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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Comparative Biochemistry and Physiology, Part A 162 (2012) 139–145 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa Stress responses of the endemic freshwater cururu stingray (Potamotrygon cf. histrix) during transportation in the Amazon region of the Rio Negro☆ R.P. Brinn a,⁎, J.L. Marcon b, D.M. McComb c, L.C. Gomes d, J.S. Abreu e, B. Baldisseroto f a Florida International University, 3000 NE 151 st. 33181, Miami, FL, USA b Universidade Federal do Amazonas (UFAM), Av. General Rodrigo Octávio Jordão Ramos, 3000, Campus Universitário, Coroado I, 69077-000, Manaus, AM, Brazil c Harbor Branch Oceanographic Institute at Florida Atlantic University, 34946, Fort Pierce, FL, USA d Centro Universitário Vila Velha, Vila Velha, ES, Brazil, Rua Comissário José Dantas de Melo, 21, Boa Vista, ,29101-770 Vila Velha, ES, Brazil e Universidade Federal de Mato Grosso, Faculdade de Agronomia e Medicina Veterinária (FAMEV), Avenida Fernando Corrêa da Costa, 2367, Boa Esperança, 78060-900, Cuiaba, MT, Brazil f Universidade Federal de Santa Maria, Campus Camobi, 97105-900, Santa Maria, RS, Brazil article info abstract Article history: Potamotrygon cf. histrix (cururu stingray) are endemic freshwater stingrays from the middle region of the Rio Received 28 February 2011 Negro in the Brazilian Amazon basin and are exported worldwide as ornamentals caught by artisanal Received in revised form 2 July 2011 fishermen. The transport process from capture to final destination is long and stressful. This study quantified Accepted 5 July 2011 stress related changes in corticosterone, blood and water samples (baseline, pre-transport, 3 h, 12 h and 24 h) Available online 13 July 2011 analyzed during a transport experiment which tested two water additives (tetracycline and the probiotic fi fi Keywords: E nol®). There was a signi cant stepwise increase in corticosterone levels in stingrays over transport time in Amazonian stingrays combination with osmoregulatory disturbances suggesting a stress related role of this corticosteroid. There + + Corticosterone were significant increases in water conductivity, Na and K losses and ammonia excretion. Blood Elasmobranch parameters such as glucose, hematocrit, red blood count and urea did not change significantly during the Osmoregulation experiment. Glucose levels did not increase significantly during transport and this may be due to the fact that Transport stress other elasmobranchs have been shown to rely more on ketone bodies for energy rather than glucose and produce ammonia as their main nitrogenous waste. The mineralocorticoid action of this hormone has been shown in elasmobranchs and most likely plays a role in osmotic homeostasis. The use of probiotic and especially antibiotic should be avoided since no beneficial effects were observed. © 2011 Elsevier Inc. All rights reserved. 1. Introduction The Brazilian Amazon region is home to a pigmy freshwater stingray that is endemic to a small portion of the Rio Negro, a large There are over 800 extant elasmobranch species most of which affluent of the Amazon River. Five stingray species are legally exported inhabit marine environments (Heinicke et al., 2009). Of the 400 batoid from the Brazilian Amazon basin under government managed annual (skates and rays) species at least 20 Potamotrygonid rays are quotas, including Potamotrygon cf histrix, regionally called “Cururu exclusively freshwater and distributed among four genera: Heliotrygon Ray”, which has yet to be fully described. Freshwater stingrays are (two species), Paratrygon (one species), Plesiotrygon (two species) and targeted by the ornamental industry primarily due to high economic Potamotrygon (18 valid species) (Carvalho et al., 2003; Martin, 2005; value making them vulnerable to fishing pressure. The city of Barcelos, Carvalho and Lovejoy, 2011; Carvalho and Ragno, 2011). In contrast to within the Brazilian Amazon region, is an artisanal fishing community marine species, freshwater stingrays possess unique morphological which collects and exports thousands of ornamental fishes from the and physiological specializations including low plasma urea levels, a Amazon including P.cf histrix (Chao et al., 2001). The Rio Negro is a reduction in rectal gland size (Thorson et al., 1983) and a reduction in warm, ion poor, oxygen