Aquaculture Research, 2009, 40, 157^165 doi:10.1111/j.1365-2109.2008.02077.x

s Benefits of using the probiotic Efinol Lduring transportation of cardinal , axelrodi (Schultz), in the Amazon

Levy Carvalho Gomes1, Richard Philip Brinn2, Jaydione Luiz Marcon3, Lucelle Arau¤jo Dantas1, Franmir Rodrigues Brandao1,JanessaSampaiodeAbreu4, Paulo Evandro Mendes Lemos3, Dawn Michelle McComb5 & Bernardo Baldisserotto6 1Embrapa AmazoŒ nia Ocidental, Manaus, AM, 2Florida International University, Miami, FL, USA 3Universidade Federal do Amazonas, Manaus, AM, Brazil 4Universidade Estadual Paulista, Jaboticabal, SP,Brazil 5Florida Atlantic University,Boca Raton, FL, USA 6Universidade Federal de Santa Maria, Santa Maria, RS, Brazil

Correspondence: Present address: L C Gomes, Centro UniversitaŁ rio deVilaVelha, Programa de Mestrado em Ecologia de Ecossistemas, Rua ComissaŁ rio Jose¤ Dantas de Melo 21, BoaVista,VilaVelha, ES 29102-770, Brazil. E-mail: [email protected]

Abstract Keywords: probiotic, survival, Amazon, orna- mental ¢sh, ion £ux, body cortisol The objective of this experiment was to test the pro- s biotic E¢nol L during transportation of , Paracheirodon axelrodi (Schultz). For the trans- Introduction portation, ¢sh were distributed in 18 plastic tanks, of s which nine contained the E¢nol L (10mg L À 1;pro- The cardinal tetra, Paracheirodon axelrodi (Schultz), is biotic treatment) and the remaining had no probiotic an endemic ¢sh from the middle (control treatment). Transport lasted 24 h and three Basin widely used as an ornamental ¢sh in aqua- di¡erent boxes of each treatment were sampled at 3, riums worldwide (Walker 2004). The cardinal tetra 12 and 24 h. Up to the 12-h sampling period, no sig- is the most important commercial species of orna- ni¢cant di¡erence in the survival was observed; on mental ¢sh exported from the Brazilian Amazon, the other hand, survival was higher at the end of the contributing to about 80% of exports. Some 12^15 transport (24 h) in the probiotic treatment. No signif- million wild specimens are legally exported annually icant di¡erence was found in dissolved oxygen and and accounted by the Brazilian government (Chao temperature between treatments. Conductivity, pH 2001). However, Chao and Prada-Pedreros (1995) esti- and alkalinity increased along the transport, but mate that at least 24^32 million cardinals are taken without a di¡erence between treatments. Ammonia from the Rio Negro tributaries each year. The orna- increased in all treatments, although it was signi¢- mental industry generates 60% of the total income cantly lower in the probiotic group at 24 h. Cortisol le- of the middle Rio Negro region’s economy (Chao & vels were signi¢cantly higher in all transported ¢sh Prang 1997), with between 6000 and 8000 ¢sher- when compared with the basal values. At 24 h, cortisol men involved in this activity (Chao & Prada-Pedreros levels in control ¢sh were signi¢cantly higher than 1995; Pra ng 2001). those in the probiotic treatment.With the observed re- Transportation is an important part of all phases sults, we are able to conclude that the probiotic of the export process. Cardinals are normally caught s E¢nol L is e⁄cient during cardinal transport, lowering in small streams (igarape¤ ). After this, ¢sh are typi- the mortality and helping maintain water quality pos- cally transported in 40-L À 1 plastic boxes to Barcelos, sibly by lowering metabolic wastes. a base city for the ornamental industry in the middle

r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd 157 s Cardinal transportation with E¢nol L LCGomeset al. Aquaculture Research, 2009, 40, 157 ^ 165

