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Short-term effects of 17β-estradiol on physiological responses in stellate sturgeon Acipenser stellatus
Short-term effects of 17β-estradiol on physiological responses in stellate sturgeon Acipenser stellatus Bahram Falahatkar, Samaneh Poursaeid, Bahman Meknatkhah, Iraj Efatpanah PII: DOI: Reference:
S1532-0456(17)30014-5 doi:10.1016/j.cbpc.2017.01.006 CBC 8278
To appear in:
Comparative Biochemistry and Physiology Part C
Received date: Revised date: Accepted date:
19 November 2016 22 January 2017 24 January 2017
Please cite this article as: Falahatkar, Bahram, Poursaeid, Samaneh, Meknatkhah, Bahman, Efatpanah, Iraj, Short-term effects of 17β-estradiol on physiological responses in stellate sturgeon Acipenser stellatus, Comparative Biochemistry and Physiology Part C (2017), doi:10.1016/j.cbpc.2017.01.006
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ACCEPTED MANUSCRIPT Short-term effects of 17β-estradiol on physiological responses in stellate sturgeon Acipenser stellatus
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a
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Bahram Falahatkar a,*, Samaneh Poursaeid b, Bahman Meknatkhah c, Iraj Efatpanah c
Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, P.O.
b
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Box 1144, Guilan, Iran
Fisheries Department, Faculty of Marine Science, Tarbiat Modares University, Noor,
Dr. Yousefpour Fish Hatchery Center, Siahkal, Guilan, Iran
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c
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Mazandaran, Iran
*
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Running title: Effect of 17β-estradiol in sturgeon
Corresponding author.
E-mail address: [email protected] (B. Falahatkar).
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ACCEPTED MANUSCRIPT ABSTRACT There is no evidence of the effect of 17β-estradiol (E2) administration on stress response and
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related metabolites after a short period in chondrostean. In this study, we examined whether E2
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is capable of inducing the hypothalamus-pituitary-interrenal axis activity. To accomplish this, sexually immature Acipenser stellatus were injected intraperitoneally with saline or E2 (5 mg/kg
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body mass) and sampled 0, 1, 3, 6, 12, 24, 48, and 72 h later. Plasma E2, cortisol, glucose,
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lactate, hematocrit (Hct), total protein (TP), cholesterol, triglyceride, alkaline phosphatase activity (ALP), calcium (Ca), and phosphorus concentrations were examined. Plasma levels of
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E2 significantly increased after 1 h in E2-injected fish and remained high after 12 h. Injection of E2 enhanced plasma TP and Ca concentrations, but had no effect on other parameters. Injection
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of either saline or E2 enhanced plasma glucose, lactate, TP, and Ca concentrations, but had no effect on plasma Hct, cholesterol, triglyceride, and ALP. The results demonstrated that acutely
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elevated level of E2 did not lead to intense changes of stress metabolites and altered biochemical changes compared to the fish in control or saline treatment groups. Therefore, we found no exhibition of E2 on the rate of cortisol synthesis or stress indicators in this species, while E2 could modulate some other related metabolites on vitellogenesis pathway. Keywords: Estrogen, Stress, Cortisol, Metabolites, Sturgeon, Acipenser stellatus
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ACCEPTED MANUSCRIPT 1. Introduction Depletion of sturgeon stocks in the native habitats, mainly the Caspian Sea, coupled with the
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strong demand of international markets for black caviar have led to the introduction of sturgeon
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species into aquaculture in which those are raised mainly for production of precious caviar. Sex control to produce all-female populations for commercial culture is of interest to aquaculturists
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(Omoto et al., 2005; Flynn and Benfey, 2007). Since sex steroids have important and distinct
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roles in modulating sex differentiation in the natural process, the basis for controlling this
2001; Devlin and Nagahama, 2002).
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process is the administration of exogenous steroids to sexually undifferentiated fish (Piferrer,
Of the sex steroids, 17β-estradiol (E2), the most important of estrogens secreted by ovaries, is
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widely used for feminization purposes. The potency of E2 in the induction of feminization has been reported in a variety of teleostean fish (Piferrer, 2001; Specker and Chandlee, 2003; Flynn
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and Benfey, 2007; Wang et al., 2008). Similarly, E2 has been shown to skew sex ratios toward female in paddlefish, Polyodon spathula (Shelton and Mims, 1998), bester (Huso huso ♀ × Acipernser ruthenus ♂) (Omoto et al., 2005), and shortnose sturgeon, Acipenser brevirostrum (Flynn and Benfey, 2007). In most studies, the chronic administration of estrogen through inclusion in the diet has been used for sex reversal. Our previous studies have shown that continuous exogenous feeding of E2 or its inclusion in a slow-release formulation had adverse effects on the growth and welfare (Khara et al., 2014; Akhavan et al., 2015). In contrast, the administration of E2 through injection into the body cavity did not result in any secondary adverse effects in juvenile stellate sturgeon, Acipenser stellatus (Falahatkar et al., 2014). Therefore, injection may be a promising alternative to former methods for the feminization of sturgeon without having any negative effects on the survival and growth performance
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ACCEPTED MANUSCRIPT (Falahatkar et al., 2014). However, the effect of E2 injection even in short term on the metabolic hormones such as cortisol and carbohydrate, lipid and calcium metabolism could not be
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excluded.
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In vertebrates, it has been demonstrated that sex steroids are responsible for hyperactivation of the hypothalamus-pituitary-adrenal axis (Jasnow et al., 2006; Handa et al., 2009). The
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involvement of E2 in the regulation of cortisol production has also been reported in some teleosts
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(Young et al., 2001; McQuillan et al., 2003; Teles et al., 2005, 2006; Lerner et al., 2007; Fuzzen et al., 2011a). However, there is no evidence for the effects of E2 administration on cortisol
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secretion and stress response in sturgeon species. The results on other species raise questions regarding the role that E2 plays in the modulation of hypothalamus-pituitary-interrenal (HPI)
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axis and physiological parameters in sturgeon as an ancient species. Previous studies have shown that long-term E2 feeding had adverse effects on growth and welfare, but it is not clear that is it
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mediated via a cortisol-dependent mechanism or not. It is possible that E2 injection might induce cortisol synthesis and secretion. It is also unknown if cortisol secreted in response to E2 exogenous can interfere with the effectiveness of this hormone in feminization. Fish are exposed to E2 in both their rearing or natural environments. Stellate sturgeon has lower puberty age of 57 years and spawning intervals of 2-3 years, and 15-20% gonadosomatic index (Chebanov and Galich, 2011). These characters make it as a good candidate for caviar production in aquaculture systems. Also in wild habitats and also during the releasing of juveniles into the rivers for stock rehabilitation program, fish are subjected to different pollutants like domestic effluents which are included natural female estrogens. Therefore, the present study aimed to access the short-term effects of E2 on juvenile immature stellate sturgeon at different levels including stress indicators and other related metabolites.
