ABSTRACT

The objective of this study was to evaluate menthol and eugenol as anesthetics for early juveniles of curimba, Prochilodus lineatus. Juveniles of 4.0±0.5 g and a total length of 8.8±0.1 cm were exposed to the anesthetics menthol (20, 30, 40, 50, 60, 70, and 80 mg L⁻¹) and eugenol (20, 30, 40, 50, 60, and 70 mg L⁻¹) up to deep anesthesia. The anesthetic effects were evaluated measuring the induction time to deep anesthesia (characterized by loss of equilibrium, absence of swimming, reduction of opercular movements, and responses only to intense tactile stimuli), recovery time, time to appetite return, and mortality rate after 96 h of procedure. The concentrations between 60 to 80 mg of menthol L⁻¹ provided the lowest time of induction. Increased concentrations led to a decrease in recuperation time. The concentrations between 50 to 70 mg of eugenol L⁻¹ provided the lowest induction times; however, recovery time was not affected by eugenol concentrations. The return to appetite was observed 24 h after anesthesia, while the survival after 96 h was >90%. Concentrations of 60 and 50 mg L⁻¹ of menthol and eugenol, respectively, are recommended for effective anesthesia with limited side effects.

Key Words:
aquaculture; physiology; Prochilodus lineatus; stress

Introduction

In Brazil, anesthesia has been studied aiming to minimize stress and, thereby, decrease suffering of animals during handling procedures (Bertozi Junior et al., 2014Bertozi Junior, M.; Diemer, O.; Neu, D. H.; Bittencourt, F.; Boscolo, W. R. and Feiden, A. 2014. Benzocaína e eugenol como anestésicos para juvenis de Pimelodus britskii (mandi-pintado). Revista Brasileira de Ciência Agrária 9:134-138.; Ribeiro et al., 2015aRibeiro, P. A. P.; De Melo, D. C.; Santo, A. H. E.; Silva, W. S.; Santos, A. E. H. and Luz, R. K. 2015a. Tricaine as an anaesthetic for larvae and juveniles of Lophiosilurus alexandri, a carnivorous freshwater fish. Aquaculture Research 46:1788-1792. https://doi.org/10.1111/are.12316
https://doi.org/10.1111/are.12316... ,bRibeiro, P. A. P.; Miranda-Filho, K. C.; De Melo, D. C. and Luz, R. K. 2015b. Efficiency of eugenol as anesthetic for the early life stages of Nile tilapia (Loreochromis niloticus). Anais da Academia Brasileira de Ciências 87:529-535. https://doi.org/10.1590/0001-3765201520140024
https://doi.org/10.1590/0001-37652015201... ). The concentration and efficacy required for induction may vary in relation to species, age, size, and water quality parameters (Walsh and Pease, 2002Walsh, C. T. and Pease, B. C. 2002. The use of clove oil as an anesthetic for the long finned eel, Anguilla reinhardtii (Steindachner). Aquaculture Research 33:627-635.; Woody et al., 2002Woody, C. A.; Nelson, J. and Ramstad, K. 2002. Clove oil as an anesthetic for adult sockeye salmon: field trails. Journal of Fish Biology 60:340-347.; Gomes et al., 2011Gomes, D. P.; Chaves, B. W. C.; Becker, A. G. and Baldisserotto, B. 2011. Water parameters affect anaesthesia induced by eugenol in silver catfish, Rhamdia quelen. Aquaculture Research 42:878-886. https://doi.org/10.1111/j.1365-2109.2011.02864.x
https://doi.org/10.1111/j.1365-2109.2011... ).