Rio Negro region, then onto exporters’ facilities in tion in metabolic wastes and reduction in stress re- Manaus and ¢nally to importing countries, mainly sponses. Therefore, the objective of this experiment s Europe, Asia and North America (Santos & Santos was to test the e⁄ciency of E¢nol L solution during 2005). According to Waichman, Silva and Marcon the transport of the cardinal tetra between Barcelos (2001), mortality during the transportation between and Manaus. Barcelos and Manaus is a negative factor in the ex- porting process, which can be attributed to the pro- blem of bad management practices and poor water Material and methods quality leading to disease outbreaks, mostly bacteria based. This work was carried out in December 2005 during a Most recently,probiotics have appeared in the mar- scienti¢c expedition to the middle Rio Negro region, ket as a method to increase the quality and health of close to Barcelos, AM, Brazil, aboard the Miss Bebel several ¢sh species during some processes involved in vessel, with the main objective of studying the biology, management (Gatesoupe 1999; Gomez-Gil, Roque & ¢shing and management of several ornamental ¢sh Turnbull 2000). These products are primarily com- captured and exported from this region. posed of highly concentrated bacteria, vitamins and Cardinals (0.032 Æ 0.011g; mean Æ SD) were cap- nutrients (Gatesoupe 1999; Verschuere, Rombaut, tured by professional ¢shermen in a small stream Sorgeloos & Verstraete 2000) and were created to sti- called igarape¤ Puxurituba. After capture, the ¢sher- mulate the immune system and help increase the men used a standard procedure for keeping the ¢sh water quality in the environment.The use of probiotic before transport to Barcelos. were kept in plastic helps increase ¢sh health during stressful processes boxes containing 20 L of water before the actual such as severe changes in environmental conditions transport. Between the capture and the experimen- (Taoka, Maeda, Jo, Jeon, Bai, Lee, Yuge & Koshio tal period, ¢sh were maintained for a maximum of 2006). Carnevali, De Vivo, Sulpizio, Gioacchini, 10 days in boxes and fed with a commercial feed Olivotto, Silvi and Cresci (2006) observed an inverse for omnivorous ¢sh (32% crude protein and correlation between gut colonization with lactic acid 4180 kcal CE kg À 1 of crude energy) twice a day. Fish bacteria strain and cortisol levels; thus, Rollo, Sulpi- were starved 2 days before the transport. zio, Nardi, Silvi, Orpianesi, Caggiano, Cresci and Car- For the transport experiment,18 plastic boxes were nevali (2006) suggest that a probiotic is able to utilized with a total capacity of 40 L but ¢lled with modulate cortisol responses in ¢sh, lowering the 10L of local stream water. Fish were randomly dis- magnitude of stress response. tributed between the boxes following the procedure Most probiotics are designed to be mixed with food, used by local ¢shermen, with a ¢nal density of but recently some probiotic products have been for- 578 Æ 26 cardinals per box or 1.84 Æ 0.19 g of ¢sh mulated that can be dissolved in water and can per litre of water. The density used was in the range help during management and transport operations. (500^1000 per box) commercially used by local ¢sh- s E¢nol L is a water-soluble commercial product sold ermen. In half of the boxes, a solution of 10mg L À 1 s as a proven anti-stress formula for aquaculture probiotic E¢nol L (Bentoli Agrinutrition, Austin, TX, hatcheries, useful for larval transport and to improve USA) was utilized, while the rest were maintained the health and survival of aquatic larvae.This with probiotic-free water (control group). The probio- product is formulated with Bacillus subtilis, Bacillus tic concentration is in accordance with the manufac- licheniformes, Lactobacillus acidophilus and Saccharo- turer’s instructions. A 24-h transport experiment myces cerevisiae, along with selected amino acids, proceeded with the vessel simulating conditions that vitamins, minerals, free-£ow and anti-caking agents local ¢shermen use to take their ¢sh through Barcelos (calcium carbonate and silica). This combination to Manaus. Survival, water quality, net ion £ux and characterizes the product as a probiotic enriched body cortisol were monitored from three di¡erent with some nutrients. Previous results obtained by boxes of each treatment at 3,12 and 24 h of transport. Gomes, Brinn, Marcon, Dantas, Brandao, Abreu, Each box was sampled only once and then discarded McComb and Baldisserotto (2008) during transpor- from the experiment. tation of another Amazonian ornamental ¢sh, the For whole-body cortisol analysis, samples of un- marbled hatchet¢sh, Carnegiella strigata (Gˇnther), stressed ¢sh were caught directly from the local s with E¢nol L demonstrated that this probiotic was streams to be compared with the transported ¢sh. e⁄cient in improving the water quality by the reduc- They were caught and anaesthetized with a lethal