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2. Methods
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2.1. Fish and holding system
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Two hundred and sixteen stellate sturgeons, Acipenser stellatus, were used for this experiment. The fish were originally hatched at Shahid Dr. Beheshti Sturgeon Fish Propagation
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and Rearing Complex (Rasht, Guilan, Iran) from a pair of wild Caspian stock and then reared at
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Dr. Yousefpour Fish Hatchery Center (Siahkal, Guilan, Iran). During the year, the fish were held in circular concrete tanks and fed with commercial pellet (Biomar, no. 3, Nersac, France). When
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the average weight and length of fish were 281.5 ± 3.0 g and 50.0 ± 0.2 cm, respectively, they were distributed into nine circular concrete tanks (1.85 m diameter, 30 cm depth, 806 L volume),
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supplied with flow-through filtered river water at a rate of 20 ± 0.7 L/min. During 10 days of acclimation and experiment, water temperature, dissolved oxygen, and pH were 28.5 ± 0.2°C,
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6.1 ± 0.2 mg/L, and 7.7 ± 0.1, respectively.
2.2. Experimental protocol
Three experimental groups with three replicates were considered. Twenty four fish were introduced per tank. Fish received a single intraperitoneal (i.p.) injection with dose of 5 mg 17βestradiol (E2; Sigma-Aldrich, St. Luis, MO, USA) per kg body weight (BW), using sterile injectable physiological saline solution (0.9% NaCl) as a carrier with a volume of 0.5 mL/100g BW. The second groups of fish were similarly injected with physiological solution, and the third group was kept as control without any treatment and handling. The fish in two injected groups were injected with 2-mL plastic syringe (gauge 25). E2 was prepared through dissolving in 100% ethanol and then in saline solution according to Falahatkar et al. (2014).
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ACCEPTED MANUSCRIPT 2.3. Sampling For sampling, eight different time points, 0 (as resting time before injection), 1, 3, 6, 12, 24,
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48, and 72 h following the injection, were considered. At each time point, two fish were
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randomly captured from each tank. After anaesthetizing with 400 mg/L clove powder extract solution in 2 min, the blood was taken from the caudal vein by a 2-mL heparinized syringe.
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Blood sampling lasted 30 sec for each fish. The fish were returned to the recovery tanks (with the
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same volume) after sampling.
A small part of blood for each fish was transferred to two capillary microhematocrit tubes,
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and the remaining part was transferred to a tube for plasma extraction at 1500 g for 10 min. Afterwards, the separated plasma was stored at -70°C for later analysis. Hematocrit (Hct) was
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2.4. Plasma measurements
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determined by microhematocrit method at 3500 g for 7 min.
Plasma E2 and cortisol concentrations were measured by radioimmunoassay (Special kits from Immunotech, Marseille, France) based on the methods described by Pankhurst and Carrager (1992) and Pankhurst et al. (2008), respectively. The efficiency and inter assay variability and cortisol were 78 and 7.4% for E2 and 74 and 12.3% for cortisol, respectively. Glucose levels were determined in accordance to the colorimetric method considering the glucose-peroxidase reaction (Pars Azmun, Karaj, Iran) (Bayunova et al., 2002). Plasma lactate levels were determined enzymatically with an available commercial kit (Sigma, St. Louis, MO, USA).
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ACCEPTED MANUSCRIPT Plasma cholesterol and triglyceride concentrations were analyzed using commercial kits (Pars Azmun, Karaj, Iran) by CHOD-PAP and GPO-PAP methods, respectively (Chatzifortis et al.,
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2010). Total protein was analyzed by Biuret method (Ziestchem, Tehran, Iran).
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Plasma total calcium (Ca) and phosphorus concentrations were determined by colorimetric method, based on Sigma-Aldrich procedures no. 587 and 360, respectively. Alkaline phosphatase
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activity (ALP) was determined by means of the commercial kit instruction (Pars Azmun, Karaj,
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Iran) with auto analyzer (RA-1000, Technicon, USA).
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2.5. Statistics
Before any analysis, all data were expressed to Kolmogorov-Smirnov and Levene’s tests for
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checking the normality of data and homogeneity of variances, respectively. The data were transformed to log10 when needed. Two-way analysis of variance (ANOVA) was used to find
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the effect of different treatments and times.Tukey’s post-hoc test was used to identify the significant differences among the various means. All statistical procedures were analyzed by SPSS software (SPSS, version 13.0, Chicago, IL). The accepted significance level of P value was considered at P < 0.05. All data throughout the text are expressed as means ± standard errors of the mean (mean ± SE).
3. Results The highest concentration of E2 was measured in E2 injected fish, compared to the other groups, with a significant elevation 1h after injection, and a sharp decrease to basal level after 12h (Fig. 1). The saline and control groups had similar concentrations of plasma E2 with no changes.
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ACCEPTED MANUSCRIPT The results of cortisol levels in different treatments showed higher significant levels in injected groups, 1 h and 72 h following injection than non-injected control (Fig. 2a). Glucose
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levels increased 6 h after injection in saline and E2 groups. Also, significant differences were
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observed among the groups from the injection time to 6 h, but no changes were observed for other times (Fig. 2b). Lactate levels revealed the highest values 1 h after injection and reached to
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the basal level after 6 h in saline and E2 injected groups (Fig. 2c).
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Hct levels decreased in the saline group 48 h after injection, compared to the resting time. No significant changes were observed among the groups and throughout the sampling times (Fig. 3).
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TP concentration increased 48 h after injection for E2-treated fish, while no changes were made in other groups. Also, no significant changes were observed among the groups except at
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the end of the sampling time between E2 and saline groups (Fig. 4a). Cholesterol levels were similar among all experimental groups and during the sampling (Fig. 4b), while only E2 group
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showed the highest value of triglyceride 48 h after injection (Fig. 4c). No changes were observed in control and saline groups throughout the experiment, but the highest significant value of Ca concentration was recorded at 72 h following the injection of E2 group. Also highest concentrations were observed at this time compared to other groups (Fig. 5a). Phosphorus concentrations significantly rose 1 h after injection in saline group, but no changes were observed after 12 h among other groups (Fig. 5b). No changes were observed in ALP levels except 24 h after injection with the highest value in the saline group in comparison with the control fish (Fig. 6).
