Physiology & Behavior 128 (2014) 232–236
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Puberty influences stress reactivity in female catfish Rhamdia quelen Leonardo J.G. Barcellos a,⁎, Viviane M. Woehl b, Gessi Koakoski c, Thiago A. Oliveira c, Daiane Ferreira c, João Gabriel S. da Rosa c, Murilo S. de Abreu c, Rosmari Mezzalira Quevedo a, Michele Fagundes a a b c
Universidade de Passo Fundo (UPF), Programa de Pós-Graduação em Bioexperimentação, Campus I, Bairro São José, Caixa Postal 611, Passo Fundo, RS, Brazil Universidade Federal de Santa Catarina (UFSC), Centro de Ciências Biológicas, Departamento de Morfologia, Campus Universitário Reitor João David Ferreira Lima, Trindade, Florianópolis, SC, Brazil Universidade Federal de Santa Maria (UFSM), Programa de Pós-Graduação em Farmacologia, Campus Universitário, Camobi, Santa Maria, RS, Brazil
H I G H L I G H T S • • • • •
Sexual maturation alters stress reactivity in mammals and salmonid fish. Puberty seems to be the key event promoting these changes. Prepubertal female Rhamdia quelen presented a protracted stress response. Puberty seems to be the key event in stress reactivity changes in Rhamdia quelen. These changes have physiological and productive implications.
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Article history: Received 12 August 2013 Received in revised form 9 October 2013 Accepted 5 February 2014 Available online 15 February 2014 Keywords: Silver catfish Puberty Stress reactivity Reproductive cycle HPA responsiveness
a b s t r a c t We investigated a group of Rhamdia quelen females during their entire first reproductive cycle and beginning of the 2nd cycle by evaluating the stress response at different phases of gonadal maturation. In mammals, including humans, pubertal development modulates stress response reactivity due to the maturation of the neuroendocrine stress axis. These shifts in the stress reactivity were also detected in salmonid fishes. This effect comes from changes in the sensitivity of the stress axis glands or in the capacity of the adrenal tissue to synthesise glucocorticoids. Here, for the first time, we show that similar to mammals and salmonid fishes, pre-pubertal female R. quelen exhibit a protracted stress response compared to adult fish, pointing to puberty as a key event on HPI axis modulation. © 2014 Elsevier Inc. All rights reserved.
1. Introduction The effect of life stage on the responsiveness of fish to stressors has been previously described in the scientific literature. These former studies have focused on the effects of different phases of the reproductive cycle of adult fish on the neuroendocrine stress axis responsiveness and/or on sex related differences in salmonid fish [1–4]. Past research has focused also on ontogenic aspects, aiming to determine the age at which fish become responsive to stress [5–7] or the phases at which fish are particularly sensitive [8]. To our knowledge, no studies have pointed to puberty as a key event on changes in the stress axis reactivity in non-salmonid fish as the bagrid Rhamdia quelen. ⁎ Corresponding author. Tel.: +55 54 316 8100; fax: +55 54 316 8487. E-mail addresses:
[email protected] (L.J.G. Barcellos),
[email protected] (V.M. Woehl),
[email protected] (G. Koakoski),
[email protected] (T.A. Oliveira),
[email protected] (D. Ferreira),
[email protected] (J.G.S. da Rosa),
[email protected] (M.S. de Abreu),
[email protected] (M. Fagundes).
http://dx.doi.org/10.1016/j.physbeh.2014.02.023 0031-9384/© 2014 Elsevier Inc. All rights reserved.
As in mammals, including humans [9,10], both the magnitude and the duration of the hormonal stress response change dramatically throughout a fish's life span. For example, fingerlings and juveniles attained peak cortisol earlier when compared to adult fish [11]. These differences are mainly related to fish age and not to fish size or weight [12]. Few studies have compared the reactivity of the neuroendocrine stress axis in prepubertal and adult mammals and concluded that prepubertal animals have a significantly prolonged hormonal stress response compared with adults [13–16]. Although the stress responsiveness in fish of different ages has been characterised [11,12] as well in salmonid fish across their reproductive cycles [1–4], little is known about how stress reactivity changes during female sexual maturation, especially before and after puberty in other fish species. We investigated a group of jundiá (R. quelen) females during their first and second reproductive cycles to identify possible differences in the responsiveness of the stress axis during their ovarian cycle, especially before and after puberty. This species is a nocturnal Heptapteridae
L.J.G. Barcellos et al. / Physiology & Behavior 128 (2014) 232–236
bagrid fish important in fishery production in southern South America. It shares several similarities with other families of fish and thus might be used as a research model for other bagrid fish throughout the world. 2. Materials and methods 2.1. Ethical note This study was approved by the Ethics Commission for Animal Use (CEUA) of Universidade de Passo Fundo, UPF, Passo Fundo, RS, Brazil (Protocol#3/2011-CEUA, July 2009) and met the guidelines of the Brazilian College for Animal Experimentation (COBEA; http://www.cobea.org.br).