r 2008 The Authors 158 Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 40, 157^ 165 s Aquaculture Research, 2009, 40, 157^165 Cardinal transportation with E¢nol L LCGomeset al. concentration of benzocaine (200 mg L À 1) and im- ment groups presented homogeneous variances and mediately frozen and stored in liquid nitrogen. survival was compared using a t-test, while water During transport, ¢sh samples were taken from each quality and net ion £uxes were compared using a box at each sampling time and frozen in liquid two-way ANOVA (time and treatment as variables) and nitrogen. the Tukey test. Di¡erences between treatments and Water analysis of temperature, dissolved oxygen body cortisol at di¡erent samplings were compared (YSI 55), pH (YSI pH-100) and conductivity with a di- with unstressed ¢sh (basal values) using one-way gital meter (Bernauer, Blumenau, Brazil) was per- ANOVA and Dunnett’stest (Po0.05) and between treat- formed directly in the box.Water samples were taken ments at the same sample time using a t-test. All sta- from the box for further analysis of alkalinity by titra- tistical tests were conducted using software STATISTICA tion (American Public Health Association, American (version 5.1). Data were expressed as means Æ SEM, WaterWorks Association,Water Environment Federa- and the minimum signi¢cance level was set at Po0.05. tion 1992), total ammonia by the salicylate method (Verdouw, Vanechted & Dekkers 1978) and net ion £uxes of Na1,K1 and Cl À. Water Na1 and K1 levels were measured directly Results in a B462 £ame photometer (Micronal, Brazil) and Cl À levels using the colorimetric assay described by After12h of transport, less than1% of ¢sh died in

Zall, Fisher and Garner (1956). Net ion £uxes (Jnet) both the treatments; on the other hand, signi¢cant were calculated based on changes in the ion concen- mortality between treatments occurred in the 24-h tration of transport water over the sampling periods sampling period, with the probiotic treatment having according to the equation by Gonzalez, Wood, the higher survival (99.71 Æ 0.29%) when compared Wilson, Patrick, Bergman, Narahara and Val (1998): with control (94.62 Æ 1.44%) (Table1). À 1 Jnet 5V([ion]1 À [ion]2) Â (Mt) , where [ion]1 and No signi¢cant di¡erences were observed in dis-

[ion]2 are the bath ion concentrations at the begin- solved oxygen levels and temperature (Fig.1)between ning and the end of the £ux period, respectively,V is treatments, although levels of oxygen decreased to the bath volume in litres, M is the mass of the ¢sh in levels close to or below 2 mg L À 1 after 3 h of trans- kg and t is the duration of the £ux period in hours. port, maintaining these levels throughout the experi- Initial waterborne Na1 levels in the ¢sh boxes were ment. Temperature varied between 28.5 and 30.2 1C. (mmol L À 1, mean Æ SEM) 16.5 Æ 0.4 and 43.4 Æ 2.1, Conductivity, pH and alkalinity increased signi¢- Cl À 32.0 Æ 6.0 and 84.6 Æ 12.4 and K 1 2.6 Æ 0.3 cantly during the transport process (Fig. 1). At the and 18.2 Æ 1.7 for control and probiotic groups end of the transport, conductivity was 37.87 Æ 0.76 respectively. and 38.43 Æ 4.14 mscmÀ 1 in the probiotic and con- Whole-body cortisol extractions were performed trol treatment respectively (Fig. 1). The pH did not according to de Jesus, Hirano and Inui (1991) with a change signi¢cantly between treatments; however, it few modi¢cations. Brie£y,pools of10 ¢sh were homo- increased signi¢cantly along the transport periods. genized with phosphate-bu¡ered saline with gelatin The initial pH was 4.9^5.0 and reached 6.50^6.58 (PBS-G) and a 0.5 g sample was taken for extraction. at the end (Fig. 1). Alkalinity was signi¢cantly The samples underwent a triple extraction using higher in the probiotic treatment after 24 h diethyl ether, followed by evaporation in a water bath and further resuspension of the pellet with PBS-G before assay. Radioimmunoassay was performed Table 1 Survival of cardinal tetra (Paracheirodon axelrodi) s s using a commercial kit (DPC ) for cortisol in hu- during transportation with (probiotic E¢nol Ltreatment) mans. A thorough validation through parallel curves and without addition of probiotic to the water (control and extraction e⁄ciency process was performed for treatment) this species. The limit of detection was 0.2 mgdLÀ 1 Time of transport (h) Control (%) Probiotic (%) and intra-assay and inter-assay coe⁄cients of varia- tion were below 8%. The average extraction e⁄cien- 3 99.82 Æ 0.18 99.77 Æ 0.14 cies varied from 49% to 57%, and the values reported 12 99.40 Æ 0.35 99.94 Æ 0.06 Ã in this article were not corrected for e⁄ciency. 24 94.62 Æ 0.83 99.71 Æ 0.16 Homogeneity of variances among the di¡erent N 5 3 boxes per treatment at each sample time. Ã groups was tested using the Levene test. Data of treat- Indicates signi¢cant di¡erence from control by t-test (Po0.05).

r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 40, 157^ 165 159 s Cardinal transportation with E¢nol L LCGomeset al. Aquaculture Research, 2009, 40, 157 ^ 165