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ACCEPTED MANUSCRIPT 4. Discussion In the present study, the acute effects of E2 on the stress indicators and some other
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biochemical parameters of stellate sturgeon were investigated. Various i.p. doses of E2 were
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investigated, showing some significant physiological effects (Zaroogian et al., 2000; Teles et al., 2005, 2006; Carrera et al., 2007; Flynn and Benfey, 2007; Maria et al., 2007; Moura Costa et al.,
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2010; Falahatkar et al., 2014). The dose of 5 mg E2/kg BW in the current study was mostly
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similar to other studies and a previous study by the authors (Falahatkar et al., 2014). In the current experiment, a significant increase of E2 concentration was recognized
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following E2 injection. The increase continued 12 h after injection, and afterwards, the levels were similar to control and basement levels. Similarly, the previous study on Asian swamp eel
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(Monopteus albus) fed by E2 showed an enhanced level of plasma E2 (Yuan et al., 2011). Longterm effect of E2 in female brook trout (Salvelinus fontinalis) and beluga sturgeon caused an
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increase of plasma E2 concentration (Schafhauser-Smith and Benfey, 2003; Akhavan et al., 2015). Repeated injection of 5 mg E2/kg BW in stellate sturgeon increased the level of plasma E2 (Falahatkar et al., 2014). The current study showed that exogenous E2 caused an elevation in plasma E2 within 12 h in treated fish. Returning to pre-injection levels after 12 h suggests that exogenous E2 was likely cleared from the circulation and accumulated in some tissues. In the current study, plasma cortisol showed two different patterns of response to handling. In first phase (from 1 to 6 h after handling), a transient elevation in plasma cortisol level was observed in both saline and E2-injected groups. This increase is a typical response to handling in sturgeon fish for allowing them to cope with handling in order to maintain their homeostatic states (Barton et al., 1998, 2000; Falahatkar et al., 2009). It also suggests that injection procedure has induced an acute stress in stellate sturgeon. Interestingly, the elevated cortisol levels in the
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ACCEPTED MANUSCRIPT E2-treated fish were lower than those in the saline group, indicating that there is an interaction between the HPI and exogenous E2. It seems that exogenous E2 is capable of altering the
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endocrine stress response in stellate sturgeon. Plasma cortisol levels returned to prestress levels
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by 6 h in the saline group, while plasma levels of fish injected with E2 increased again after 6 h and were approximately maintained throughout the sampling times. The second manner of
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response following E2 injection was associated with the clearance of excess E2 from circulation.
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These observations confirm our concept in which E2 can affect the rate of cortisol synthesis in stellate sturgeon probably by acting on components of the HIP axis, as described previously by et
al.
(2011)
for
zebrafish.
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Fuzzen
However,
further
experiments
would be required to clarify the possible mechanisms involved in mediating the effect of
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exogenous E2 on the HPI axis in chondrostean fish. There are inconsistent results regarding fish stress responses to E2 treatment; Teles et al. (2004,
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2005) showed a decrease in plasma cortisol following E2 exposure in immature gilthead sea bream (Sparus aurata) and juvenile seabass (Dicentrarchus labrax).
While, other studies
reported elevated levels of cortisol and adrenocorticotropic hormone in salmonids (Pottinger et al., 1996) and gilthead sea bream (Carrera et al., 2007) after exposure to E2. Similar to our results, i.p. injection method of E2 to tilapia, Oreochromis mossambicus (Vijayan et al., 2001), and Atlantic salmon, Salmo salar (McCormick et al., 2005) showed no significant changes. These contrary findings may be explained by different administration methods of E2, species specificity of response to E2 injection, anatomy of interrenal tissue and sampling times following E2 exposure. The mechanism involved in such a response in such a time is not clear, but it is clear that such changes do not occur in a short period of 48 h following E2 injection.
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ACCEPTED MANUSCRIPT Considering the glucose as a secondary response to stressors, the injection stress stimulated the releasing of glucose in short-term injection of E2 or saline to 6 h, while no changes were
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made in the control group. The highest value of glucose after stress was recorded in many
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sturgeons after 3-6 h (Barton et al., 2000; Falahatkar et al., 2009; Falahatkar and Poursaeid, 2013; Eslamloo and Falahatkar, 2014), which is similar to the current work. It seems that E2
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cannot induce the metabolic changes of gluconeogenesis or glycogenolysis, and these pathways
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are affected by injection and handling stressors to supply the energy demand. However, given the more clear alignment between the time frame of the plasma glucose increase and decrease of
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circulating E2, possibly the response is related to the energy demands of metabolic removal of the injected E2, rather than to the primary stress response. Exposure of E2 in sea bass showed
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induction of plasma glucose increase (Teles et al., 2004), but not in gilthead sea bream (Teles et al., 2005) or sea bass (Teles et al., 2006). A significant increase was observed in stellate sturgeon
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for glucose concentration when injected with 5mg E2/kg BW (Falahatkar et al., 2014). Since glucose is an important product of gluconeogenesis process for different activities to cope with energy needs, such changes in the study were just affected by handling, and E2 could not affect carbohydrate mechanism for glucose transmission or absorption (Mommsen et al., 1999) as fuel energy.
In the present study, although the level of lactate changed through the trial with the highest value 1 h after injection, these changes are correlated with stress but not E2 injection. Lactate levels in juvenile sea bass remained unchanged after E2 exposure (Teles et al., 2006); however, another study on gilthead sea bream showed increasing of lactate level (Teles et al., 2005). Different administration methods or exposure times are possible reasons for such results.
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ACCEPTED MANUSCRIPT Generally, stress can stimulate to release primary (cortisol) and secondary (glucose and lactate) responses of the fish (Wendelaar Bonga, 1997; Pankhurst, 2011). In this study, despite
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cortisol change in fish injected E2, 72 h following injection, the responses were directly affected
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by stress, and E2 could not mobilize stress substrates such as glucose and lactate as fuel energy. Considering different sampling times, no significant differences were observed in Hct values,
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but the lowest level was observed 48 h after injection in fish treated with saline compared to the
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resting time. Although Hct value varies after different handling procedures, it was observed that i.p. administration has not induced or reduced the rate of erythropoiesis or even the blood
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volume. Previous studies revealed that dietary administration of E2 in triploid female brook trout causes Hct decline (Schafhauser-Smith and Benfey, 2003). Similar decrease was observed in
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stellate sturgeon when fed by different levels of E2 (25 or 50 mg/kg diet) or i.p. injected fish by 5 mg/kg BW (Khara et al., 2014; Falahatkar et al., 2014). Previous studies demonstrated that the
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administration of E2 has an inhibitory effect on erythropoiesis, but the present inquiry revealed that this way of administration has no effect (at least in short time) on blood index or being affected by the handling stressor.
At the end of the sampling, 48 and 72 h after E2 injection, the fish showed higher TP compared to the resting time. Similar effect of E2 was observed in plasma TP in red sea bream, Chrysophrys major (Woo et al., 1993), and stellate sturgeon (Falahatkar et al., 2014). This increase might be due to the role of E2 in stimulating vitellogenic process and mobilization of different lipoproteins in blood circulation (Sharpe and MacLatchy, 2007). In addition, the results revealed no cholesterol changes among the treatments and during the experiment, but significant elevation of triglyceride was observed in E2-injected fish 48 h following injection. Hyperlipidimic effect was observed in rainbow trout, Oncorhynchus mykiss
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ACCEPTED MANUSCRIPT (Wallaert and Babin, 1992), goldfish, Carassius auratus (Sharpe and MacLatchy, 2007), stellate sturgeon (Falahatkar et al., 2014), and beluga sturgeon (Akhavan et al., 2015) when exposed to
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E2. In our study, the short period of E2 exposure with a single dose of injection could not
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provide similar circulating hyperlipidemia. Also, no changes in cholesterol and triglyceride levels in control and saline-injected fish were demonstrated, without handling effects in blood
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lipid sources, but the induction of triglyceride was observed at the end of the sampling times.