Eugenol and menthol are among the main natural anesthetics used in aquaculture. Eugenol is a natural product obtained from the distillation of the extract of clove (Eugenia caryophyllata) leaves, stems, and roots (Inoue and Moraes, 2007Inoue, L. A. K. A. and Moraes, G. 2007. Óleo de cravo: um anestésico alternativo para o manejo de peixes. Embrapa Amazônia Ocidental, Manaus-AM. Available at: <https://www.embrapa.br/web/mobile/publicacoes/-/publicacao/681655/oleo-de-cravo-um-anestesicoalternativo-para-o-manejo-de-peixes>. Accessed on: Aug. 22, 2017.
https://www.embrapa.br/web/mobile/public... ). The efficiency of eugenol has been observed for several species, such as rainbow trout (Oncorhynchus mykiss; Keene et al., 1998Keene, J. I.; Noakes, D. I. G.; Moccia, R. D. and Soto, G. C. 1998. The efficacy of clove oil as an anesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture Research 29:89-101.), pacamã (Lophiosilurus alexandri; Ribeiro et al., 2013Ribeiro, P. A. P.; Miranda Filho, K. C.; Melillo Filho, R.; Santos, A. E. H.; Silva, W. S.; Rodrigues, L. A. and Luz, R. K. 2013. Efeito anestésico do eugenol em juvenis de pacamã. Pesquisa Agropecuária Brasileira 48:1136-1139. https://doi.org/10.1590/S0100-204X2013000800048
https://doi.org/10.1590/S0100-204X201300... ), Nile tilapia (Oreochromis niloticus; Vidal et al., 2008Vidal, L. V. O.; Albinati, R. C. B.; Albinati, A. C. L.; Lira, A. D.; Almeida, T. R. and Santos, G. B. 2008. Eugenol como anestésico para a tilápia-do-nilo. Pesquisa Agropecuária Brasileira 43:1069-1074. https://doi.org/10.1590/S0100-204X2008000800017
https://doi.org/10.1590/S0100-204X200800... ; Ribeiro et al., 2015bRibeiro, P. A. P.; Miranda-Filho, K. C.; De Melo, D. C. and Luz, R. K. 2015b. Efficiency of eugenol as anesthetic for the early life stages of Nile tilapia (Loreochromis niloticus). Anais da Academia Brasileira de Ciências 87:529-535. https://doi.org/10.1590/0001-3765201520140024
https://doi.org/10.1590/0001-37652015201... ), matrinxã (Brycon cephalus; Barbosa et al., 2007Barbosa, L. G.; Moraes, G. and Inoue, L. A. K. A. 2007. Respostas metabólicas do matrinxã submetido a banhos anestésicos de eugenol. Acta Scientiarum Biological Science 29:255-260. https://doi.org/10.4025/actascibiolsci.v29i3.474
https://doi.org/10.4025/actascibiolsci.v... ; Vidal et al., 2007Vidal, L. V. O.; Furuya, W. M.; Graciano, T. S.; Schamber, C. R.; Silva, L.; C. R.; Santos, L. D. and Souza, S. R. 2007. Eugenol como anestésico para juvenis de matrinxã (Brycon cephalus). Revista Brasileira de Saúde Produção Animal 8:335-342.), pacu (Piaractus mesopotamicus; Gonçalves et al., 2008Gonçalves, A. F. N.; Santos, E. C. C.; Fernandes, J. B. K. and Takahashi, L. S. 2008. Mentol e eugenol como substitutos da benzocaína na indução anestésica de juvenis de pacu. Acta Scientiarum Animal Science 30:339-344. https://doi.org/10.4025/actascianimsci.v30i3.1081
https://doi.org/10.4025/actascianimsci.v... ), lambari (Astyanax altiparanae; Pereira-da-Silva et al., 2009Pereira-da-Silva, E. M.; Oliveira, R. H. F.; Ribeiro, M. A. R. and Coppola, M. P. 2009. Efeito anestésico do óleo de cravo em alevinos de lambari. Ciência Rural 39:1851-1856. https://doi.org/10.1590/S0103-84782009005000127
https://doi.org/10.1590/S0103-8478200900... ), silver catfish (Rhamdia quelen; Cunha et al., 2010Cunha, M. A.; Zeppenfeld, C. C.; Garcia, L. O.; Loro, V. L.; Fonseca, M. B.; Emanuelli, T.; Veeck, A. P. L.; Copatti, C. E. and Baldisserotto, B. 2010. Anesthesia of silver catfish with eugenol: time of induction, cortisol response and sensory analysis of fillet. Ciência Rural 40:2107-2114. https://doi.org/10.1590/S0103-84782010005000154
https://doi.org/10.1590/S0103-8478201000... ; Gomes et al., 2011Gomes, D. P.; Chaves, B. W. C.; Becker, A. G. and Baldisserotto, B. 2011. Water parameters affect anaesthesia induced by eugenol in silver catfish, Rhamdia quelen. Aquaculture Research 42:878-886. https://doi.org/10.1111/j.1365-2109.2011.02864.x
https://doi.org/10.1111/j.1365-2109.2011... ), and fat snook (Centropomus parallelus; Souza et al., 2012Souza, R. A. R.; Carvalho, C. V. A.; Nunes, F. F. N.; Scopel, B. R.; Guarizi, J. D.; Tsuzuki, M. Y. 2012. Efeito comparativo da benzocaína, mentol e eugenol como anestésicos para juvenis de robalo peva. Boletim Instituto de Pesca 38:247-255.).

