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Effects of prebiotics on growth and survival of silver catfish (Rhamdia quelen) juveniles/Efectos de prebioticos sobre el crecimiento y la supervivencia de juveniles de Rhamdia quelen/Efeito de prebioticos no crescimento e sobrevivencia de juvenis de jundia, Rhamdia quelen.

SUMMARY

Recently, proof of the advantages of employing beneficial microorganisms or organic additives as growth promoters, replacing chemoterapeutic and antimicrobial agents, has been obtained. The aim of this work was to evaluate the effect of two experimental diets, one supplemented by a flavonoid-based metabolic antioxidant (FLAVOXIN[R]) and the other supplemented by a combination of organic acids and yeast wall (UNIWALL MOS 50[R]) on growth and survival parameters of Rhamdia quelen juveniles, in contrast to a control diet. The experiment consisted of three dietary treatments: a control consisting of a commercial diet with 280g of proteins/kg and 120g of lipids/kg, the commercial diet supplemented with FLAVOXIN[R] (FL) and commercial diet supplemented with UNIWALL MOS[R] (MOS), both products added on a 2g/kg basis. Nine batches consisting of 42 specimens distributed in outdoor cement tanks of 16[m.sup.3] were assigned according to a 3x3 Latin square design. The experiment lasted a total of 60 days. Weight gain was determined in all treatments, without statistical differences between them (p>0.05). The survival rate was 89% for the control diet and 97 and 95% for FL and MOS, respectively. Both FL and MOS treatments resulted superior and statistically different to the control diet (p<0.05). The results show that the use of FLAVOXIN[R] and UNIWALL MOS 50[R] at the dose employed for this study has a positive effect on survival of R. quelen juveniles, without modifying growth parameters.

RESUMEN

Recientemente se han obtenido evidencias que demuestran las ventajas de la utilizacion de microorganismos bencficos o aditivos organicos como promotores del crecimiento en reemplazo de agentes quimioterapeuticos y antimicrobianos. El objetivo del presente trabajo fue evaluar el efecto de un alimento suplementado con un reparador metabolico a base de flavonoides (FLAVOXIN[R]) y otro con una combinacion de acidos organicos y pared de levadura (UNIWALL MOS 50[R]), sobre parametros de crecimiento y supervivencia en juveniles de Rhamdia quelen, contrastados con un alimento control. Se utilizaron tres tratamientos alimentarios." control consistente en dieta comercial con 280g de proteinas/kg y 120g de lipidos/kg, dieta comercial suplementada con FLAVOXIN[R] (FL), y dieta comercial suplementada con UNIWALL MOS[R] (MOS), ambos productos adicionados a razon de 2g/kg. Nueve lotes de 42 ejemplares distribuidos en estanques de mamposteria a cielo abierto de 16[m.sup.3] fizeron asignados de acuerdo a un diseho en cuadrado latino 3 x 3. La experiencia tuvo una duracion total de 60 dias, verificandose un incremento de peso en todos los tratamientos alimentarios, aunque sin diferencias estadisticamente significativas entre los mismos (p>0,05). El porcentaje de supervivencia fue de 89% en la dieta control y de 97 y 95% para FL y MOS, respectivamente, resultando ambos tratamientos estadisticamente superiores al control (p<0,05). Los resultados demuestran que el uso de FLAVOXIN[R] y UNIWALL MOS 50[R] a la dosis empleada en el presente estudio ejerce un efecto positivo sobre la supervivencia de juveniles de R. quelen sin modificar los parametros de crecimiento.

RESUMO

Em estudos recentes, tem sido provada a vantagem do emprego de microorganismos beneficos ou aditivos organicos como promotores de crescimento, substituindo agentes quimioterapicos e antimicrobianos. O objetivo deste trabalho foi avaliar o efeito de duas dietas experimentais, uma suplementada por um reparador metabolico baseada em flavonoides (FLAVOXIN[R]) e outra suplementada com uma combinacao de acidos organicos e parede de levedura (MOS UNIWALL 50[R]) sobre parametros de crescimento e sobrevivencia para juvenis de Rhamdia quelen, em contraste com uma dieta controle. O experimento consistiu em tres tratamentos alimentares: controle consistente em dieta comercial contendo 280g de proteinas/kg e 120g de lipidios/kg, dieta comercial suplementada com FLAVOXIN[R] (FL), e dieta comercial suplementada com UNIWALL MOS[R] (MOS), ambos os produtos adicionados em base a 2g/ kg de racao. Nove lotes de 42 animais foram distribuidos em tanques de cimento de 16[m.sup.3], atribuidas de acordo com um desenho em quadrado latino 3x3. O experimento teve duracao de 60 dias. Ao final do ensaio, foi observado ganho de peso em todos os tratamentos, sem diferenca estatistica entre eles (p>0,05). A taxa de sobrevivencia foi de 89% para a dieta controle e 97 e 95%o para FL e MOS, respectivamente. Os tratamentos FL e MOS foram superiores e estatisticamente diferentes ao controle (p<0,05). Os resultados mostram que o uso de FLAVOXIN[R] e UNIWALL MOS 50[R] na dose empregada para este estudo tem um efeito positivo na sobrevivencia de juvenis de R. quelen, sem modificar os parametros de crescimento.