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In the current study, elevated level of plasma Ca was observed in E2-treated fish compared to other treatments and basal level after 72 h. The level of phosphorus 1 h after exposure was
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affected by E2 and saline treatments, but the trends were not clear during the sampling times. Previous observations have revealed that E2 can stimulate vitellogenesis which is accompanied
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by an enhance in plasma Ca and phosphorus (Persson et al., 1998; Guerreiro et al., 2002; Linares-Casenave et al., 2003; Akhavan et al., 2015). It is clearly demonstrated that vitellogenin
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is a calcium-binding lipophosphoprotein, and E2 is implicated in regulation and elevation of plasma Ca through an increase in its uptake from water or mobilization from internal sources of Ca such as scutes or cartilage on sturgeons. Therefore, the elevation of Ca in E2-treated fish might indicate vitellogenin synthesis in juvenile stellate sturgeon. ALP activity was unaltered through the experiment and among the treatments except for the 24 h with the lowest level in control group. The results of hepatic enzymes in gilthead seabream showed further reduction compared to the control or non-injected fish (Carrera et al., 2007). Most previous studies also show depletion of hepatic metabolizing enzymes by estrogens or estrogen-like compounds (Vaccaro et al., 2005; Teles et al., 2005). Although the levels of hepatic enzymes showed damage to hepatic tissue, the unchanged results of ALP activity revealed no
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ACCEPTED MANUSCRIPT effect of different treatments. Higher activity of ALP 24 h after injection demonstrates the ability of such stress procedure at this delayed time.
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Our previous results demonstrated all feminization of stellate sturgeon which was
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periodically injected with E2 (Falahatkar et al., 2014), but no data was collected in this study regarding to the single injection of this hormone. The simulatory manipulation of plasma E2
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concentrations used in our study proved a valuable tool for finding the effects of E2 on stress
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response and biochemical parameters. We compared the response of E2-injected fish with control and saline-injected fish at rest and different times following injection. All the other
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parameters in TP, triglyceride, Hct, and Ca are very episodic, they concern mainly the 48 h (or the 72 h) samples, suggesting that there might be some specific changes in these samples.
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Although i.p. injections are not the most revealed mode of exposure in environment, but it is an effective way to provide a means of determining whether they are relevant for fish (Moura Costa
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et al., 2010; Falahatkar et al., 2014). Also, treatment with pellet application to achieve longer and physiologically more realistic profiles of elevated E2 can be further investigated.
5. Conclusions
In conclusion, stress responses and metabolite parameters after administration of different treatments showed some related changes in glucose and lactate as a fuel sources of stress response which they are not related to E2-induction, while E2 treated fish exhibited elevation of E2, TP, triglyceride, and Ca concentrations in the juvenile stellate sturgeon. Therefore, it is demonstrated that some blood indices can be modulated by E2 injection for a short time not exactly by handling stressor, but E2 cannot change the response of HPI axis in terms of secondary stress response, at least. E2 injection in saline is an effective way of getting a short-
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ACCEPTED MANUSCRIPT lived but high level increase in plasma E2, and that other than perhaps cortisol, it does not perturb physiological response for too long and that the possible effect on cortisol might need
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some extra study. Further research is needed to find how E2 acts as a modulator in positive of
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negative feedbacks of the stress. In fact, there is a need for immature fish to access the effects of E2 exposure regarding genotoxic response, antioxidants status, immune system, hepatic
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condition indicators, biotransformation, and the difference between two sexes.
Acknowledgements
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The authors sincerely thank staff at the Dr. Yousefpour Fish Hatchery Center, for kindly supplying fish and facilities. We also thank to NW Pankhurst for performing the steroid analyses
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ACCEPTED MANUSCRIPT Lerner, D.T., Bjornsson, B.T., McCormick, S.D., 2007. Aqueous exposure to 4-nonlphenol and
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ACCEPTED MANUSCRIPT Omoto, N., Maebayashi, M., Mitsuhashi, E., Yoshitomi, K., Adachi, S., Yamauchi, K., 2002. Effects of estradiol-17β and 17α-methyltestosterone on gonadal sex differentiation in the F2
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ACCEPTED MANUSCRIPT Sharpe, R.L., MacLatchy, D.L., 2007. Lipid dynamics in goldfish (Carassius auratus) during a period of gonadal recrudescence: Effects of β-sitosterol and 17β-estradiol exposure.
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Specker, J.L., Chandlee, M.K., 2003. Methodology for estradiol treatment in marine larval and
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endocrine and genotoxic responses to β-naphthoflavone, 4-nonylphenol and 17β-estradiol: comparison of individual and combined chemical exposures. Chemosphere 57, 147–158.
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Teles, M., Pacheco, M., Santos, M.A., 2005. Sparus aurata L. liver EROD and GST activities, plasma cortisol, lactate, glucose and erythrocytic nuclear anomalies following short-term
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exposure either to 17β-estradiol (E2) or E2 combined with 4-nonylphenol. Science Total of Environment 336, 57–69.
Teles, M., Pacheco, M., Santos, M.A., 2006. Biotransformation, stress and genotoxic effects of 17β-estradiol in juvenile sea bass (Dicentrarchus labrax L.). Environment International 32, 470–477.
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ACCEPTED MANUSCRIPT Vijayan, M.M., Takemura, A., Mommsen, T.P., 2001. Estradiol impairs hyposmoregulatory capacity in the euryhaline tilapia, Oreochromis mossambicus. American Journal of
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Wallaert, C., Babin, P.J., 1992. Effects of 17β-estradiol and starvation on trout plasma lipoproteins. Lipids 27, 1032–1041.
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Wang, H.P., Gao, Z., Beres, B., Ottobre, J., Wallat, G., Tiu, L., Rapp, D., O'Bryant, P., Yao, H.,
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2008. Effects of estradiol-17β on survival, growth performance, sex reversal and gonadal structure of bluegill sunfish Lepomis macrochirus. Aquaculture 285, 216–223.
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Wendelear Bonga, S.E., 1997. The stress response in fish. Physiol. Rev. 11, 591–625. Woo, N.Y.S., Chung, A.S.B., Ng, T.B., 1993. Influence of oral administration of estradiol-17β
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and testosterone on growth, digestion, food conversion and metabolism in the underlying read sea bream, Chrysophrys major. Fish Physiology and Biochemistry 10, 377–387.
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Young, E.A., Altemus, M., Parkison, V., Shastry, S., 2001. Effects of estrogen antagonists and agonists on the ACTH response to restraint stress in female rats. Neuropsychopharmacology 25, 881–891.
Yuan, H.W., Xu, Q.Q., Gong, S.Y., Yuan, Y.C., Chu, Z.J., Yang, D.Q., 2011. Effects of different exogenous estradiol contents on steroid hormones, GSI, survival rate and sex reversal in the Asian swamp eel. Advance Marine Research 382, 481–485. Zaroogian, G., Gardner, G., Borsay Horowitz, D., Gutjahr-Gobell, R., Haebler, R., Mills, L., 2001. Effect of 17bestradiol, o, p_-DDT, octylphenol and p, p_-DDE on gonadal development and liver and kidney pathology in juvenile male summer flounder (Paralichthys dentatus). Aquatic Toxicology 54, 101–112.