Menthol is an essential oil extracted from plants of the genus Mentha (Patel et al., 2007Patel, T.; Ishiuji, Y. and Yosipovitch, G. 2007. Menthol: a refreshing look at this ancient compound. American Academy of Dermatology 57:873-878. https://doi.org/10.1016/j.jaad.2007.04.008
https://doi.org/10.1016/j.jaad.2007.04.0... ). This drug may be used for fish anesthesia, as observed for pacu (Gonçalves et al., 2008Gonçalves, A. F. N.; Santos, E. C. C.; Fernandes, J. B. K. and Takahashi, L. S. 2008. Mentol e eugenol como substitutos da benzocaína na indução anestésica de juvenis de pacu. Acta Scientiarum Animal Science 30:339-344. https://doi.org/10.4025/actascianimsci.v30i3.1081
https://doi.org/10.4025/actascianimsci.v... ), tambaqui (Colossoma macropomum; Façanha and Gomes, 2005Façanha, M. F. and Gomes, L. C. 2005. A eficácia do mentol como anestésico para tambaqui (Colossoma macropomum, Characiformes: Characidae). Acta Amazonica 35:71-75. https://doi.org/10.1590/S0044-59672005000100011
https://doi.org/10.1590/S0044-5967200500... ), fat snook (Souza et al., 2012Souza, R. A. R.; Carvalho, C. V. A.; Nunes, F. F. N.; Scopel, B. R.; Guarizi, J. D.; Tsuzuki, M. Y. 2012. Efeito comparativo da benzocaína, mentol e eugenol como anestésicos para juvenis de robalo peva. Boletim Instituto de Pesca 38:247-255.), dourado (Salminus brasiliensis; Pádua et al., 2010Pádua, S. B.; Pietro, P. S.; Iglesias-Filho, P. S.; Ishikawa, M. M. and Hisano, H. 2010. Mentol como anestésico para dourado (Salminus brasiliensis). Boletim do Instituto de Pesca 36:143-148.), and Nile tilapia (Simões and Gomes, 2009Simões, L. N. and Gomes, L. C. 2009. Eficácia do mentol como anestésico para juvenis de tilápia-do-nilo (Oreochromis niloticus). Arquivos Brasileiros Medicina Veterinária Zootecnia 61:613-620. https://doi.org/10.1590/S0102-09352009000300014
https://doi.org/10.1590/S0102-0935200900... ).