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their first spawning, at the onset of the second vitellogenesis period and after their second spawning (see Fig. 1) totalizing seven sample times. In each of these sample times, we sampled 30 females. First, six females were measured for testosterone (T) and 17β-estradiol (E2) and for the pre-stress concentration of serum cortisol. Then, we performed a protocol that had been previously reported to induce stress in R. quelen [18]: active persecution with a pen net for 120 s. We then sampled six females at 1, 4, and 24 h post stress. For each phase, three females were weighed and then killed by spinal section and decapitation, for histological examination and gonadosomatic index (GSI) calculation. The GSI was calculated as the weight of the gonads divided by the total body weight times 100.
2.4. Analysis 2.2. Subjects We examined a group of 250 R. quelen females at 60 days of age (20.8 ± 3.1 g). Fish were followed during their first reproductive cycle from May to December 2011 until the onset of their second cycle period in September and December 2012, under natural photoperiod varying from 14 h light:10 h dark to 10 h light:14 h dark. They were distributed in seven 15,000-L concrete tanks that were 1 m deep (35 fish per tank). This independent group distribution aimed to prevent cumulative effects of stress induced by sequential sampling. In each tank, five adult males (325.8 ± 25 g) were introduced when the females reach 200 g of mean weight, to permit natural progression through the reproductive cycle. The tanks were observed twice a day to check for group spawning and the presence of fry and larvae at the surface. The spawning behaviour of R. quelen was associated with mild agitation in the tank and some of the fish swimming near the water surface [17]. The water flow rate was 6 L/min, the level of dissolved oxygen varied between 5.0 and 7.0 mg/L, and the pH varied between 7.0 and 7.2. The fish were fed once a day ad libitum with commercial fish pellets (30% crude protein). 2.3. Study strategy and sample protocol Our investigative strategy consisted of evaluating the acute stress response in a group of R. quelen females cultured in the same conditions. We evaluated the cortisol response two times before puberty, during early vitellogenesis, during late vitellogenesis in mature females, after
For blood sample collection, buffered MS222 (Finquel®; Argent Chemical Laboratories, Redmond, WA, USA, 300 mg/L) was applied as an anaesthetic prior to the withdrawal of 1 to 2 mL of blood from the caudal vein into heparinised syringes. The collected blood samples were centrifuged for 10 min at 10,000 ×g and stored at −20 °C. Cortisol, E2 and T levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (EIAgen ™ Cortisol Test, EIAgen™ Estradiol and EIAgen™ Testosterone; BioChem ImmunoSystems) previously validated for R. quelen serum and plasma samples [12,17]. Both ovaries were removed, weighed (0.001 g), and fixed in Bouin's solution for 24 h for histological examination. Tissues were embedded in Paraplast, cut into 5-mm sections, and stained with hematoxylin– eosin. The reproductive stages were defined according to Barcellos et al. [17].
2.5. Statistics The mean ± SEM values for each group were calculated using the GraphPad Prism statistical package (GraphPad Software, San Diego, CA, USA). Testosterone and E2 concentrations for each group were compared using one-way analysis of variance (ANOVA) followed by Tukey's test. Cortisol concentrations were compared using a two-way ANOVA, with ovarian development stages and time after stressor as factors, followed by Bonferroni post-test to discriminate means of column (ovarian development stages) factor. Within each time course curve, the different time points were compared by one-way ANOVA followed by Tukey's test.
Fig. 1. Schematic view of the experimental design.
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Homogeneity of variance was determined using Hartley's test, and normality was determined using the Kolmogorov–Smirnov test. Differences with P values b0.05 were considered statistically significant.
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3.3. Cortisol concentrations There were significant differences in the response to acute stress related to ovarian development stage (P b 0.0001; F6,35 = 35.42), and time after acute stressor (P b 0.0001, F3,20 = 742.2) and there was a significant ovarian development stage ∗ time after stress interaction (P b 0.0001, F18,140 = 19.40). Cortisol levels remained low in all pre-stress measurements (Fig. 3). Cortisol levels were significantly elevated after 60 min of exposure to the standard stressor in all groups in relation to the controls. However, when we compared the seven peaks, we perceived that the values measured in both groups of pre-pubertal females were higher than in the other groups. Four hours after stress, cortisol levels of prepubertal fish with 60 days of age are higher than all other fish groups while cortisol levels of prepubertal fish with 180 days of age are higher than the subsequent ovarian development stage groups. Twenty-four hours after the stressor, all groups had returned to the basal pre-stress levels, without differences between females of different ovarian development stages. Within the time course curves, only in prepubertal fish with 180 days
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