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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Plasma 17β-estradiol in juvenile stellate sturgeon Acipenser stellatus at the
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control, saline and 17β-estradiol injected groups at rest and different times following
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intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Lower
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cases indicate significant differences in an experimental group over time (ANOVA followed by Tukey’s post hoc test, P<0.05), and capital letters indicate significant differences among treatments for a given time (ANOVA followed by Tukey’s post
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hoc test, P < 0.05).
Fig. 2. Plasma concentrations of cortisol (a), glucose (b) and lactate (c) in juvenile
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stellate sturgeon Acipenser stellatus at the control, saline and 17β-estradiol injected
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groups at rest and different times following intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Lower cases indicate significant differences in an
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experimental group over time (ANOVA followed by Tukey’s post hoc test, P < 0.05), and capital letters indicate significant differences among treatments for a given time
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(ANOVA followed by Tukey’s post hoc test, P < 0.05).
Fig. 3. Hematocrit values in juvenile stellate sturgeon Acipenser stellatus at the control, saline and 17β-estradiol injected groups at rest and different times following intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Lower cases indicate significant differences in an experimental group over time (ANOVA followed by Tukey’s post hoc test, P < 0.05).
Fig. 4. Plasma concentrations of total protein (a), cholesterol (b) and triglyceride (c) in juvenile stellate sturgeon Acipenser stellatus at the control, saline and 17β-estradiol
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given time (ANOVA followed by Tukey’s post hoc test, P < 0.05).
Fig. 5. Plasma concentrations of total calcium (a) and phosphorus (b) in juvenile
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stellate sturgeon Acipenser stellatus at the control, saline and 17β-estradiol injected
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groups at rest and different times following intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Lower cases indicate significant differences in an
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experimental group over time (ANOVA followed by Tukey’s post hoc test, P < 0.05),
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(ANOVA followed by Tukey’s post hoc test, P<0.05).
Fig. 6. Plasma alkaline phosphatase activity (ALP) in juvenile stellate sturgeon
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Acipenser stellatus at the control, saline and 17β-estradiol injected groups at rest and different times following intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Capital letters indicate significant differences among treatments for a given time (ANOVA followed by Tukey’s post hoc test, P < 0.05).
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S1532-0456(17)30014-5 doi:10.1016/j.cbpc.2017.01.006 CBC 8278
To appear in:
Comparative Biochemistry and Physiology Part C
Received date: Revised date: Accepted date:
19 November 2016 22 January 2017 24 January 2017
Please cite this article as: Falahatkar, Bahram, Poursaeid, Samaneh, Meknatkhah, Bahman, Efatpanah, Iraj, Short-term effects of 17β-estradiol on physiological responses in stellate sturgeon Acipenser stellatus, Comparative Biochemistry and Physiology Part C (2017), doi:10.1016/j.cbpc.2017.01.006
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ACCEPTED MANUSCRIPT Short-term effects of 17β-estradiol on physiological responses in stellate sturgeon Acipenser stellatus
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a
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Bahram Falahatkar a,*, Samaneh Poursaeid b, Bahman Meknatkhah c, Iraj Efatpanah c
Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, P.O.
b
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Box 1144, Guilan, Iran
Fisheries Department, Faculty of Marine Science, Tarbiat Modares University, Noor,
Dr. Yousefpour Fish Hatchery Center, Siahkal, Guilan, Iran
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c
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Mazandaran, Iran
*
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Running title: Effect of 17β-estradiol in sturgeon
Corresponding author.
E-mail address: [email protected] (B. Falahatkar).
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ACCEPTED MANUSCRIPT ABSTRACT There is no evidence of the effect of 17β-estradiol (E2) administration on stress response and
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related metabolites after a short period in chondrostean. In this study, we examined whether E2
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is capable of inducing the hypothalamus-pituitary-interrenal axis activity. To accomplish this, sexually immature Acipenser stellatus were injected intraperitoneally with saline or E2 (5 mg/kg
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body mass) and sampled 0, 1, 3, 6, 12, 24, 48, and 72 h later. Plasma E2, cortisol, glucose,
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lactate, hematocrit (Hct), total protein (TP), cholesterol, triglyceride, alkaline phosphatase activity (ALP), calcium (Ca), and phosphorus concentrations were examined. Plasma levels of
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E2 significantly increased after 1 h in E2-injected fish and remained high after 12 h. Injection of E2 enhanced plasma TP and Ca concentrations, but had no effect on other parameters. Injection
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of either saline or E2 enhanced plasma glucose, lactate, TP, and Ca concentrations, but had no effect on plasma Hct, cholesterol, triglyceride, and ALP. The results demonstrated that acutely
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elevated level of E2 did not lead to intense changes of stress metabolites and altered biochemical changes compared to the fish in control or saline treatment groups. Therefore, we found no exhibition of E2 on the rate of cortisol synthesis or stress indicators in this species, while E2 could modulate some other related metabolites on vitellogenesis pathway. Keywords: Estrogen, Stress, Cortisol, Metabolites, Sturgeon, Acipenser stellatus
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ACCEPTED MANUSCRIPT 1. Introduction Depletion of sturgeon stocks in the native habitats, mainly the Caspian Sea, coupled with the
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strong demand of international markets for black caviar have led to the introduction of sturgeon
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species into aquaculture in which those are raised mainly for production of precious caviar. Sex control to produce all-female populations for commercial culture is of interest to aquaculturists
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(Omoto et al., 2005; Flynn and Benfey, 2007). Since sex steroids have important and distinct
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roles in modulating sex differentiation in the natural process, the basis for controlling this
2001; Devlin and Nagahama, 2002).
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process is the administration of exogenous steroids to sexually undifferentiated fish (Piferrer,
Of the sex steroids, 17β-estradiol (E2), the most important of estrogens secreted by ovaries, is
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widely used for feminization purposes. The potency of E2 in the induction of feminization has been reported in a variety of teleostean fish (Piferrer, 2001; Specker and Chandlee, 2003; Flynn
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and Benfey, 2007; Wang et al., 2008). Similarly, E2 has been shown to skew sex ratios toward female in paddlefish, Polyodon spathula (Shelton and Mims, 1998), bester (Huso huso ♀ × Acipernser ruthenus ♂) (Omoto et al., 2005), and shortnose sturgeon, Acipenser brevirostrum (Flynn and Benfey, 2007). In most studies, the chronic administration of estrogen through inclusion in the diet has been used for sex reversal. Our previous studies have shown that continuous exogenous feeding of E2 or its inclusion in a slow-release formulation had adverse effects on the growth and welfare (Khara et al., 2014; Akhavan et al., 2015). In contrast, the administration of E2 through injection into the body cavity did not result in any secondary adverse effects in juvenile stellate sturgeon, Acipenser stellatus (Falahatkar et al., 2014). Therefore, injection may be a promising alternative to former methods for the feminization of sturgeon without having any negative effects on the survival and growth performance
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ACCEPTED MANUSCRIPT (Falahatkar et al., 2014). However, the effect of E2 injection even in short term on the metabolic hormones such as cortisol and carbohydrate, lipid and calcium metabolism could not be
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excluded.