Curimba (Prochilodus lineatus) has social, ecological, and economic importance in South America (Jorge et al., 2011Jorge, L. C.; Sanchez, S. and Moreira Filho, O. 2011. Chromosomal characterization of Prochilodus lineatus from Paraná River. Corrientes, Argentina. I. B Chromosomes and NOR banding. Cytologia 76:219-222. https://doi.org/10.1508/cytologia.76.219
https://doi.org/10.1508/cytologia.76.219... ). The species has excellent growth performance and potential for aquaculture (Graeff and Tomaselli, 2011Graeff, A. and Tomaselli, A. 2011. Policultivo de carpas com introdução crescente do curimatã (Prochilodus scropha) como espécie principal. REDVET Revista Electónica de Veterinaria 12:1-11.). However, studies on the effects of eugenol and menthol in early juveniles of curimba were not tested yet in curimba. Therefore, the present study aimed to evaluate the efficiency of eugenol and menthol as anesthetics for early juveniles of the species.

Material and Methods

Early juveniles of curimba were purchased from a commercial hatchery and transported by car to the laboratory rearing facilities in Cordeiro, RJ, Brazil (22°01′03.88″ S and 42°21′24.14″ W). All procedures were carried out according to the international practices for animal use and care under the control of a local ethical committee on animal use (case no. 003/2016).

A total of 150 juveniles of curimba, weighing 4.0±0.5 g, with a total average length of 8.8±0.1 cm, were used for anesthetic induction by menthol and eugenol. The juveniles of curimba were acclimated for a period of 30 days in a recirculation system in 120-L water tanks. They were fed three times a day (at 8:00, 12:00, and 17:00 h) a commercially formulated diet containing 400 g protein kg⁻¹, 350 mg kg⁻¹ vitamin C, 80 g ether extract kg⁻¹, and 100 g moisture kg⁻¹ (levels and guarantees made available by the manufacturer). The experimental units were cleaned prior to the first and after the last feed for the withdrawal of excreta and food residues. The water quality indicators remained in the range considered adequate for the maintenance of tropical fish species (Arana, 2004Arana, L. 2004. Princípios químicos de qualidade da água em aquicultura. UFSC, Florianópolis.). Water temperature was kept at 25.2±0.8 °C; dissolved and saturated oxygen at 7.0±0.4 mg L⁻¹ and 86.0±3.9 %, respectively; pH at 6.7±0.3; electric conductivity at 0.6±0.1 μS cm⁻¹; total dissolved solids at 0.3±0.1 ppt; and unionized ammonia nitrogen (NH₃-N) at 0.15±0.03 mg L⁻¹. Physical and chemical monitoring of water was carried out using a HI83203-01 Hanna Photometer, HI9146-04 Hanna oxygen meter, and HI98130 Hanna multi-parameter Combo.

The experiments were performed in a completely randomized design, in which the treatments were menthol (20, 30, 40, 50, 60, 70, and 80 mg L⁻¹) and eugenol (20, 30, 40, 50, 60, and 70 mg L⁻¹) concentrations. Ten fish randomly chosen were used in each treatment. Eugenol (Escamaforte^®) and menthol (CRQ−1006310100) were first diluted (1:10) in ethanol 98.8% to reduce their hydrophobic character, and later added to the experimental units in accordance with the predetermined concentrations. Fish feed was suspended for a 24-h period before the anesthetic procedure. The hyperactivity of fish subjected to immersion in an anesthetic should not be attributed to ethanol used as a solvent. Therefore, early juveniles of curimba were subjected to an ethanol (98.8%) solution at similar quantity of that used in the highest concentrations of anesthetics to evaluate the possible hyperactivity.

