Physiological response of American bullfrog tadpoles to stressor conditions of capture and hypoxia

Pesquisa Veterinária Brasileira

Antonucci, Antonio M⁴  Marcantonio, Adriana S³  Rocha, Guilherme C¹  Lauretto, Marcelo⁵  Dias, Danielle C²  Teixeira, Patrícia C²  França, Fernanda M⁴  Ferreira, Cláudia M¹ 

[1] Universidade Estadual Paulista (Unesp), Jaboticabal, Brazil;Agência Paulista de Tecnologia dos Agronegócios (APTA), Pindamonhangaba, Brazil;Instituto de Pesca, São Paulo, Brazil;Universidade de São Paulo (USP),, São Paulo

关键词: Frogculture;    stress;    cortisol;    Lithobates catesbeianus;    frog;    amphibian.;   

DOI  :  10.1590/S0100-736X2010001000014

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

The American bullfrog (Rana catesbeiana), recently named Lithobates catesbeianus is currently farmed for commercial purposes throughout various regions of Brazil. Stressful situations such as problems of management, inadequate facilities and environmental changes with consequent reduction of immunity are common in intensive production. The assessments of these situations of stress allow us detect these problems decreasing the injuries causes by confinement. The main objective of this study was to use the biological markers of plasma cortisol and glucose level and hematological parameters to evaluate the response of bullfrog tadpoles submitted to stressed mechanisms of capture and hypoxia. The animals were subjected to three treatments: stress due to individual capture with a hand net; stress due to batch capture with a hand net; and stress due to capture by emptying. The results obtained demonstrated that there were no statistically significant differences in the parameters tested when comparing the treatments with and without exposure to air (normoxia and hypoxia). Based on these results we can conclude that the stressful stimuli tested were not adequate to alter the biomarker tested. For the cortisol, probably this should have occurred due to the synergistic action between this hormone and thyroxin, which induces metamorphosis in these animals.INDEX TERMS: Frogculture, stress, cortisol, Lithobates catesbeianus, frog, amphibian.RESUMOA rã-touro americana (Rana catesbeiana) recentemente denominada Lithobates catesbeianus é criada com propósito comercial em várias regiões do Brasil. Situações estressantes tais como problemas de manejo, criação inadequada e alterações ambientais com consequente redução da imunidade são comuns em produções intensivas. A avaliação destas situações de estresse permite-nos detectar estes probemas e diminuir as injurias causadas pelo confinamento. O principal objetivo deste estudo foi utilizar os marcadores biológicos de cortisol, glicemia e dados hematológicos para avaliar a resposta de girinos de rã-touro submetidos aos mecanismos estressores de captura e hipóxia. Os animais foram distribuídosem três tratamentos: estresse por captura individual com puçá; estresse por captura em massa com puçá e estresse por captura por escoamento. Os resultados obtidos demostraram não haver diferenças estatisticamente significativas entre os parametros avaliados quando comparou-se os grupos com e sem exposição ao ar (normoxia e hipoxia). Com base nestes resultados pode-se concluir que os estímulos estressores avaliados não foram adequados para alterar os valores plasmáticos dos marcadores biológicos testados. Para o cortisol, isto ocorreu provavelmente em virtude da ação sinérgica deste hormônio e a tiroxina, que induz a metamorfose nestes animais.TERMOS DE INDEXAÇÃO: Ranicultura, estresse, cortisol, Lithobates catesbeianus, rã, anfíbios.     IntroductionThe American bullfrog (Rana catesbeiana), recently named Lithobates catesbeianus (Frost et al. 2006), was introduced to Brazil in 1935 and became a farmed commodity to this day. When compared to native Brazilian anurans, the American bullfrog has several zootechnical advantages including ease of handling, rapid growth, high fecundity and the fact that it is rarely affected by epidemic-type pathological causes of death (Ferreira et al. 2002). However, various factors can lead to undesirable physiologic responses, such as temperature, water quality, age, physiological conditions, social circumstances, different species and strains, abrupt or extreme changes in physical environment, social interactions and human interference (including physical, prophylactic and sanitary handling, and inadequate feeding) (Wendelaar Bonga 1997, Ferreira et al. 2001). These factors, individually or in unison, can cause stress to the organisms with regard to reproductive dysfunction, growth and health, for example. Stress can be defined as a condition in which the dynamic equilibrium of the animal (homeostasis) is disturbed, as a result of an intrinsic and/or extrinsic stimulus, called a stressor). Animals submitted to stressful conditions can undergo adaptations to a perturbed homeostasis by compensatory physiological changes (Wendelaar Bonga 1997). We can be divided the response to stress into three steps. In the first step, there is the activation of brain system center, which culminates in an intense release of catecholamines and corticoids (mainly cortisol). In the second step, the immediate actions and effects of these hormones occur which are released into the blood stream and tissues. In the third step, the states of exhaustion are characterized which cause a fall in performance and decrease in resistance to diseases (Barcelos et al. 2000). The latter can lead to infection of the animal by opportunistic agents, including bacteria and fungi that could potentially lead to death (Barton & Iwama 1991). Some indicators of stress can be detected in the blood. Plasma cortisol and glucose, for example, are the markers most utilized today and are thought to be accurate indicators of stress. Cortisol was chosen as a biological marker in this study because it is a hormone used in routine diagnostic tests. It is therefore easily accessible and cheaper than other available markers