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In vertebrates, it has been demonstrated that sex steroids are responsible for hyperactivation of the hypothalamus-pituitary-adrenal axis (Jasnow et al., 2006; Handa et al., 2009). The
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involvement of E2 in the regulation of cortisol production has also been reported in some teleosts
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(Young et al., 2001; McQuillan et al., 2003; Teles et al., 2005, 2006; Lerner et al., 2007; Fuzzen et al., 2011a). However, there is no evidence for the effects of E2 administration on cortisol
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secretion and stress response in sturgeon species. The results on other species raise questions regarding the role that E2 plays in the modulation of hypothalamus-pituitary-interrenal (HPI)
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axis and physiological parameters in sturgeon as an ancient species. Previous studies have shown that long-term E2 feeding had adverse effects on growth and welfare, but it is not clear that is it
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mediated via a cortisol-dependent mechanism or not. It is possible that E2 injection might induce cortisol synthesis and secretion. It is also unknown if cortisol secreted in response to E2 exogenous can interfere with the effectiveness of this hormone in feminization. Fish are exposed to E2 in both their rearing or natural environments. Stellate sturgeon has lower puberty age of 57 years and spawning intervals of 2-3 years, and 15-20% gonadosomatic index (Chebanov and Galich, 2011). These characters make it as a good candidate for caviar production in aquaculture systems. Also in wild habitats and also during the releasing of juveniles into the rivers for stock rehabilitation program, fish are subjected to different pollutants like domestic effluents which are included natural female estrogens. Therefore, the present study aimed to access the short-term effects of E2 on juvenile immature stellate sturgeon at different levels including stress indicators and other related metabolites.
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2. Methods
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2.1. Fish and holding system
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Two hundred and sixteen stellate sturgeons, Acipenser stellatus, were used for this experiment. The fish were originally hatched at Shahid Dr. Beheshti Sturgeon Fish Propagation
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and Rearing Complex (Rasht, Guilan, Iran) from a pair of wild Caspian stock and then reared at
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Dr. Yousefpour Fish Hatchery Center (Siahkal, Guilan, Iran). During the year, the fish were held in circular concrete tanks and fed with commercial pellet (Biomar, no. 3, Nersac, France). When
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the average weight and length of fish were 281.5 ± 3.0 g and 50.0 ± 0.2 cm, respectively, they were distributed into nine circular concrete tanks (1.85 m diameter, 30 cm depth, 806 L volume),
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supplied with flow-through filtered river water at a rate of 20 ± 0.7 L/min. During 10 days of acclimation and experiment, water temperature, dissolved oxygen, and pH were 28.5 ± 0.2°C,
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6.1 ± 0.2 mg/L, and 7.7 ± 0.1, respectively.
2.2. Experimental protocol
Three experimental groups with three replicates were considered. Twenty four fish were introduced per tank. Fish received a single intraperitoneal (i.p.) injection with dose of 5 mg 17βestradiol (E2; Sigma-Aldrich, St. Luis, MO, USA) per kg body weight (BW), using sterile injectable physiological saline solution (0.9% NaCl) as a carrier with a volume of 0.5 mL/100g BW. The second groups of fish were similarly injected with physiological solution, and the third group was kept as control without any treatment and handling. The fish in two injected groups were injected with 2-mL plastic syringe (gauge 25). E2 was prepared through dissolving in 100% ethanol and then in saline solution according to Falahatkar et al. (2014).
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48, and 72 h following the injection, were considered. At each time point, two fish were
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randomly captured from each tank. After anaesthetizing with 400 mg/L clove powder extract solution in 2 min, the blood was taken from the caudal vein by a 2-mL heparinized syringe.
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Blood sampling lasted 30 sec for each fish. The fish were returned to the recovery tanks (with the
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same volume) after sampling.
A small part of blood for each fish was transferred to two capillary microhematocrit tubes,
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and the remaining part was transferred to a tube for plasma extraction at 1500 g for 10 min. Afterwards, the separated plasma was stored at -70°C for later analysis. Hematocrit (Hct) was
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2.4. Plasma measurements
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determined by microhematocrit method at 3500 g for 7 min.
Plasma E2 and cortisol concentrations were measured by radioimmunoassay (Special kits from Immunotech, Marseille, France) based on the methods described by Pankhurst and Carrager (1992) and Pankhurst et al. (2008), respectively. The efficiency and inter assay variability and cortisol were 78 and 7.4% for E2 and 74 and 12.3% for cortisol, respectively. Glucose levels were determined in accordance to the colorimetric method considering the glucose-peroxidase reaction (Pars Azmun, Karaj, Iran) (Bayunova et al., 2002). Plasma lactate levels were determined enzymatically with an available commercial kit (Sigma, St. Louis, MO, USA).
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ACCEPTED MANUSCRIPT Plasma cholesterol and triglyceride concentrations were analyzed using commercial kits (Pars Azmun, Karaj, Iran) by CHOD-PAP and GPO-PAP methods, respectively (Chatzifortis et al.,
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2010). Total protein was analyzed by Biuret method (Ziestchem, Tehran, Iran).
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Plasma total calcium (Ca) and phosphorus concentrations were determined by colorimetric method, based on Sigma-Aldrich procedures no. 587 and 360, respectively. Alkaline phosphatase
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activity (ALP) was determined by means of the commercial kit instruction (Pars Azmun, Karaj,
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Iran) with auto analyzer (RA-1000, Technicon, USA).
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2.5. Statistics
Before any analysis, all data were expressed to Kolmogorov-Smirnov and Levene’s tests for
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checking the normality of data and homogeneity of variances, respectively. The data were transformed to log10 when needed. Two-way analysis of variance (ANOVA) was used to find
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the effect of different treatments and times.Tukey’s post-hoc test was used to identify the significant differences among the various means. All statistical procedures were analyzed by SPSS software (SPSS, version 13.0, Chicago, IL). The accepted significance level of P value was considered at P < 0.05. All data throughout the text are expressed as means ± standard errors of the mean (mean ± SE).
3. Results The highest concentration of E2 was measured in E2 injected fish, compared to the other groups, with a significant elevation 1h after injection, and a sharp decrease to basal level after 12h (Fig. 1). The saline and control groups had similar concentrations of plasma E2 with no changes.
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ACCEPTED MANUSCRIPT The results of cortisol levels in different treatments showed higher significant levels in injected groups, 1 h and 72 h following injection than non-injected control (Fig. 2a). Glucose
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levels increased 6 h after injection in saline and E2 groups. Also, significant differences were
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observed among the groups from the injection time to 6 h, but no changes were observed for other times (Fig. 2b). Lactate levels revealed the highest values 1 h after injection and reached to
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the basal level after 6 h in saline and E2 injected groups (Fig. 2c).
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Hct levels decreased in the saline group 48 h after injection, compared to the resting time. No significant changes were observed among the groups and throughout the sampling times (Fig. 3).
