PERFORMANCE OF Centropomus parallelus FINGERLINGS FED A DIET SUPPLEMENTED WITH CITRATE AND ACETATE

This study aimed to evaluate the addition of sodium acetate and sodium citrate in the diets of fingerling fat snook (Centropomus parallelus), through growth performance and modification of the intestinal microbiota. In the experiment, 3,000 fat snook fingerlings were used, with an initial average weight of 0.57 ± 0.10 g. The fish were stocked into 12 tanks (50 L) at a density of 5 fingerlings L-1 per tank distributed in three treatments with four replicates. The treatments were as follows: control group (without supplementation), one group supplemented with sodium acetate, and another group supplemented with sodium citrate. The diets supplemented with organic salts were sprayed with cod liver oil previously homogenized with 3% sodium acetate or sodium citrate. The fish supplemented with sodium acetate showed higher final weight, length, and yield, compared with the control group fish. The intestine of fingerling fat snook showed lower counts of total heterotrophic marine bacteria in the fish supplemented with acetate and citrate in relation to fish of the control group. Lower counts of sucrose non-fermenting vibrios were also observed in fish supplemented with acetate. Through this study, it was possible to evaluate for the first time the dietary inclusion of organic salts in the diets of marine fish in Brazil, with the dietary inclusion of 3% sodium acetate showing benefits for performance of fingerling fat snook.


INTRODUCTION
In recent decades, extractive fishing has severely reduced the fish stocks of marine ecosystems.An alternative to reduce fishing activity toward preserving the natural stocks of fish is fish farming (WARD and MYERS, 2005).
The snook genus Centropomus, with its present high commercial value, stands out for its refined quality of meat and resistance to adversity in culture (CERQUEIRA, 2002).The species Centropomus parallelus, popularly known as fat snook, consists of diadromous and euryhaline marine fishes that can be found in coastal areas, bays, estuaries and brackish lagoons, freshwater environments, and occasionally in hypersaline lagoons (CERVIGÓN et al., 1992).They are carnivorous fish in the natural environment, feeding mainly on fish and crustaceans (CERQUEIRA, 2004).
As with other marine fish, the most critical step in fat snook production is the hatchery, because high mortality makes it difficult to obtain juveniles, and a major factor of mortality is bacterial disease (SOUZA et al., 2010).
Antibiotics in aquaculture have traditionally been used as a prophylactic treatment.However, extensive use of antibiotics causes the selection of resistant bacterial strains and environmental pollution.Thus, there is a need to search for alternative feed additives that aid in the nutrition and health of cultivated aquatic animals (DEFOIRDT et al., 2011).In this context, research with organic acids and their salts has been highlighted up (LÜCKSTÄDT, 2008;NG and KOH, 2011).
Organic acids and their salts affect the growth performance of aquatic animals through distinct mechanisms.In the diet, they act as preserving agents, inhibiting microbial growth and diminishing a possible intake of pathogenic organisms (LÜCKSTÄDTS, 2007).In the intestinal tract, they inhibit the growth of pathogenic bacteria; they may modify intestinal microbiota (DEFOIRDT et al., 2009;SCHRYVER et al., 2010;MINE and BOOPATHY, 2011) and thereby increase survival (DEFOIRDT et al., 2006;DEFOIRDT et al., 2007;LIU et al., 2010;NHAN et al., 2010).In animal metabolism, organic acids can also affect the action of digestive enzymes; organic acids can serve as a source of energy through the citric acid cycle or the carboxylic acid cycle (LÜCKSTÄDT, 2008).In addition, organic acids might alter the availability and absorption of certain nutrients, such as phosphorus, lipids, and amino acids (BARUAH et al., 2005(BARUAH et al., , 2007;;MORKEN et al., 2011;KHAJEPOUR and HOSSEINI, 2012;SARKER et al., 2012aSARKER et al., , 2012b)).
The current study aimed to evaluate the dietary inclusion of sodium acetate and sodium citrate for fat snook fingerlings (C.parallelus), through their effects on growth performance and intestinal bacteria counts.