For the induction analysis and recovery from anesthesia, fish were randomly captured one at a time and placed in a 2-L beaker containing the concentration of anesthetic to be tested. The behavioral characteristics of the deep anesthesia (stage 4 of anesthesia) used in this study followed the recommendations suggested by Ross and Ross (2008)Ross, L. G. and Ross, B. 2008. Anaesthetic and sedative techniques for aquatic animals. 3rd. ed. Blackwell Science, Oxford., basically characterized by loss of equilibrium, absence of swimming, reduction of opercular movements, and responses only to intense tactile stimuli. The non-reaction to stimuli was verified by touching the fish on the side using a glass rod. When the individuals reached the stage 4 of anesthesia, their biometry and weight (model BL320H Shimatzu scale) measurements (total length using a ruler) were carried out to simulate the actual handling conditions in fish farming. For recovery, all individuals were placed in anesthetic-free water in 2-L beakers. To assess the anesthetic recovery stages, the criteria proposed by Hikasa et al. (1986)Hikasa, Y.; Takasa, K.; Ogasawara, T. and Ogasawara, S. 1986. Anesthesia and recovery with tricaine methanesulfonate, eugenol and thiopental sodium in the cap, Cyprinus carpio. Nippon Juigaku Zasshi 48:341-351. were adopted. After the experiments, fish from each replicate were pooled and kept in 50-L tanks in a recirculating water system to observe the return to appetite and the survival rate after 96 h.

An ANOVA and the Tukey's test for the comparison of means were performed using the SAS software (Statistical Analysis System, version 8.0). The results of the induction and recovery time were analyzed using a non-linear regression for the function with best adjustment of data.

Results

Exposure to ethanol solvent did not provoke any behavioral change. Menthol and eugenol concentrations proved efficient in promoting induction at the deep anesthetic phase in early juveniles of curimba. As concentrations increased, there was a decrease (P<0.05) in the induction and recovery time. Juveniles of curimba did not reach deep anesthesia after 30 min (1800 s) of exposure to the 20 mg menthol L⁻¹. Thus, this concentration was withdrawn from the statistical analysis. The longest induction time (P<0.05) was verified at the lowest concentration of 30 mg menthol L⁻¹, whereas the shortest times (P<0.05) were observed in the 60 to 80 mg menthol L⁻¹ concentrations (P<0.05). Juveniles subjected to anesthesia showed a recovery time inversely proportional to menthol concentrations, i.e., it was verified that, as the anesthetic concentrations increased, the recovery time decreased (P<0.05) (Figure 1).

Eugenol proved to be efficient in anesthetizing early juveniles of curimba, with concentrations presenting statistical differences for the time required to induce the deep anesthesia phase (Figure 1). The longest induction time (P<0.05) occurred at lower concentrations of 20 mg eugenol L⁻¹, whereas the shortest induction time (P<0.05) occurred at a concentration of 70 mg eugenol L⁻¹. Regarding the recovery period, no difference was detected (P>0.05) relative to the tested concentration (Figure 1).

In all treatments, the early juveniles of curimba showed a return to appetite in 24 h after the anesthesia test. After 96 h following the recovery from anesthesia, a 10% mortality occurred for 30, 50, 60, and 80 mg menthol L⁻¹ concentrations. For eugenol, a 10% mortality was observed in 20 to 70 mg L⁻¹ concentrations. In our observations, this mortality resulted from fish interactions (bites) and was not directly related to anesthetic toxicity.

Discussion

Hyperactivity was observed in fish subjected to immersion in an anesthetic and should not be attributed to ethanol, generally used as a solvent, but to the anesthetic itself (Vidal, et al., 2008Vidal, L. V. O.; Albinati, R. C. B.; Albinati, A. C. L.; Lira, A. D.; Almeida, T. R. and Santos, G. B. 2008. Eugenol como anestésico para a tilápia-do-nilo. Pesquisa Agropecuária Brasileira 43:1069-1074. https://doi.org/10.1590/S0100-204X2008000800017
https://doi.org/10.1590/S0100-204X200800... ; Readman, et al., 2013Readman, G. D.; Owen, S. F., Murrell, J. C. and Knowles, T. G. 2013. Do fish perceive anaesthetics as aversive? Plos One 8:e73773. https://doi.org/10.1371/journal.pone.0073773
https://doi.org/10.1371/journal.pone.007... ). Therefore, early juveniles of curimba were subjected to ethanol solution; these animals showed no reactions or hyperactivity. The present study provides insights into adequate techniques for the use of the anesthetics menthol and eugenol for sedation of early juveniles of curimba.