7 33 Probiotic Probiotic 6 Control Control 32 5 31 4 a a a 30 a 3 a a 29 2 a

1 a 28 a a a a

0 27 0 3 6 9 12 15 18 21 24 0639121518 21 24

50 7.5 45 c 7.0 40 c b * 35 6.5 b b c 30 6.0 a 25 b 5.5

20 pH (units) a a 15 5.0 a 10 a 4.5 5 0 4.0 0 3 6 9 12 15 18 21 24 0639121518 21 24

50 2.0 45 c * b 40 1.5 35

30 b c * 25 1.0 a 20 b b 15 ab 0.5 10 a 5 a b a 0 3 6 9 12 15 18 21 24 0639121518 21 24 Time of transport (h) Time of transport (h)

s Figure 1 Water parameters during transportation of cardinal tetra (Paracheirodon axelrodi) with (Probiotic E¢nol Ltreat- ment) and without addition of a probiotic to the water (control treatment). N 5 3 boxes per treatment at each sample time. Ã Asterisk ( ) indicates signi¢cant di¡erences from control by two-way ANOVA and theTukey test (Po0.05). Di¡erent letters indicate signi¢cant di¡erences among times of transport within the same group by two-way ANOVA and the Tukey test (Po0.05).

(32.27 Æ 9.16mg L À 1) while it reached 22.00 Æ Control cardinal tetras presented a net Na1 in£ux 4.40 mg L À 1 in the control group (Fig. 1). Total and net Cl À and K1 e¥uxes after 3 h of transport. ammonia increased with transport time, reaching After 12 h of transport, net Na1 in£ux reverted to a signi¢cantly higher levels in the control group at the net e¥ux, while net Cl À e¥ux reverted to a net in- 24-h sampling. At 24 h, ammonia concentrations £ux (but much lower than the previous net e¥ux). were 1.56 Æ 0.17mg L À 1 in the control group and These £uxes remained constant up to 24 h (Fig. 2a 1.32 Æ 0.09 mg L À 1 in the probiotic group (Fig.1). and b). Net K1 £ux did not change throughout the

r 2008 The Authors 160 Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 40, 157^ 165 s Aquaculture Research, 2009, 40, 157^165 Cardinal transportation with E¢nol L LCGomeset al.

Figure 3 Body cortisol concentrations of cardinal tetra (Paracheirodon axelrodi) during transportation with (Pro- s biotic E¢nol L treatment) and without addition of a pro- biotic to the water (control treatment). N 5 3 boxes per treatment at each sample time. Asterisk (Ã) indicates a sig- ni¢cant di¡erence from the basal values by one-way ANOVA ÃÃ and Dunnett’s test (Po0.05). Double asterisk ( )indi- cates signi¢cant di¡erences between treatments at each sampling time using a t-test (P 5 0.049).

than those of control ¢sh, while Cl À presented a very high net e¥ux. All ion £uxes tended to zero and were not signi¢cantly di¡erent from ion £uxes in control cardinal tetras after 24 h (Fig. 2). Whole-bodycortisol levels were signi¢cantly lower in the baseline group when compared with all sam- ples from the transport experiment. The baseline va- lue was 13.75 Æ 0.47 ng g À 1 (Fig. 3). All transported ¢sh samples had cortisol levels at least fourfold high- er than the baseline levels, some reaching an eight- to nine-fold di¡erence. Body cortisol was signi¢cantly greater in control ¢sh than in ¢sh from the probiotic treatment at 24 h of transport (Fig.3). Figure 2 Net Na1 (a), Cl À (b) and K1 (c) £uxes of cardi- nal tetra (Paracheirodon axelrodi) during transportation s with (Probiotic E¢nol L treatment) and without addition of a probiotic to the water (control treatment). Positive Discussion values indicate net in£uxes and negative values indicate Wild-caught ¢sh represent only 10% of the fresh- net e¥uxes. N 5 3 boxes per treatment at each sample water ornamental ¢sh commerce in the world (Tlusty time. Asterisk (Ã) indicates signi¢cant di¡erences from 2002), and the is one of the main control by two-way ANOVA and the Tukey test (Po0.05). Di¡erent letters indicate signi¢cant di¡erences among sources (Gerstner, Ortega, Sanchez & Graham 2006). times of transport within the same group by two-way Cardinal tetra is one of the most exploited freshwater ANOVA and theTukey test (Po0.05). ornamental ¢sh species in the world and the wild stocks are declining (Bayley & Petrere Jr 1989). The s use of E¢nol L improved ¢sh survival during trans- transport (Fig. 2c). Cardinal tetras exposed to a pro- portation, which may represent an additional survi- biotic showed a very high net in£ux of all measured val of 1.2^1.6 million ¢sh per year. This result is ions after 3 h of transport. These net ion in£uxes re- important from a commercial and environmental duced signi¢cantly after 12 h of transport, but net point of view, because it could potentially improve Na1 and K1 in£uxes were still signi¢cantly higher the gain of ¢shermen and reduce ¢sh exploitation,