MATERIALS AND METHODS
This work was performed at the Laboratório de Piscicultura Marinha (LAPMAR), Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.The fat snook fingerlings were obtained through natural spawning by hormonal induction (CERQUEIRA, 2010), cultured for 69 days after hatching (DAH), to temperature and salinity averages of 23ºC and 35‰, respectively.
In the experiment, 3,000 fat snook fingerlings were used, with an initial average weight of 0.57 ± 0.10 g and an average length of 3.65 ± 0.19 cm.After the initial biometry, the fingerlings were randomly distributed into 12 tanks of 50 L useful volume at a density of 5 fingerlings•L-1 with three treatments containing four replicates each.The fish were acclimated for one week.The water of the tanks was maintained under controlled temperature by thermostats and heaters (100 W), Bol.Inst.Pesca, São Paulo, 42(1): 65-72, 2016 constant aeration, and daily renewal of 65% total volume of each tank.The photoperiod during the experiment was 9 h light.Temperature, pH and dissolved oxygen, and weekly total ammonia were measured daily using methods described by APHA (1995).
The treatments were as follows: control group (without supplementation), one group supplemented with sodium acetate, and another group supplemented with sodium citrate.The diet used in the experiment was a commercial diet containing 57% crude protein (INVE Aquaculture, U.S.A NRD 0.8/1.2 and NRD 1.2/2.0)(Table 1).The diets supplemented with organic salts were sprayed with cod liver oil (10 mL per kg of feed) previously homogenized with 30 g sodium acetate or sodium citrate.The final concentration of organic salts in the diets was 3%.The control diet also received the cod live oil without the addition of organic salts.The growth performance experiment lasted 30 days.At the end of the trial performance parameters were calculated using the following formulas: number of final fish number of stocked fish Final weight (g) = final biomass(g) number of fish Feed conversion ratio = amount of feed(g) final biomass (g) At the end of the experiment 10 fingerlings were randomly collected from each tank for microbiological analysis of gut.Fingerlings were washed with 70% alcohol to remove bacteria attached to the external body surface without affecting the internal bacteria.The fish guts were then excised with the use of forceps and scalpel, and were homogenized in sterile saline solution in 3% of NaCl, in a grail.The samples were then serially diluted (1/10), seeded in Agar Marine culture medium (for total heterotrophic marine bacteria) and Agar Thiosulfate, Citrate, Bile and Sucrose (TCBS; selective medium for vibrios).After an incubation of 24 h at 26ºC, total counts of colony forming units (CFU) were performed.
The bacteriological count data were transformed into log10 (x+1).Data homoscedasticity was assessed by the test of Bartlett.The data were then subjected to unifactorial variance analysis.Where necessary, performance of medium separation was carried out by the test of Tukey.All the statistical tests were evaluated with a significance level of 5% (ZAR, 2010).

RESULTS
During the experimental period, the water of all tanks showed average values of pH 7.6 ± 0.1, dissolved oxygen 4.1 ± 0.6 mg L-1, temperature 25.85 ± 0.35 ºC, and total ammonia 0.07 ± 0.01 mg L-1.These data did not differ among the different treatments.
Bol. Inst.Pesca, São Paulo, 42(1): [65][66][67][68][69][70][71][72]2016 Table 2 shows the observed results of zootechnical parameters.Fish supplemented with sodium acetate showed higher final weight, length, and yield, compared with the control group fish.The fish supplemented with sodium citrate did not differ from the fish under other treatments.Survival, feed conversion ratio, and condition factor showed no significant differences between the fish of different groups.The intestine of fat snook fingerling showed lower counts of total heterotrophic marine bacteria (Marine Agar) in fish supplemented with acetate and citrate in relation to the fish of the control group.A lower count of green colonies on TCBS agar, sucrose non-fermenting bacteria, was also observed in fish supplemented with sodium acetate compared with the fish supplemented with sodium citrate or the fish without supplementation (Table 3).

DISCUSSION
Following CERQUEIRA (2010), the water quality remained adequate throughout the experiment.All parameters were within those acceptable for fat snook cultivation.
Changes in the gut microbiota community, aimed at reducing the concentration of potentially pathogenic bacteria, constitute a major benefit from the addition of feed additive growth promoters used in aquaculture.Like in this study, dietary supplementation of a probiotic (Lactobacillus plantarum) decreased the concentration of total heterotrophic bacteria and Vibrio spp. in the fat snook gut (BARBOSA et al., 2011).Studies with lactic acid bacteria have demonstrated their potential inhibition, especially against Gram-negative bacteria, due to the production of organic acids such as acetic acid and lactic acid (VAZQUEZ et al., 2005).
Other studies on supplementation with organic acids or their salts in fish observed changes in intestinal microbiota.European sea bass (D. labrax) supplemented with 2% to 5% polyhydroxy-butyrate (PHB) showed changes in the gut bacterial community compared with nonsupplemented fish, with the fish receiving supplementation obtaining better weight gain and feed conversion ratio (SCHRYVER et al., 2010).Hybrid tilapia (Oreochromis sp.) fed a diet supplemented with 0.1% to 0.3% of a mixture of organic acids (not identified by the authors) showed a lower total count of bacteria in feces and intestinal tract (NG et al., 2009).Macrobrachium rosenbergii larvae fed brine shrimp enriched with PHB decreased total bacteria and Vibrios, besides presenting better development (NHAN et al., 2010).Juvenile Litopenaeus vannamei supplemented with 0.4% to 2% of a commercial inorganic acid, based on calcium sulfate, also showed changes in the intestinal microbiota (ANUTA et al., 2010).

CONCLUSION
Through the results of this study, it was possible to evaluate for the first time dietary supplementation of organic salts for Brazilian native marine fish, where the inclusion of 3% sodium acetate to fat snook fingerlings (Centropomus parallelus) increased the final weight, final length, and yield, and decreased the total heterotrophic bacteria and sucrose nonfermenting vibrionaceas in the gut.

Table 1 -
Proximal composition of the commercial diet (INVE) used in the experiment.
Different letters represent statistically significant differences(p <0.05) between treatments on the Tukey test.
YC -yellow colonies on TCBS Agar, sucrose-fermenting; GC -green colonies on TCBS Agar, sucrose non-fermenting.Different letters represent statistically significant differences(p <0.05) between treatments on the Tukey test.