The concentrations tested in this study fall within the suggestions given by Marking and Meyer (1985)Marking, L. L. and Meyer, F. P. 1985. Are better fish anesthetics needed in fisheries? Fisheries, Bethesda 10:2-5., who considered a good anesthetic as that which induces deep anesthesia from 1 to 3 min (60 to 180 s), and recovery at 5 min (300 s). In general, there were differences in methods and fish size during the anesthesia process. Smaller fish have a larger gill surface area ratio in relation to body weight than larger fish and, thus, have a superior area for anesthetic absorption through the gills (Woody et al., 2002Woody, C. A.; Nelson, J. and Ramstad, K. 2002. Clove oil as an anesthetic for adult sockeye salmon: field trails. Journal of Fish Biology 60:340-347.; Ross and Ross, 2008Ross, L. G. and Ross, B. 2008. Anaesthetic and sedative techniques for aquatic animals. 3rd. ed. Blackwell Science, Oxford.). The present study provides information about the use of menthol and eugenol as anesthetics for early juveniles of curimba (4 g).

The results of the present study demonstrated that the increase in menthol concentrations led to a decrease in induction and recovery times for early juveniles of curimba. A similar response was observed in S. brasiliensis (Pádua et al., 2010Pádua, S. B.; Pietro, P. S.; Iglesias-Filho, P. S.; Ishikawa, M. M. and Hisano, H. 2010. Mentol como anestésico para dourado (Salminus brasiliensis). Boletim do Instituto de Pesca 36:143-148.), O. niloticus (Mello et al., 2012Mello, R. A.; Costa, L. S.; Okamura, D.; Araújo, F. G.; Ribeiro, P. A. P.; Corrêa, F. M. and Rosa, P. V. 2012. Avaliação de 2-fenoxietanol e mentol como agentes anestésicos em tilápias. Boletim do Instituto de Pesca 38:53-59.), and fat snook (Souza et al., 2012Souza, R. A. R.; Carvalho, C. V. A.; Nunes, F. F. N.; Scopel, B. R.; Guarizi, J. D.; Tsuzuki, M. Y. 2012. Efeito comparativo da benzocaína, mentol e eugenol como anestésicos para juvenis de robalo peva. Boletim Instituto de Pesca 38:247-255.). For fat snook, increased menthol concentration did not affect recovery time. Pádua et al. (2010)Pádua, S. B.; Pietro, P. S.; Iglesias-Filho, P. S.; Ishikawa, M. M. and Hisano, H. 2010. Mentol como anestésico para dourado (Salminus brasiliensis). Boletim do Instituto de Pesca 36:143-148. anesthetized juveniles of S. brasiliensis and found that the longest anesthetic induction time was observed at the concentration of 60 mg menthol L⁻¹, and the shortest time was verified at the concentration of 150 mg menthol L⁻¹. For lambari, induction time decreased, and recovery time increased linearly with the increase in the menthol concentration (Pereira-da-Silva et al., 2014Pereira-da-Silva, E. M.; Oliveira, R. H. F. and Nero, B. D. 2014. Menthol as anaesthetic for lambari Astyanax altiparanae (Garutti & Britski 2000): attenuation of stress responses. Aquaculture Research 47:1413-1420. https://doi.org/10.1111/are.12599
https://doi.org/10.1111/are.12599... ). In addition, menthol has an anesthetic effect and attenuates the stress response in lambari; 50 mg L⁻¹ was the most effective concentration for inducing deep anesthesia within 60 s, and was safe up to 360 s exposure. The 60 mg menthol L⁻¹ concentration suggested for P. lineatus showed that animals can reach deep anesthesia within 180 s and recover 332 s after being induced.