r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 40, 157^ 165 161 s Cardinal transportation with E¢nol L LCGomeset al. Aquaculture Research, 2009, 40, 157 ^ 165 which will help to maintain the wild stock. One addi- 1997; Gonzalez & Preest 1999). The same was ob- s tional advantage of the use of E¢nol L is the low cost; served in the Na1 transport system of cardinal tetras, a dosage used in one plastic box ¢lled with 10L of which is specialized for high rates of Na1 uptake even water (100 mg) costs less than two cents in US dollars. in the dilute waters of Rio Negro (Gonzalez & Wilson The explanation for the negligible mortality of pro- 2001). At the beginning of the transport, cardinal tet- biotic-treated ¢sh at 24 h is probably related to corti- ra presented a net in£ux (gain) of Na1 and a very sol. Basal cortisol levels are at least eightfold smaller high net e¥ux (loss) of Cl À. Both £uxes tended to than any other value during the entire experimental near-zero values after 24 h, i.e., equilibrium between period, demonstrating a high stress response in all in£ux and e¥ux. Cardinal tetras exposed to probiotic treatments and times of transport, with greater va- showed avery high net in£ux of ions at the beginning lues in control ¢sh at 24 h. High levels of cortisol lead of the transport.This was expected because the addi- to immunosuppression and consequent exposure to tion of probiotic to the water increased Na1 and Cl À pathogens could lead to mortality [reviewed by Rollo levels by 2.6-fold and K1 levels by ninefold, and ac- s et al.(2006)].E¢nolL might have worked in two cording to Gonzalez, Wilson, Wood, Patrick and Val ways: by diminishing the stress stimulus or a¡ecting (2002), at least for Na1, the increase in waterborne the physiology of stress response by modifying clear- Na1 levels led to a higher increase in the Na1 in£ux ance in cortisol in the body or by decreased produc- compared with e¥ux in cardinal tetras, which would tion from the interrenal gland. result in a higher net Na1 in£ux. In spite of this, net The two principal mechanisms of action of the pro- ion £uxes in cardinal tetras maintained in water with biotics described in ¢sh are (1) as an antagonist to a probiotic also tended to equilibrium after 24 h, de- pathogens (Verschuere et al. 2000) and (2) through monstrating a fast adjustment to the higher water- stimulation of the immune system (Taoka et al. borne ion levels. 2006). Water-diluted probiotics have been demon- Before transport, dissolved oxygen levels were 5.7^ strated to improve the survival of halibut Hippoglos- 6.0 mg L À 1, values similar to those measured in the sus hippoglossus (Linnaeus), turbot, Scophthalmus igarape¤ where ¢sh were collected during our expedi- maximus Linnaeus, and Atlantic salmon, Salmo salar tion. However, Geisler and Annibal (1984) reported Linnaeus, when exposed to pathogenic bacteria and oxygen concentrations between 1 and 5.7 mg L À 1 in channel cat¢sh, Ictalurus punctatus (Ra¢nesque), some igarape¤ s where cardinal tetras occur. After 3 h during the grow-out period in ponds [reviewed by of transportation, oxygen levels declined to 2 mg L À 1 Verschuere et al. (2000)]. and these levels were maintained throughout till the According to Gatesoupe (1999), several commer- end of transportation. These oxygen concentrations cial products have sought to exploit the same idea can cause mortalities in some ¢sh species during that bacteria, which improve water quality, may be transportation (Berka 1986), but not for cardinals, as bene¢cial to animal health. These products are re- these values are similar to those observed in the wild. ferred to as ‘probiotics’ and most of them contain To support this, the low oxygen concentrations were nitrifying bacteria and/or Bacillus spp. These two similar in both treatments, and ¢sh from probiotic kinds of bacteria are quite di¡erent. The nitrifying treatment presented negligible mortality. The main- bacteria have strict ecological niches, their actions tenance of dissolved oxygen levels at a constant level interfere directly with the water quality and they through 3^24 h of transport could be explained by have not been detected in the gastrointestinal tract the huge water surface in the ¢sh boxes, which al- of .The strains of Bacillus spp. used as probio- lows enough gaseous exchange to maintain these tics may be active in the intestine during transit, with oxygen levels. an indirect action on the water quality (Gournier- The temperatures measured during transportation Chateau, Larpent, Castellanos & Larpent1994). are in accordance with the ones reported by Froese The water used for ¢sh transportation was charac- (1998) during transportation of tropical ¢sh, and prob- terized as black water, and some studies demonstrate ably did not cause any adverse e¡ect. The increase in that the black waters from Rio Negro Basin are pH, alkalinity and conductivity during transportation among those with the lowest mineral levels in the is mainly associated with ammonia excretion and ion world (Mortatti & Probst 2003). In several characid £uxes, which contribute to the growth of salt availabil- species that live in this environment, ion regulation ity in the water.The increase in pH levels in the present is characterized by high-a⁄nity and high-capacity work is in contrast with those reported when ¢sh ion transport systems (Gonzalez, Dalton & Patrick are transported in closed systems (plastic bags into