at this time (e.g. corticosterone).Cortisol or hydrocortisone is a glucocorticoid that is related to metabolism, inflammation and stress (Voet et al. 2000). It exerts an effect on various functions of the body, among them are effects on the metabolism of carbohydrates, proteins and fats. When in excess, it causes a decrease in muscle protein, immune function, performance of the animal, and at the same time disrupts hydromineral balance and increases levels of hepatic glycogen and fatty acids. Glucose is a metabolite whose main biological function is to serve as an energy substrate for cells. It is stored in the form of glycogen in the liver. In situations where blood cortisol levels are high, gluconeogenesis occurs, that is, hepatic cells convert proteins and glycerol into glucose (Feder & Burggren 1992).There are not many scientific reports on stress in captive amphibians such as L. catesbeianus, but these physiological processes probably also occur in this type of animal. Based on experimental studies on the behavior of frogs Duellman & Trueb (1994) reported that these animals are easily stressed. According to Ferreira et al. (2001), stress can be the cause of one of the main problems in frog farming diseases. In various phases of development, characteristics as a consequence of exposure to stress factors can be observed. Symptoms most often noted in tadpoles include a lack of appetite, lethargy and disorientation, among others. In the pre-growing and growing phases, the animals pile on each other in the corners of the pens and jump in a disoriented manner. Additionally, excess skin is seen in moist areas and sometimes mucus can be observed in the form of froth. In the reproductive period, females, when stressed, abort and produce characteristic cries, while males quit croaking (Lima & Agostinho 1992, Ferreira et al. 2001).The aim of the present study was to evaluate the physiological response of bullfrog tadpoles (Lithobates catesbeianus) to stress caused by different means of capture and by hypoxia. Plasma cortisol and glucose and hematological parameters were determined.  Material and methodsBullfrog tadpoles (Lithobates catesbeianus) in the pro-metamorphosis stage were obtained from the Frog Farming of the Aquacultural Center of São Paulo State University, Jaboticabal, SP (CAUnesp). The tadpoles utilized in the collections had a mean weight of 9.05±1.69g and mean length of 11.96 ±1.31cm. For estimate values form control group the blood was drawn from each individual tadpole upon immediate removal from their original tanks. Following transportation to the lab, animals were acclimated for 7 days in polyethylene tanks (500 liters), at a density of 1 tadpole/liter at the Experimental Frog Farming of Aquacultural Center (PRDTAVP), Agricultural Department of São Paulo State, Pindamonhangaba, Brazil. These tanks were conditioned using an agricultural oven, and the surface was covered with polyethylene plastic and the sides with nylon screen. The treatments performed were: stress due to individual capture with a hand net (Treatment 1); stress due to batch capture with a hand net (Treatment 2); and stress due to capture by emptying (Treatment 3), which is the handling traditionally used in Brazil frog farming. Each treatment was carried out in simultaneous triplicate in a random order, and 12 individuals were used (6 normoxia - blood collected immediately, and 6 hypoxia - blood collected after 15 min exposure to air). Time to hypoxia was determined through preliminary tests.There were two sets of collections with an interval of 5 days between them (totaling 72 tadpoles for the whole study). Following exposure to each of the stress inducing conditions, prior to the collection of blood, animals were placed in water for 30 min to facilitate the release of cortisol. Results of the blood samples obtained at the end of the study were compared those samples collected before the onset of the study.The animals that eventually died during the study period were removed from the tanks in the morning so as not to degrade the quality of the water. Also at this time, the animals were fed rations containing 45% crude protein, 6% crude fiber 9% ether extract, once a day, in an amount of 1% of live weight. The water of each tank was changed completely at various intervals throughout the day (continuous flow system). The ambient temperature, relative humidity of the air and physical-chemical parameters of the water (temperature, electrical conductivity, pH, oxygen) was monitored daily. Total ammonia and nitrite were measured weekly.For physiological analyses, an aliquot of blood was obtained from the rupture of the caudal vessel with the aid of disposable needles and heparinized tips, after the application of Lidocaine for local anesthesia of tails of the tadpoles. In accordance with the circadian rhythm of the animals, blood collections took place in the morning hours. The animals were subsequently anesthesiated with benzocaine (1:10) and sacrificed. The cortisol and glucose levels were determined in the plasma obtained by centrifugation. Cortisol was assayed by an ELISA method (Active-Cortisol EIA DSL10, Diagnostic System Labs USA) with limit of detection of 0.1ìg/dL and coefficient of variation for intra- and inter-assay precision of 5.9 and 8.7%, respectively. Glucose was determined by means of the LABTEST® kit (GLUCOSE PAP Liquiform).The hematological parameters determined and the methods used were those recognized internationally: total number of erythrocytes (RBC) counted in a Neubauer chamber using Hayem's solution as diluent; hematocrit (Ht), hemoglobin (Hb) total and differential leukocyte counts in blood smears stained with May-Grünwald and Giemsa.Physiological and hematological variables were statistically described. After normality (D'Agostino-Pearson) and homocedasticity (Bartlett's test) verifications, a two-way analysis of variance was conducted (both factors fixed), followed by a Tu

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