The concentration suggested in this study for the anesthetic induction of 4 g curimba was 50 mg eugenol L⁻¹. Concentrations of 20 mg eugenol L⁻¹ provided the longest time for curimba juvenile sedation, more than 30 min. For silver catfish, eugenol concentrations between 20 to 30 mg L⁻¹ induced stage 4 anesthesia within 15 min (900 s) (Cunha et al., 2010Cunha, M. A.; Zeppenfeld, C. C.; Garcia, L. O.; Loro, V. L.; Fonseca, M. B.; Emanuelli, T.; Veeck, A. P. L.; Copatti, C. E. and Baldisserotto, B. 2010. Anesthesia of silver catfish with eugenol: time of induction, cortisol response and sensory analysis of fillet. Ciência Rural 40:2107-2114. https://doi.org/10.1590/S0103-84782010005000154
https://doi.org/10.1590/S0103-8478201000... ). In addition, rapid induction to stage 4 of anesthesia was observed with limited effects on the fish at a concentration of 50 mg eugenol L⁻¹. This value was similar to that suggested by Diemer et al. (2012)Diemer, O.; Neu, D. H.; Bittencourt, F.; Signor, S.; Boscolo, W. R. and Feiden, A. 2012. Eugenol como anestésico para jundiá (Rhamdia voulezi) em diferentes pesos. Semina: Ciências Agrárias 33:1495-1500. https://doi.org/10.5433/1679-0359.2012v33n4p1495
https://doi.org/10.5433/1679-0359.2012v3... for juveniles of jundiá (Rhamdia voulezi). In early life stages of Nile tilapia, with an average weight of 2 and 11 g, 50 mg eugenol L⁻¹ induced anesthesia within 3 min (Ribeiro et al., 2015bRibeiro, P. A. P.; Miranda-Filho, K. C.; De Melo, D. C. and Luz, R. K. 2015b. Efficiency of eugenol as anesthetic for the early life stages of Nile tilapia (Loreochromis niloticus). Anais da Academia Brasileira de Ciências 87:529-535. https://doi.org/10.1590/0001-3765201520140024
https://doi.org/10.1590/0001-37652015201... ). Pereira-da-Silva et al. (2009)Pereira-da-Silva, E. M.; Oliveira, R. H. F.; Ribeiro, M. A. R. and Coppola, M. P. 2009. Efeito anestésico do óleo de cravo em alevinos de lambari. Ciência Rural 39:1851-1856. https://doi.org/10.1590/S0103-84782009005000127
https://doi.org/10.1590/S0103-8478200900... studied the effectiveness of eugenol on early juveniles of lambari and found that concentrations between 50 and 150 mg L⁻¹ promoted deep anesthesia in <3 min; however, mortalities following higher dosages were documented (150 mg L⁻¹).