depleted, acidic river reaching 3.5 pH in some the tubule length of the Ampullae of Lorenzini (Raschi and Mackanos, areas (Matsuo and Val, 2003; Mortatti and Probst, 2003). Small 1989; McGowan and Kajiura, 2009). streams (igarapés) feed the main Rio Negro system with tannin rich waters supporting high ichthyological diversity. The Rio Negro Basin has a high seasonal variation in water level and the stingrays have a ☆ This paper stems from a presentation in the Symposium "The Physiological Stress predetermined reproductive cycle strongly linked to the river pulse Response in Elasmobranch Fishes", at the 26th annual meeting of the American (Charvet-Almeida et al., 2005). Local fishermen catch, collect and Elasmobranch Society, held on July 11, 2010, in Providence, Rhode Island (USA). transport stingrays with rudimentary techniques and in many cases ⁎ Corresponding author. Tel.: +1 305 919 5348; fax: +1 305 919 4040. add unmeasured and untested compounds including tetracycline to E-mail addresses: brinnr@fiu.edu (R.P. Brinn), [email protected] (J.L. Marcon), [email protected] (D.M. McComb), [email protected] (L.C. Gomes), the water in an effort to minimize stress and mortality. Once taken [email protected] (J.S. Abreu), [email protected] (B. Baldisseroto). from the wild and transported to Barcelos, the rays are carried by 1095-6433/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2011.07.004 Author's personal copy 140 R.P. Brinn et al. / Comparative Biochemistry and Physiology, Part A 162 (2012) 139–145 freight boat to Manaus in transport that can last over 24 h dependent and 24 h of transport. Each box was sampled only once and then upon river conditions. The transport occurs in overcrowded plastic discarded from the experiment thereafter. totes with little water to optimize space and cost, creating unfavorable conditions to fish. Techniques developed to minimize stress during transport in teleosts have been successful in other species and include 2.3. Blood collection and analyses the addition of probiotic products (Gomes et al., 2008; 2009). Once arriving in Manaus, fish are held in quarantine at local exporters prior After anesthesia and tail immobilization stingrays were kept in to international exportation. Many stingrays are subsequently flown to ventral position and a cardiac blood sample taken with an insulin-type Miami (FL, USA) where they are held for weeks or months prior to final hypodermic 1 mL syringe coated with ETDA 10% as anticoagulant. To shipment worldwide. The transport process is lengthy and therefore ensure no mortality of experimental animals due to excessive blood acute and chronic stress strongly impacts survivorship. loss, the volume removed was less than 1% of the individual body mass. Measuring stress in elasmobranchs has been a challenge because The same procedures were applied for all samples collected in all they produce a unique stress hormone identified as 1 α hydroxicorti- treatment groups. Blood was used for hematocrit (Htc) determination costerone (Idler and Truscott, 1966) which has no commercially by the microhematocrit centrifugation technique, and for red blood available measuring technique. Some species of elasmobranch have cell counts (RBC) performed by manual counting in a Neubauer also been shown to produce corticosterone however with no well- hemocytometer with 1/200 dilution of blood in phormol-citrate defined stress related role (Truscott and Idler, 1972; Rasmussen and solution. MCV (mean cellular volume) was calculated based on the Gruber, 1990; Manire et al., 2007). obtained Htc and RBC values. The remaining blood was centrifuged at The primary objective of this experiment was to investigate stress 2500 g for 3 min and plasma was immediately stored in a liquid related osmoregulatory, blood parameter alterations and costicoster- nitrogen container (Cryo Diffusion S.A., France). one responses of P. cf. histrix (cururu stingray) to two different water Plasma samples were analyzed for glucose, urea, chloride levels additives during the transport process. The secondary objective was and osmolarity. Glucose levels were measured by the enzymatic to contribute to the general understanding of the stress response in method of glucose oxidase at 510 nm and Urea was determined by freshwater elasmobranchs. an enzymatic method (600 nm) based on the reaction with urease in alkaline medium. The concentration of chloride ions was