KEYWORDS/Diet/Juveniles/Prebiotics/Rhamdia quelen/

Introduction

In the last decade, growth in worldwide fish consumption and decline in the fishing extraction rate from natural watercourses has stimulated the development of aquatic organism production in captivity, with fish farming as one of the fastest growing productive activities (Luchini and Panne Huidobro, 2008; FAO, 2010).

Rhamdia quelen is a commercially relevant freshwater fish both in Argentina and Brazil, mainly reared under semi-intensive and intensive production systems (Baldisserotto and Radunz Neto, 2004; Luchini and Panne Huidobro, 2008). An increase in its production has been observed in the last couple of years, becoming the most cultivated native species in southern Brazil (Silveira and Muller, 2011).

Intensification of production exposes fish to a great variety of stress factors, such as high-density stock and inadequate water quality, which adversely affect the productive performance (Canario et al., 1998; Barton, 2002). Theretbre, constant stress may result in a decline in growth rate and alimentary efficiency, favoring also the appearance of diseases (Barcellos et al., 2004; Baldwin, 2010), and limiting the economic development of aquaculture (Sado et al., 2008).

In order to avoid such inconveniencies, several studies have focused on the use of food supplements as tools to promote the welfare of farmed organisms. Among the most widely employed alternatives are the addition of beneficial microorganisms or probiotics and/or their metabolites (organic acids, bacteriocins), prebiotics (oligosaccharides), organic acidifiers, yeast extracts and other growth stimulants. All of them were aimed at identifying alternatives favoring growth (Apun-Molina et al., 2009), health (Gatesoupe, 2007) and the capability to resist diseases, replacing chemotherapeutic and antimicrobial agents (Fritts and Waldroup, 2003), which cause numerous adverse effects and participate in the dissemination of crossed resistance in indigenous microbiota and potentially pathogen microorganisms (Balcazar et al., 2006; Salinas et al., 2008).

Although probiotics have been widely studied in aquaculture, their application is limited due to high costs, potential environment impact and food-processing difficulties. In contrast, the use of prebiotics is rising due to the fact that it is more practical and allows intestinal microbiota management in aquatic organisms (Yousefian and Amiri, 2009; Ringo et al., 2010).

Flavonoid prebiotics are natural food additives present in plants. They have antioxidative, anti-inflammatory and anti-carcinogenic effects (Knekt et al., 1997; Nowakowska, 2007; Fariba et al., 2009). Several studies on humans have shown that flavonoids can be used therapeutically for their antioxidative actions by reducing free radicals which induces tissue injuries (Barreea et al., 2009). Moreover, it is well-known that flavonoids reduce or inhibit the growth of many microorganisms and their toxins (Cushnie and Lamb, 2005; Lee et al., 2010).

Non-digestible oligosaccharides are another important group of prebiotics which, administered in animal feed, are metabolized by specific intestinal health-promoter bacteria such as Lactobacillus and Bifidobacterium. Within this group, inulin, fructooligosaccharides (FOS), short-chain fructooligosaccharides (scFOS) and mannan oligosaccharides (MOS) are of special relevance (Yousefian and Amiri, 2009; Ringo et al., 2010). MOS, obtained from the cell wall of Saccharomyces cerevisiae, present a favorable effect on intestinal health due to improvements in absorption and immune response modulation in productive species (Genc et al., 2007; Dimitroglou et al., 2009).

Even though several studies on R. quelen nutritional requirements in different categories (Meyer and Fracalossi, 2004; Piedras et al., 2006; Tronco et al., 2007) or food supplements (MontesGirao and Fracalossi, 2006; Trombetta et al., 2006) have been carried out, there are still few evaluations regarding use of nutritional additives (Souza et al., 2009). Therefore, the aim of this work was to evaluate the use of two commercial additives, FLAVOXIN[R] and UNIWALL MOS 50[R], in contrast to control food, on growth and survival parameters for Rhamdia quelen juveniles.