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TP concentration increased 48 h after injection for E2-treated fish, while no changes were made in other groups. Also, no significant changes were observed among the groups except at
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the end of the sampling time between E2 and saline groups (Fig. 4a). Cholesterol levels were similar among all experimental groups and during the sampling (Fig. 4b), while only E2 group
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showed the highest value of triglyceride 48 h after injection (Fig. 4c). No changes were observed in control and saline groups throughout the experiment, but the highest significant value of Ca concentration was recorded at 72 h following the injection of E2 group. Also highest concentrations were observed at this time compared to other groups (Fig. 5a). Phosphorus concentrations significantly rose 1 h after injection in saline group, but no changes were observed after 12 h among other groups (Fig. 5b). No changes were observed in ALP levels except 24 h after injection with the highest value in the saline group in comparison with the control fish (Fig. 6).
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ACCEPTED MANUSCRIPT 4. Discussion In the present study, the acute effects of E2 on the stress indicators and some other
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biochemical parameters of stellate sturgeon were investigated. Various i.p. doses of E2 were
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investigated, showing some significant physiological effects (Zaroogian et al., 2000; Teles et al., 2005, 2006; Carrera et al., 2007; Flynn and Benfey, 2007; Maria et al., 2007; Moura Costa et al.,
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2010; Falahatkar et al., 2014). The dose of 5 mg E2/kg BW in the current study was mostly
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similar to other studies and a previous study by the authors (Falahatkar et al., 2014). In the current experiment, a significant increase of E2 concentration was recognized
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following E2 injection. The increase continued 12 h after injection, and afterwards, the levels were similar to control and basement levels. Similarly, the previous study on Asian swamp eel
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(Monopteus albus) fed by E2 showed an enhanced level of plasma E2 (Yuan et al., 2011). Longterm effect of E2 in female brook trout (Salvelinus fontinalis) and beluga sturgeon caused an
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increase of plasma E2 concentration (Schafhauser-Smith and Benfey, 2003; Akhavan et al., 2015). Repeated injection of 5 mg E2/kg BW in stellate sturgeon increased the level of plasma E2 (Falahatkar et al., 2014). The current study showed that exogenous E2 caused an elevation in plasma E2 within 12 h in treated fish. Returning to pre-injection levels after 12 h suggests that exogenous E2 was likely cleared from the circulation and accumulated in some tissues. In the current study, plasma cortisol showed two different patterns of response to handling. In first phase (from 1 to 6 h after handling), a transient elevation in plasma cortisol level was observed in both saline and E2-injected groups. This increase is a typical response to handling in sturgeon fish for allowing them to cope with handling in order to maintain their homeostatic states (Barton et al., 1998, 2000; Falahatkar et al., 2009). It also suggests that injection procedure has induced an acute stress in stellate sturgeon. Interestingly, the elevated cortisol levels in the
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ACCEPTED MANUSCRIPT E2-treated fish were lower than those in the saline group, indicating that there is an interaction between the HPI and exogenous E2. It seems that exogenous E2 is capable of altering the
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endocrine stress response in stellate sturgeon. Plasma cortisol levels returned to prestress levels
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by 6 h in the saline group, while plasma levels of fish injected with E2 increased again after 6 h and were approximately maintained throughout the sampling times. The second manner of
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response following E2 injection was associated with the clearance of excess E2 from circulation.
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These observations confirm our concept in which E2 can affect the rate of cortisol synthesis in stellate sturgeon probably by acting on components of the HIP axis, as described previously by et
al.
(2011)
for
zebrafish.
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Fuzzen
However,
further
experiments
would be required to clarify the possible mechanisms involved in mediating the effect of
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exogenous E2 on the HPI axis in chondrostean fish. There are inconsistent results regarding fish stress responses to E2 treatment; Teles et al. (2004,
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2005) showed a decrease in plasma cortisol following E2 exposure in immature gilthead sea bream (Sparus aurata) and juvenile seabass (Dicentrarchus labrax).
While, other studies
reported elevated levels of cortisol and adrenocorticotropic hormone in salmonids (Pottinger et al., 1996) and gilthead sea bream (Carrera et al., 2007) after exposure to E2. Similar to our results, i.p. injection method of E2 to tilapia, Oreochromis mossambicus (Vijayan et al., 2001), and Atlantic salmon, Salmo salar (McCormick et al., 2005) showed no significant changes. These contrary findings may be explained by different administration methods of E2, species specificity of response to E2 injection, anatomy of interrenal tissue and sampling times following E2 exposure. The mechanism involved in such a response in such a time is not clear, but it is clear that such changes do not occur in a short period of 48 h following E2 injection.
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ACCEPTED MANUSCRIPT Considering the glucose as a secondary response to stressors, the injection stress stimulated the releasing of glucose in short-term injection of E2 or saline to 6 h, while no changes were
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made in the control group. The highest value of glucose after stress was recorded in many
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sturgeons after 3-6 h (Barton et al., 2000; Falahatkar et al., 2009; Falahatkar and Poursaeid, 2013; Eslamloo and Falahatkar, 2014), which is similar to the current work. It seems that E2
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cannot induce the metabolic changes of gluconeogenesis or glycogenolysis, and these pathways
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are affected by injection and handling stressors to supply the energy demand. However, given the more clear alignment between the time frame of the plasma glucose increase and decrease of
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circulating E2, possibly the response is related to the energy demands of metabolic removal of the injected E2, rather than to the primary stress response. Exposure of E2 in sea bass showed
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induction of plasma glucose increase (Teles et al., 2004), but not in gilthead sea bream (Teles et al., 2005) or sea bass (Teles et al., 2006). A significant increase was observed in stellate sturgeon
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for glucose concentration when injected with 5mg E2/kg BW (Falahatkar et al., 2014). Since glucose is an important product of gluconeogenesis process for different activities to cope with energy needs, such changes in the study were just affected by handling, and E2 could not affect carbohydrate mechanism for glucose transmission or absorption (Mommsen et al., 1999) as fuel energy.
In the present study, although the level of lactate changed through the trial with the highest value 1 h after injection, these changes are correlated with stress but not E2 injection. Lactate levels in juvenile sea bass remained unchanged after E2 exposure (Teles et al., 2006); however, another study on gilthead sea bream showed increasing of lactate level (Teles et al., 2005). Different administration methods or exposure times are possible reasons for such results.
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ACCEPTED MANUSCRIPT Generally, stress can stimulate to release primary (cortisol) and secondary (glucose and lactate) responses of the fish (Wendelaar Bonga, 1997; Pankhurst, 2011). In this study, despite
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cortisol change in fish injected E2, 72 h following injection, the responses were directly affected
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by stress, and E2 could not mobilize stress substrates such as glucose and lactate as fuel energy. Considering different sampling times, no significant differences were observed in Hct values,
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but the lowest level was observed 48 h after injection in fish treated with saline compared to the
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resting time. Although Hct value varies after different handling procedures, it was observed that i.p. administration has not induced or reduced the rate of erythropoiesis or even the blood
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volume. Previous studies revealed that dietary administration of E2 in triploid female brook trout causes Hct decline (Schafhauser-Smith and Benfey, 2003). Similar decrease was observed in
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stellate sturgeon when fed by different levels of E2 (25 or 50 mg/kg diet) or i.p. injected fish by 5 mg/kg BW (Khara et al., 2014; Falahatkar et al., 2014). Previous studies demonstrated that the
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administration of E2 has an inhibitory effect on erythropoiesis, but the present inquiry revealed that this way of administration has no effect (at least in short time) on blood index or being affected by the handling stressor.
