orthogonal polynomials for trend analysis.

3 Results

The one way ANOVA of survival, mean weight and produced biomass of larvae administered with the microbial mixture of LAB does not indicate significant differences between treatments and control group (p>0.05) (Table 2). On the other hand, the two ways ANOVA analysis does not show neither interaction between doses and stages nor significant effect of dose or stage over any of the variables analyzed (p>0.05) (Figure 1). However, the low parameters in some of the treatments with dose 7 and 10 allowed to select the LAB suspension (composed by E. faecium strains CRL1940 and CRL1941 and P. acidilactici CRL 1939), containing 6x10⁴ CFU L-1 of each strain as the most suitable for its incorporation to a composite probiotic formula to be tested in future assays (Figure 2).

Figure 2 Mean weight, Survival percentage and Biomass of Piaractus mesopotamicus after bacterial treatments with LAB suspension on day 15

The one-way ANOVA of the administration of ENT suspension (Table 3) shows no significant differences in the survival percentage of the specimens (p>0.05), with similar values after all the treatments (Figure 3). The two-ways ANOVA analysis shows neither dose-phase interaction nor significant effect of the stage of administration (p>0.05) (Figure 4). However, the orthogonal polynomials for trend analysis of the dose effect detected a significant effect of the linear and quadratic contrasts of the mean weight and biomass (p>0.05) (Table 4). The quadratic contrast showed to be the best model to describe the polynomial function for both variables due to the fact that the highest estimated values belonged to dose 6x10⁷ CFU L-1.

Table  3  One way ANOVA of mean weight, survival and biomass of P. mesopotamicus larvae administered with ENT mixture in different doses and stag

The macroscopic evaluation of the animals showed no visible alterations of the normal development and/or behavior of the fishes in any of the doses of bacterial administered. The histological evaluation indicated no modifications of the normal structure of major organs, aggrupation of microorganisms or translocation to any of the organs under evaluation when comparing each treatment with control. Histology of stomach, liver and intestine showed no significant differences between control animals and those treated with the dose 4 of LAB and dose 7 of ENT during the three different stages assayed. They are resumed in Figure 5.

4 Discussion

The use of probiotics in aquaculture has increased exponentially in the last years (Verschuere et al., 2000; Reid et al., 2003; Romalde et al., 2005). However, commercial probiotics in fish culture are relatively ineffective. Most products include non-autochthonous strains unable to survive or remain viable in the intestinal environment (Ridha and Azad, 2015). Different authors have shown from long time ago (Kotarsky and Savage, 1979, Marteau, 2011) and demonstrated lately by the application of molecular biology techniques, the specie-specificity of the microbioma, or autochtonous microbiota, and also related to each specific tract of mucosa (Chi et al., 2014). It is essential the isolation and study of putative native probiotic microorganisms that are part of the microbiome of each specific host. Such strains have higher possibilities to survive and remain because they have already resisted these environmental conditions (Ghosh et al., 2007). Based on previous in vitro evaluations, our research group has selected five autochthonous lactic acid bacteria (three E. faecium and one P. acidilactici strains) as novel putative probiotics (Guidoli et al., 2015). However, the definitive application and clinical evidence of their beneficial effects should be evaluated through in vivo assays (ISAPP, 2011). Therefore, in this work we evaluated the effect of the administration of two different formulae composed by beneficial autochthonous microorganisms isolated on two seasons on the survival, mean weight, biomass and histological parameters of P. mesopotamicus larvae.

There are three critical items related to probiotic application: the stage of the biological cycle, the optimal dose and the way in which they should be administered to the host. Pasteris et al. (2012) suggested that the colonization of the skin (or scams) and the gastrointestinal tract of fishes occurs together with the ontogeny, as a way to be incorporated into the microbioma of each specie. The microbial adhesion to the eggs’ surface is, then, the main factor to determine the development of the epibiota. Afterwards, the ingestion of microorganisms in larval stages results in the establishment of a dominant intestinal microbiota that seems to persist during the fish life. Unfortunately, there is no general consensus on the other two critical items cited before. Bibliographic references suggest doses of administration from 1x10³ to 1x10⁹ CFU and different ways of administration such as balanced food or environmental water (Brunt and Austin, 2005; Bagheri et al., 2008). The last critical item is the way of administration, being widely used the food (Brunt and Austin, 2005; Bagheri et al., 2008). In this work, the selected beneficial microorganisms were added at different doses, stages and systems of administration.

Although ANOVA analysis did not show significant differences or interactions of different treatments with ENT suspension and control, the trend analysis of the dose effect showed significant differences for the mean weight and biomass. This test showed the quadratic contrast as the best model to describe the polynomial function for both variables, indicating the dose 7 (6x10⁷ CFU L-1 of each strain) as the most effective one. The better effect of medium doses (quadratic effect) was described by several authors in aquaculture (Bagheri et al., 2008; Faramarzi et al., 2011; Kapareiko et al., 2011).

Statistical evaluations of the results of administering BAL suspension did not show significant differences. None of the treatments with dose 4, regardless stage of administration, showed values of survival, mean weight and biomass higher than the control group. Then, we selected dose 4 as the most adequate to be incorporated in future assays. This result, although not expected, support the concept that a higher number of probiotic cells included in diets and host intestine does not necessarily result in a highest or improved growth, survival and protein efficiency ratio of animals (Bagheri et al., 2008).

The macroscopic observations of the animals did not show modifications in their behavior and/or development of those treated with the probiotic strains when compared with control. The histological evaluation demonstrated no translocation or aggrupation of microorganisms, studies that needs to be performed to determine the safety of the strains used as probiotics (Pasteris et al., 2009; ISAPP, 2011). Histological studies of major organs and tissues were comparable to those described as normal for each stage (Mendoza et al., 2013). The results obtained indicate that the administration of the two formulae in the doses herein evaluated did not affect the normal development and function of animals.

Then, the dose 7 (6x10⁷ CFU L-1 of each strain) of the ENT suspension (composed by strains E. faecium CRL 1937 and E. faecium CRL 1938) and dose 4 (6x10⁴ CFU L-1 of each strain) of the LAB suspension (composed by strains E. faecium CRL 1940 and CRL 1941 and P.acidilactici CRL 1939) were selected to be included in a potentially probiotic mixture to be assayed in vivo in future assays. And a combination of these strains with three autochthonous Bacillus subtilis strains, were registered for patent aims (Conicet and Unne, 2013).

Acknowledgments

The Argentinian National Council of Scientific and Technical Researches (Consejo Nacional de Investigaciones Científicas y Técnicas–CONICET), projects numbers PIP 632 and PIP 744, together with the National Agency for Scientific and Technological Promotion (Agencia Nacional de Promoción Científica y Tecnológica–ANPCyT) through the Fund for Scientific and Technological Research (Fondo para la Investigación Científica y Tecnológica–FONCyT); projects numbers PICTO-UNNE-2007-161, PICTO-UNNE-2011-198, PICT-543, PICT-1088 and PICT-1187; financed this study. Some of the results of this paper were included in a patent presentation in Argentina (INPI Exp 20130103820)