Materials and Methods

The study was carried out at the Instituto de Ictiologia del Nordeste (INICNE), Facultad de Ciencias Veterinarias -Universidad Nacional del Nordeste, Corrientes, Argentina.

Juveniles from crosses of the own broodstock of INICNE, Argentina, were used. Three food treatments were evaluated: 1) Control: a commercial diet with 280g proteins/kg and 120g lipids/kg; 2) FL: a commercial diet supplemented with 2g x [kg.sup.-1] of FLAVOXIN[R] (Vetanco S.A. Laboratory, Buenos Aires, Argentina); and 3) MOS: a commercial diet supplemented with 2gx [kg.sup.-1] of UNIWALL MOS 50[R] (Vetanco S.A. Laboratory, Buenos Aires, Argentina). Rations were elaborated by grinding the commercial diet and then mixing it with the corresponding additives, adding water (400g x[kg.sup.-1]) to form a soft paste. The paste obtained was passed through a grinder in order to form pellets 4mm in diameter, which were dried with a heater at 40[degrees]C during 48h, and then kept in a refrigerator at -18[degrees]C during the experimental period. The control diet, without additives, was subjected to the same treatment. The trial was conducted in nine 16[m.sup.-3] cement ponds, assigned according to a 3x3 Latin square design. Forty two silver catfish juveniles (mean weight and standard deviation of 0.11 [+ or -]0.015g) were placed in each pond at a density of 2.62 fish/[m.sup.-3]. Fish were fed once a day, at a 5% biomass ratio, during 60 days. The water from the ponds was fertilized with commercial bales of alfalfa (300g/[m.sup.3]) one week before the beginning of the trial.

Water quality parameters such as temperature ([degrees]C), oxygen (mg x [l.sup.-1]), conductivity ([micro]S x [cm.sup.-1]) and pH were weekly monitored, using YSI[R] 55 equipment for temperature and dissolved oxygen and HANNA[R] equipment for conductivity and pH. The obtained mean values and standard deviations for water quality variables were 18.8 [+ or -]3.20[degrees]C; 9 [+ or -]1.8mg x [l.sup.-1]; 114.8 [+ or -]4.9 [micro]S.[cm.sup.-1]; and 7.32 [+ or -]0.42, for temperature, dissolved oxygen, conductivity and pH, respectively. No significant differences were observed (p>0.05) regarding water quality parameters among the different treatments during the experimental period.

[FIGURE 1 OMITTED]

Measured parameters and statistical analysis

At the end of the experiment, final mean weight (MW), absolute growth rate (AGR), specific growth rate (SGR), survival rate (S) and final biomass (FB) were calculated as MW (g)= [W.sub.t]/N, where [W.sub.t]: total weight (g) per specimen and N: number of surviving individuals; AGR= 100(([W.sub.f]-[W.sub.i])/T), where [W.sub.f]: final mean weight (g) and [W.sub.i]: initial mean weight (g) for given period T; SGR= 100((ln[W.sub.f]-ln[W.sub.i])/T), where ln[W.sub.f] and ln[W.sub.i] are the natural logarithms of [W.sub.f] (g) and [W.sub.i] (g) for a given period T; S(%)= 100(N/ [N.sub.i]), where [N.sub.i]: initial number of fish; and FB= MWxN.

The results from the R. quelen feeding trial were yzed through a three-way analysis of variance (ANOVA) without interaction, according to a Latin square design. This design was chosen to eliminate the effect of environmental variables associated to the localization of the ponds (light and wind incidence). Comparisons of treatment means were later carried out through the Tukey test (p<0.05). Data were analyzed using Statistica software for Windows, version 6.0.

Results and Discussion

At the end of the experiment, all the feeding groups presented a similar weight (p>0.05), reaching values of 6.74, 6.37 and 6.72g in the control, FL and MOS groups, respectively (Figure la). No significant differences were found among the groups for the rest of the analyzed growth variables (AGR, SGR and FB; p>0.05; Figures 1bd). The same analysis, carried out on survival values, allowed the detection of significant differences among treatments, with 97% and 95% as estimated averages for the FL and MOS groups, respectively, which did not differ between them (p>0.05) but showed a statistically significant higher than that of the control group (89%; p<0.05; Figure 1e).

Flavonoids, as well as MOS, are commonly used as additives in feed rations for fish and crustaceans (Genc et al., 2007; Yilmaz et al., 2007; Zhou et al., 2010) in order to improve growth, sanitary conditions or pathogen resistance (Ardo et al., 2008; Citarasu, 2010), but results have been inconsistent (Peterson et al., 2010).