At the end of the sampling, 48 and 72 h after E2 injection, the fish showed higher TP compared to the resting time. Similar effect of E2 was observed in plasma TP in red sea bream, Chrysophrys major (Woo et al., 1993), and stellate sturgeon (Falahatkar et al., 2014). This increase might be due to the role of E2 in stimulating vitellogenic process and mobilization of different lipoproteins in blood circulation (Sharpe and MacLatchy, 2007). In addition, the results revealed no cholesterol changes among the treatments and during the experiment, but significant elevation of triglyceride was observed in E2-injected fish 48 h following injection. Hyperlipidimic effect was observed in rainbow trout, Oncorhynchus mykiss
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ACCEPTED MANUSCRIPT (Wallaert and Babin, 1992), goldfish, Carassius auratus (Sharpe and MacLatchy, 2007), stellate sturgeon (Falahatkar et al., 2014), and beluga sturgeon (Akhavan et al., 2015) when exposed to
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E2. In our study, the short period of E2 exposure with a single dose of injection could not
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provide similar circulating hyperlipidemia. Also, no changes in cholesterol and triglyceride levels in control and saline-injected fish were demonstrated, without handling effects in blood
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lipid sources, but the induction of triglyceride was observed at the end of the sampling times.
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In the current study, elevated level of plasma Ca was observed in E2-treated fish compared to other treatments and basal level after 72 h. The level of phosphorus 1 h after exposure was
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affected by E2 and saline treatments, but the trends were not clear during the sampling times. Previous observations have revealed that E2 can stimulate vitellogenesis which is accompanied
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by an enhance in plasma Ca and phosphorus (Persson et al., 1998; Guerreiro et al., 2002; Linares-Casenave et al., 2003; Akhavan et al., 2015). It is clearly demonstrated that vitellogenin
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is a calcium-binding lipophosphoprotein, and E2 is implicated in regulation and elevation of plasma Ca through an increase in its uptake from water or mobilization from internal sources of Ca such as scutes or cartilage on sturgeons. Therefore, the elevation of Ca in E2-treated fish might indicate vitellogenin synthesis in juvenile stellate sturgeon. ALP activity was unaltered through the experiment and among the treatments except for the 24 h with the lowest level in control group. The results of hepatic enzymes in gilthead seabream showed further reduction compared to the control or non-injected fish (Carrera et al., 2007). Most previous studies also show depletion of hepatic metabolizing enzymes by estrogens or estrogen-like compounds (Vaccaro et al., 2005; Teles et al., 2005). Although the levels of hepatic enzymes showed damage to hepatic tissue, the unchanged results of ALP activity revealed no
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ACCEPTED MANUSCRIPT effect of different treatments. Higher activity of ALP 24 h after injection demonstrates the ability of such stress procedure at this delayed time.
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Our previous results demonstrated all feminization of stellate sturgeon which was
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periodically injected with E2 (Falahatkar et al., 2014), but no data was collected in this study regarding to the single injection of this hormone. The simulatory manipulation of plasma E2
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concentrations used in our study proved a valuable tool for finding the effects of E2 on stress
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response and biochemical parameters. We compared the response of E2-injected fish with control and saline-injected fish at rest and different times following injection. All the other
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parameters in TP, triglyceride, Hct, and Ca are very episodic, they concern mainly the 48 h (or the 72 h) samples, suggesting that there might be some specific changes in these samples.
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Although i.p. injections are not the most revealed mode of exposure in environment, but it is an effective way to provide a means of determining whether they are relevant for fish (Moura Costa
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et al., 2010; Falahatkar et al., 2014). Also, treatment with pellet application to achieve longer and physiologically more realistic profiles of elevated E2 can be further investigated.
5. Conclusions
In conclusion, stress responses and metabolite parameters after administration of different treatments showed some related changes in glucose and lactate as a fuel sources of stress response which they are not related to E2-induction, while E2 treated fish exhibited elevation of E2, TP, triglyceride, and Ca concentrations in the juvenile stellate sturgeon. Therefore, it is demonstrated that some blood indices can be modulated by E2 injection for a short time not exactly by handling stressor, but E2 cannot change the response of HPI axis in terms of secondary stress response, at least. E2 injection in saline is an effective way of getting a short-
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ACCEPTED MANUSCRIPT lived but high level increase in plasma E2, and that other than perhaps cortisol, it does not perturb physiological response for too long and that the possible effect on cortisol might need
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some extra study. Further research is needed to find how E2 acts as a modulator in positive of
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negative feedbacks of the stress. In fact, there is a need for immature fish to access the effects of E2 exposure regarding genotoxic response, antioxidants status, immune system, hepatic
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condition indicators, biotransformation, and the difference between two sexes.
Acknowledgements
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The authors sincerely thank staff at the Dr. Yousefpour Fish Hatchery Center, for kindly supplying fish and facilities. We also thank to NW Pankhurst for performing the steroid analyses
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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Plasma 17β-estradiol in juvenile stellate sturgeon Acipenser stellatus at the
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control, saline and 17β-estradiol injected groups at rest and different times following
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intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Lower
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cases indicate significant differences in an experimental group over time (ANOVA followed by Tukey’s post hoc test, P<0.05), and capital letters indicate significant differences among treatments for a given time (ANOVA followed by Tukey’s post
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hoc test, P < 0.05).
Fig. 2. Plasma concentrations of cortisol (a), glucose (b) and lactate (c) in juvenile
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stellate sturgeon Acipenser stellatus at the control, saline and 17β-estradiol injected
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groups at rest and different times following intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Lower cases indicate significant differences in an
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experimental group over time (ANOVA followed by Tukey’s post hoc test, P < 0.05), and capital letters indicate significant differences among treatments for a given time
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(ANOVA followed by Tukey’s post hoc test, P < 0.05).
Fig. 3. Hematocrit values in juvenile stellate sturgeon Acipenser stellatus at the control, saline and 17β-estradiol injected groups at rest and different times following intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Lower cases indicate significant differences in an experimental group over time (ANOVA followed by Tukey’s post hoc test, P < 0.05).
Fig. 4. Plasma concentrations of total protein (a), cholesterol (b) and triglyceride (c) in juvenile stellate sturgeon Acipenser stellatus at the control, saline and 17β-estradiol
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Fig. 5. Plasma concentrations of total calcium (a) and phosphorus (b) in juvenile
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stellate sturgeon Acipenser stellatus at the control, saline and 17β-estradiol injected
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groups at rest and different times following intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Lower cases indicate significant differences in an
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experimental group over time (ANOVA followed by Tukey’s post hoc test, P < 0.05),
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Fig. 6. Plasma alkaline phosphatase activity (ALP) in juvenile stellate sturgeon
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Acipenser stellatus at the control, saline and 17β-estradiol injected groups at rest and different times following intraperitoneal injection (n=6 for each treatments). Values are mean ± SE. Capital letters indicate significant differences among treatments for a given time (ANOVA followed by Tukey’s post hoc test, P < 0.05).
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