r 2008 The Authors 162 Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 40, 157^ 165 s Aquaculture Research, 2009, 40, 157^165 Cardinal transportation with E¢nol L LCGomeset al. styrofoam boxes), in which a progressive decrease in during transportation of marbled hatchet¢sh and by pH occurs due to CO2 accumulation in the water Rollo et al. (2006) during pH-induced stress in seam (Lim, Dhert & Sorgeloos 2003). Because the plastic bream, Sparus auratus Linnaeus. Cortisol levels in boxes that transport cardinal tetras are in direct ¢sh transported with probiotic were stable through- contact with air, the CO2 produced and excreted out the transport procedure, whereas in control ¢sh, by ¢sh should not contribute to the observed changes cortisol concentration increased at 24 h, which was in water pH during transportation. In addition, while in agreement with the hypothesis that high cortisol in closed systems, declines in pH and accumulated levels were the main reason for the ¢sh mortality in

CO2 and ammonia may be critical for ¢sh health and the control treatment. s survival, in the transport procedure used for Amazo- The results show that E¢nol L is e⁄cient in redu- nian ¢sh, including cardinal tetra, wasted ammonia cing ¢sh mortality,stress response and improving the accumulation might deserve more attention during water quality during transportation and that it should water quality monitoring, whether due to its own be used during transportation of ¢sh species. How- toxic e¡ect to the ¢sh or due to the resulting changes ever, it is also recommended that the amounts of ions in water pH. in the probiotic be reduced for use with ¢sh from ion- The probiotic treatment presented a lower ammo- poor waters, in an e¡ort to decrease osmoregulatory nia concentration than the control treatment. Gomes disturbances. et al. (2008) also observed a lower ammonia concen- tration in the water of marbled hatchet¢sh trans- s ported with E¢nol L. During stressful situations, the demand for energy increases and cortisol helps Acknowledgments maintain energy levels by utilizing sources such as This work was supported by a grant from National glycogen and eventually breakdown of protein that Geographic Conservation Fund (no. C47-04) to would generate ammonia. Therefore, the main rea- Christopher Brown. The scienti¢c expedition as well son for the lower ammonia excretion in the probiotic as the collection of wild cardinal tetras for cortisol treatment at 24 h is the reduction in metabolic analysis were previously authorized by the Brazilian wastes. On the other hand, the higher ammonia con- Ministry of Science and Technology (MCT/CNPq, centration in the control group at 24 h is mainly in- process no. EXC 023/05 to J.L.M.) and by the Federal duced by the increase in cortisol. The concentrations Environmental Agency (process no. 098/2005 DI- of ammonia in both treatments at the end of trans- FAP/IBAMA-DF). The authors would like to thank portation are lower than the pattern observed in the Bentoli Agrinutrition Incorporation for supplying s majority of ¢sh transportation procedures described E¢nol L. B. Baldisserotto and L.C. Gomes are re- (Berka 1986; Lim et al. 2003), and these values are as- search fellowship recipients from CNPq. sociated with the lower ¢sh density in the ¢sh boxes and an adequate fasting period before transportation. Whole-body cortisol levels of unstressed cardinal tetras were higher than those found in zebra¢sh, References Danio rerio (Hamilton) (Ramsay, Feist, Varga, West- American Public Health Association, American Water ¢eld, Kent & Schreck 2006), and in three-spined stick- Works Association,Water Environment Federation (1992) leback, Gasterosteus aculeatus aculeatus Linnaeus Standard Methods for the Examination of Water and Waste- (Pottinger, Carrick & Yeomans 2002), but similar to water, 18th edn. American Public Health Association, those in other Amazonian ornamental ¢sh, the NewYork, NY, USA. marbled hatchet¢sh (Gomes et al. 2008). The signi¢- Bayley P.B. & Petrere M. Jr (1989) Amazon ¢sheries: assess- cantly higher cortisol levels found in transported ¢sh ment methods, current status and management options. compared with levels of ¢sh caught in the environ- In: Proceedings of the International Large River Symposium s (ed. by D.P. Dodge), pp. 385^398 Canadian Fisheries and ment demonstrate that E¢nol L is not e⁄cient in sup- Aquatic Sciences Special Publications106. pressing stress responses in ¢sh in a 24-h period, Berka R. (1986) The transport of live ¢sh. A review. EIFAC which is in agreement with the ion £ux results. How- Technical Papers 48, FAO, Rome, Italy. ever, ¢sh from the probiotic treatment presented a Carnevali O., De Vivo L., Sulpizio R., Gioacchini G., Olivotto lower magnitude of stress than control ¢sh at 24-h I., Silvi S. & Cresci A. (2006) Growth improvement sampling. This lower cortisol response in probiotic- by probiotic in European sea bass juveniles (Dicen- treated ¢sh was also observed by Gomes et al.(2008) trarchus labrax, L.), with particular attention to IGF-1,