In agreement with the results from the present study, Peterson et al. (2010) did not observe improvement regarding weight gain in juvenile channel catfish (Ictalurus punctatus) by adding BioMOS[R] in similar percentages to those used in this study, but did observe a higher survival rate as compared to a control diet. Dimitroglou et al. (2010) evaluated the addition of two concentrations of MOS (2 and 4g x [kg.sup.-1]) for Sparus aurata juveniles, observing that the final mean weight, the specific growth rate, the conversion index and the protein efficiency percentage were not affected by the use of this supplement as compared to a control diet. Moreover, Sado et al. (2008) reported that Nile tilapia juveniles (Oreochromis niloticus) presented a decrease in daily food consumption and growth rate, which was proportional to the addition of MOS to the diet from 2 to 10g x [kg.sup.-1].

Shin et al. (2010) found higher weights in olive flounders, Paralichthys olivaceus, by feeding them with quercetin flavonoids at 5g x [kg.sup.-1], reporting also a hypocholesterolemic, antioxidative effect anda rise in the lysozyme activity as compared to the control group, suggesting that this flavonoid improves the immune response against external stress. Moreover, several researchers (Staykov et al., 2007; Yilmaz et al., 2007; Dimitroglou et al., 2009) obtained improvements in the growth response of rainbow trout (Oncorhynchus mykiss) by feeding them with mannan-MOS in the diet at different levels of supplementation (0.5 to 4.5g x [kg.sup.-1]). In addition, Mazlum et al. (2011) found greater growth in Astacus leptodactylus juveniles fed with MOS (3g x [kg.sup.-1]), while the survival rate was not affected. According to Peterson et al. (2010) there is a significant variation regarding the effects of derivative mannan oligosacharides in diets on growth efficiency, which probably depends upon the species, feeding duration and level of inclusion in the food, as well as type of yeast used.

In the present study, R. quelen juveniles fed with a ration supplemented with FLAVOXIN[R]and UNIWALL MOS 50[R] (2g x [kg.sup.-1]) showed no improvement in any of the growth parameters as compared with a control diet. These results would indicate that the dose employed had no influence over growth in the animal category used. Besides, low-density breeding conditions would not entail the inconveniences habitually observed in intensive or over-intensive farming, where a beneficiai effect of the additives on fish growth could be expected.

Diet and nutritional conditions are fundamental factors for fish farming exploitation due to the fact that they determine the physiological conditions of the animals, with a direct influence on energetic conditions, and an indirect one through influence on stress tolerance (Blazer, 1992). Thus, an adequate diet may favor growth, alimentary efficacy, immunologic condition and physiology of the fish, through immune system improvement and enhancement of use of nutrients (Staykov et al., 2007).

Conclusion

The results from this work show that the use of FLAVOXIN[R] and UNIWALL MOS 50[R] at a 2g x [kg.sup.-1] fatio does not modify growth parameters, while it determines improvements in survival rate. These results need to be studied in depth in order to further adjust the use of these additives in diets for R. quelen juveniles.

Received: 11/31/2011. Modified: 08/03/2012. Accepted: 08/07/2012.

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David R. Hernandez. Ph.D., Universidad Nacional dei Nordeste (UNNE), Argentina. TeacherResearcher, UNNE. Address: Instituto de Ictiologia del Nordeste (INICNE), Sargento Cabral 2139, Corrientes, Argentina. e-mail: dhernandez@vet.unne.edu.ar

Juan J. Santinon. Zoologist, UNNE, Argentina. Doctoral student in Biological Sciences, Universidad Nacional de Cordoba (UNC), Cordoba, Argentina. Teacher-Researcher, UNNE, Argentina, e-mail: jsantinon@conicet.gov.ar

Sebastian Sanchez. Geneticist, Universidad Nacional de Misiones (UNAM), Argentina. Ph.D. in Biological Sciences, UNC, Argentina. Specialist in University Teaching, UNNE, Argentina. Teacher-Researcher, INICNE-UNNE, Argentina. e-mail: sanchez@vet.unne.edu.ar

Hugo A. Domitrovic. Ph.D., Universidad de Buenos Aires, Argentina. Teacher-Researcher, UNNE, Argentina, and Director, INICNE-UNNE., Argentina. e-mail: hdomitro@vet.unne.edu.ar
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Title Annotation:REPORTS/COMUNICACIONES/COMUNICACOES
Author:Hernandez, David R.; Santinon, Juan J.; Sanchez, Sebastian; Domitrovic, Hugo A.
Publication:Interciencia
Date:Aug 1, 2012
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