Previous studies have shown that proper immersion anesthesia may decrease the incidence of adverse effects and lead to a milder recovery (Acerete et al., 2004Acerete, L.; Balasch, J. C.; Espinosa, E.; Josa, A. and Tort, L. 2004. Physiological responses in Eurasian perch (Perca fluviatilis, l.) subjected to stress by transport and handling. Aquaculture 237:167-178. https://doi.org/10.1016/j.aquaculture.2004.03.018
https://doi.org/10.1016/j.aquaculture.20... ; Zahl et al., 2009Zahl, I. H.; Kiessling, A.; Samuelsen, O. B. and Hansen, M. K. 2009. Anaesthesia of Atlantic cod (Gadus morhua): effect of preanaesthetic sedation, and importance of body weight, temperature and stress. Aquaculture 295:52-59. https://doi.org/10.1016/j.aquaculture.2009.06.019
https://doi.org/10.1016/j.aquaculture.20... ). In the present study, there were no differences in recovery time in early juveniles of curimba anesthetized with eugenol in concentrations from 20 to 70 mg L⁻¹. Eugenol was also effective in inducing deep anesthesia in juveniles (mean weight = 3.31 g) of matrinxã (Brycon cephalus). Concentrations of 50-100 mg eugenol L⁻¹ were effective in inducing anesthesia with a relatively fast recovery time (Vidal et al., 2007Vidal, L. V. O.; Furuya, W. M.; Graciano, T. S.; Schamber, C. R.; Silva, L.; C. R.; Santos, L. D. and Souza, S. R. 2007. Eugenol como anestésico para juvenis de matrinxã (Brycon cephalus). Revista Brasileira de Saúde Produção Animal 8:335-342.). For groups of Nile tilapia ranging from 0.02 to 11 g, the recovery time was between 53.07 to 184.31 s. Eugenol is an efficient anesthetic for tambaqui; juveniles exposed to 35-135 mg eugenol L⁻¹ recovered within 350 to 1235 s, respectively (Roubach et al., 2005Roubach, R.; Gomes, L. C.; Fonseca, F. A. L. and Val, A. L. 2005. Eugenol as an efficacious anaesthetic for tambaqui, Colossoma macropomum (Cuvier). Aquaculture Research 36:1056-1061. https://doi.org/10.1111/j.1365-2109.2005.01319.x
https://doi.org/10.1111/j.1365-2109.2005... ). In fact, concentrations of 50-100 mg eugenol L⁻¹ provided a recovery time higher than the ideal (300 s), however, out of the critical range (>600 s) (Marking and Meyer, 1985Marking, L. L. and Meyer, F. P. 1985. Are better fish anesthetics needed in fisheries? Fisheries, Bethesda 10:2-5.). Similarly, early juveniles of curimba showed a recovery time of 354 to 390 s.

High survival was observed in juveniles of curimba 96 h after anesthesia with menthol and eugenol (>90%); moreover, fish showed return to appetite 24 h after the procedure. For Nile tilapia between 0.02-11 g, after 24 h of testing with the eugenol concentrations from 50 to 200 mg L⁻¹, a 100% survival was observed, and all fish resumed eating (Ribeiro et al., 2015bRibeiro, P. A. P.; Miranda-Filho, K. C.; De Melo, D. C. and Luz, R. K. 2015b. Efficiency of eugenol as anesthetic for the early life stages of Nile tilapia (Loreochromis niloticus). Anais da Academia Brasileira de Ciências 87:529-535. https://doi.org/10.1590/0001-3765201520140024
https://doi.org/10.1590/0001-37652015201... ). Elevated mortality of lambari was observed when fish were exposed to surgical anesthesia using 75 and 100 mg eugenol L⁻¹. The unique safe concentrations for surgical anesthesia in lambari was 50 mg eugenol L⁻¹. However, for deep anesthesia, concentrations of 50 to 125 mg eugenol L⁻¹ resulted in no mortality for the species (Pereira-da-Silva et al., 2009Pereira-da-Silva, E. M.; Oliveira, R. H. F.; Ribeiro, M. A. R. and Coppola, M. P. 2009. Efeito anestésico do óleo de cravo em alevinos de lambari. Ciência Rural 39:1851-1856. https://doi.org/10.1590/S0103-84782009005000127
https://doi.org/10.1590/S0103-8478200900... ). Generally, 20-70 mg eugenol L⁻¹ resulted in elevated survival and demonstrated the possibility of effective handling of animals and avoiding increased mortality. The mortality observed for early juveniles of curimba after the procedure was a result of fish interaction (bites) and not a direct relation to anesthetic toxicity.

Conclusions

Menthol and eugenol proved to be efficient in inducing deep anesthetic stages in early juveniles of curimba, and the recommended concentrations are 60 and 50 mg L⁻¹, respectively.

Acknowledgments

We acknowledge the Programa de Pós-graduação em Aquicultura e Recursos Aquáticos Tropicais of Universidade Federal Rural da Amazônia (UFRA), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), for the financial support. We also thank the Fundação Instituto de Pesca do Estado do Rio de Janeiro (FIPERJ), for the space made available for the development of this research.

References

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