r 2008 The Authors Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 40, 157^ 165 163 s Cardinal transportation with E¢nol L LCGomeset al. Aquaculture Research, 2009, 40, 157 ^ 165

myostatin and cortisol gene expression. Aquaculture 258, Gonzalez R.J., Wood C.M., Wilson R.W., Patrick M.L., 430^438. Bergman H.L., Narahara A. & Val A.L. (1998) E¡ects of Chao N.L. (2001) Fisheries, diversity and conservation water pH and calcium concentration on ion balance of ornamental ¢sh of the Rio Negro River, Brazil ^ a re- in ¢sh of the Rio Negro, Amazon. Physiological Zoology view of (1989^99). In: Conservation 71, 15^22. and Management of Ornamental Fish Resources of the Gonzalez R.J., Wilson R.W., Wood C.M., Patrick M.L. & Val Rio Negro Basin, Amazonia, Brazil ^ Project Piaba (ed. by A.L. (2002) Diverse strategies for ion regulation in ¢sh col- L.N. Chao, P.Petry, G. Prang, L. Sonneschein & M. Tlusty), lected from the ion-poor, acidic Rio Negro. Physiological pp. 161^204. University of Amazonas Press, Manaus, and Biochemical Zoology 75,37^47. Brazil. Gournier-Chateau N., Larpent J.P., Castellanos I. & Larpent Chao N.L. & Prada-Pedreros S. (1995) Diversity and habitat of J.L. (1994) Les Probiotiques en Alimentation Animale et Hu- ornamental ¢shes in the Rio Negro, Brazil: exploitation maine. Technique et Documentation Lavoisier, Paris, and conservation issues. In: Proceedings ofWorld Fisheries France,192pp. Congress, Theme 3 (ed. by D.P. Philipp, J.M. Epifanio, J.E. Lim L.C., Dhert P. & Sorgeloos P. (2003) Recent develop- Marsden & J.E. Claussen), pp.241^261. Oxford & IBH Pub- ments and improvements in ornamental ¢sh packaging lishing, New Delhi, India. systems for air transport. Aquaculture Research 34, Chao N.L. & Prang G. (1997) Project Piaba ^ towards a sus- 923^935. tainable ornamental ¢shery in the Amazon. Mortatti J. & Probst J.L. (2003) Silicate rock weathering and

Sciences and Conservation1, 105 ^ 111. atmospheric/soil CO2 uptake in the Amazon basin esti- de Jesus E.G., Hirano T. & Inui Y. (1991) Changes in cortisol mated from river water geochemistry: seasonal and spa- and thyroid hormone concentrations during early devel- tial variations. Chemical Geology197,177^196. opment and metamorphosis in the Japanese £ounder, Pottinger T.G., Carrick T.R. & Yeomans W.E. (2002) The Paralichthys olivaceus. General and Comparative Endocri- three-spined stickleback as an environmental sentinel: nology 82,369^376. e¡ects of stressors on whole-body physiological indices. Froese R. (1998) Insulating properties of styrofoam Journal of Fish Biology 61,207^229. boxes used for transporting live ¢sh. Aquaculture 159, Prang G. (2001) Aviamento and the ornamental ¢shery of 283^292. the Rio Negro, Brazil: implications for sustainable use. Gatesoupe F.J. (1999) The use of probiotics in aquaculture. In: Conservation and Management of Ornamental Fish Re- Aquaculture180,147^165. sources of the Rio Negro Basin, Amazonia, Brazil ^ Project Geisler R. & Annibal S.R. (1984) Oekologie des Cardinal Tetra Piaba (ed. by L.N. Chao, P.Petry, G. Prang, L. Sonneschein Paracheirodon axelrodi (Pisces, Characoidea) im Stromgebiet & M. Tlusty), pp. 43^73. University of Amazonas Press, des Rio Negro/Brasilien, sowie zuchtrelevante Faktoren. Manaus, Brazil. Amazoniana 9, 53^86. RamsayJ.M., Feist G.W.,Varga Z.M.,West¢eld M., Kent M.L. & Gerstner C.L., Ortega H., Sanchez H. & Graham D.L. (2006) Schreck C.B. (2006) Whole-bodycortisol is an indicator of E¡ects of the freshwater aquarium trade on wild ¢sh po- crowding stress in adult zebra¢sh, Danio rerio. Aquacul- pulations in di¡erentially-¢shed areas of the Peruvian ture 258,565^574. Amazon. Journal of Fish Biology 68,862^875. Rollo A., Sulpizio R., Nardi M., Silvi S., Orpianesi C., Gomes L.C., Brinn R.P., Marcon J.L., Dantas L.A., Brandao Caggiano M., Cresci A. & Carnevali O. (2006) Live micro- F.R., Abreu J.S., McComb D.M. & Baldisserotto B. (2008) bial feed supplement in aquaculture for improvement of s Using E¢nol L during transportation of Marbled hatchet- stress tolerance. Fish Physiology and Biochemistry 32, ¢sh, Carnegiella strigata (Gˇnther). Aquaculture Research; 167^ 177. doi: 10.1111/j.1365-2109.2008.01993.x. Santos G.M. & Santos A.C.M. (2005) Sustentabilidade da pes- Gomez-Gil B., Roque A. & Turnbull J.F. (2000) The use and ca na AmazoŒ nia. Estudos Avancados19,165^182. selection of probiotic bacteria for use in the culture of lar- TaokaY.,Maeda H., Jo J., Jeon M., Bai S.C., Lee W.,Yuge K. & val aquatic organisms. Aquaculture191, 259^270. Koshio S. (2006) Growth, stress tolerance and non-speci- Gonzalez R.J. & Preest M.R. (1999) Ionoregulatory speciali- ¢c immune response of Japanese £ounder Paralichthys oli- zations for exceptional tolerance of ion-poor acidic waters vaceus to probiotics in a closed recirculating system. in the (Paracheirodon innesi). Physiological and Fisheries Science 72,310^321. Biochemical Zoology 72, 156^163. Tlusty M. (2002) The bene¢ts and risks of aquacultural Gonzalez R.J. & Wilson R.W.(2001)Patterns of ion regulation production for the aquarium trade. Aquaculture 205, in acidophilic ¢sh native to the ion-poor, acidic Rio Negro. 203^219. Journal of Fish Biology 58,1680^1690. Verdouw H., Vanechted C.J.A. & Dekkers E.M.J. (1978) Am- Gonzalez R.J., Dalton V.M. & Patrick M.L. (1997) Ion regula- monia determination based on indophenol with sodium tion in ion-poor acidic water by the blackskirt tetra (Gym- salicylate.Water Research12,399^402. nocorymbus ternetzi), a ¢sh native to the Amazon River. Verschuere L., Rombaut G., Sorgeloos P. & Verstraete W. Physiological Zoology 70,428^435. (2000) Probiotic bacteria as biological control agents in

r 2008 The Authors 164 Journal Compilation r 2008 Blackwell Publishing Ltd, Aquaculture Research, 40, 157^ 165 s Aquaculture Research, 2009, 40, 157^165 Cardinal transportation with E¢nol L LCGomeset al.

aquaculture. Microbiology and Molecular Biology Reviews pp. 279^299. University of Amazonas Press, Manaus, 64,655^671. Brazil. Waichman A.V., Silva M.P. & Marcon J.L. (2001) Water Walker I. (2004) The food spectrum of the cardinal-tetra quality monitoring during commercialization of Amazo- (Paracheirodon axelrodi, ) in its natural habitat. nian ornamental ¢sh. In: Conservation and Manage- Acta Amazonica 34,69^73. ment of Ornamental Fish Resources of the Rio Negro Zall D.M., Fisher M.D. & Garner Q.M. (1956) Photometric Basin, Amazonia, Brazil ^ Project Piaba (ed. by determination of chlorides in water. Analytical Chemistry L.N. Chao, P.Petry, G. Prang, L. Sonneschein & M. Tlusty), 28,1665^1678.

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