Mahboubeh Hosseinzadeh, Mohammad Reza Imanpoor, Ali Shabani and Hamed Nekoubin*
Department of Fishery, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
Received Date: August 18, 2012; Accepted Date: November 06, 2012; Published Date: November 16, 2012
Citation: Hosseinzadeh M, Imanpoor MR, Shabani A, Nekoubin H (2012) Seasonal Changes in Serum Calcium and 17ß-Estradiol Levels in Persian Sturgeon, Acipenser persicus. J Aquac Res Development 4:159. doi:10.4172/2155-9546.1000159
Copyright: © 2013 Hosseinzadeh M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited..
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Concentrations of total serum calcium and17β-estradiol (E2) were measured during four stages of ovarian growth in Persian sturgeon, Acipenser persicus. Total calcium and E2 increased from stage II (cortical alveolus) and reached their highest levels (6.67 ± 1.15 mg/dl and 5.33 ± 1.06 ng/ml, respectively) during stage III (vitellogenic). Levels of both total calcium and E2 decrease during stage IV (ripe). A decline in serum calcium level was observed postspawning (stage V). Total serum calcium exhibited a linear relationship (r2=0.6789) with plasma E2 (P<0.01). Both E2 and total serum calcium can be utilized to identify mature vitellogenic (stage III) females in sturgeon broodstocks
Persian sturgeon; Calcium; 17β-estradiol; Gonadal maturation
The Persian sturgeon,Acipenser persicus, is an endangered species the southern part of Caspian Sea, especially along the shores of Iran [1,2]. During the few past decades many attempts have been made to augment their stocks in terms of supportive breeding in Iran. Successful management of sturgeon populations requires knowledge of the stock composition with regard to sex and maturational status . One of the problems in sturgeon broodstock is the slow and asynchronous ovarian maturation .
The VTG particle is phosphorylated, lipidated, and glycosylated, and serves as a major supply of minerals such as calcium, magnesium, zinc, and iron to the oocyte, and therefore to the developing embryo. The increase in plasma protein, calcium, magnesium, and phosphoprotein content can be used as indicators of plasma VTG levels [7,8].
Vitellogenesis by the liver is stimulated and maintained by E2 and is accompanied by increased plasma calcium and phosphate levels . Exogenous estrogen increases serum calcium concentrations in female goldfish , Fundulus kansae , and salmonids . Others examined effects of E2 exposure on the distribution of plasma calcium, magnesium, inorganic phosphate, and plasma protein in rainbow trout, and reported that both plasma calcium and total plasma protein increased in a dose dependent manner following exposure to E2 . An early study on Atlantic cod used plasma calcium as an index of estrogenic activity in females . Concomitant increases in plasma calcium and VTG concentration following estrogenic stimulation have been reported previously in salmonid fish .
Serum sex steroid and calcium concentrations have not previously been applied to investigate the reproductive cycle of Persian sturgeon. Therefore, we investigated relationships between serum E2 and serum total calcium in Persian sturgeon female during the natural reproductive cycle.
Fish collection was conducted seasonally between May 2011 and May 2012. Thirty-five specimens of female Persian sturgeon (A. persicus) were captured in gillnets from the southeastern Caspian Sea. The captured fishes in late winter and early spring that were in stage IIIIV or IV transferred to Shahid Marjani sturgeon Propagation Center in Gorgan, Iran. Two injections of sturgeon pituitary preparation (PP) (3-5 mg/kg) were used to simulate final maturation. The first PP injection (5% of total dose) was made at 10 PM and second (95% of total dose) 12 h later at 8 AM and blood samples were taken from caudal vein with a non heparinized syringe 24 h after second injection.
Determination of sexual maturity stages
Each sturgeon was weighed and fork length measured. The gonad samples of females during each season were fixed in BouinÊ¼s fluid for 48 h and then transferred to 70% ethanol for storage until processing for light microscopy. Paraffin sections of 4-7 μm thickness were stained with hematoxylin and eosin. The developmental stages of gonads were classified according to the system of Amiri et al. .
Serum 17β-estradiol and calcium Analyses
Blood samples were centrifuged for 10 min. at 3000×g. The serum was removed and placed into a clean microcentrifuge tube and stored at ?20°C until analyzed.
Serum E2 was measured by Enzyme-Linked Immune Sorbent Assay (ELISA) according to the procedure of Semenkova et al. . Commercial kits for measurement of E2 levels in the sturgeon serum were obtained from Tehran, Iran.
Total serum calcium concentrations were measured for each female Persian sturgeon using a spectrophotometer (Model wap-S2000-UV/ VIS, Cambridge-UK).
Distribution parameters are presented as means and Standard Deviation (SD). Serum E2 and calcium concentrations were compared by the one-way Analysis of Variance (ANOVA) and Duncan’s test. Assignment of data correlation was done by Pearson tests, and the relationships between total plasma calcium and E2 were evaluated by linear regression. The accepted statistical significance level was P<0.05.
The ovary stages of Persian sturgeon are summarized in table 1. Total calcium and E2 increased and reached their highest levels in stage III (Table 2).
|Cortical alveoli stage (ΙΙ)||In this stage oocytes in the ovaries appear to contain oil droplets.|
|Vitellogenic (yolk) stage (ΙII)||Yolk granules were visible as a ring of deep eosinophilic inclusions in the cytoplasm and later incorporated the whole cytoplasmic area. The zona radiata was clearly visible.|
|Ripe (mature) stage (IV)||The oocyte has become larger and more hydrated, and the nucleus has migrated toward the periphery and is in the process of dissolution.|
|Ovulation stage (V)||During ovulation the oocytes detach from the follicle cells.|
Table 1: Ovary stages of A. persicus.
|Category||n||Calcium (mg/dl)||E2 (ng/ml)|
|Cortical alveoli stage (ΙΙ)||7||0.29 ± 0.12c||0.78 ± 0.10c|
|Vitellogenic stage (ΙII)||6||6.67 ± 1.15a||5.33 ± 1.06a|
|Ripe (mature) stage (IV)||14||2.86 ± 0.78b||1.98 ± 0.48b|
|Ovulation stage (V)||8||0.07 ± 0.05c||2.31 ± 0.35b|
Values with different superscript letters denote significantly different means within each column (P<0.05)
Table 2: Calcium and 17β-estradiol (E2) concentrations in serum (mean ± SD) of Persian sturgeon.
There was significant differences in mean plasma E2 concentration among reproductive stages (Figure 1, P<0.05). Immature (stage II) female captured in autumn 2011, had a significantly lower mean E2 (0.78 ± 0.1 ng/ml E2) than all other stages (P<0.05). Stage III had significantly higher serum E2 concentration compared to other stages. Serum E2 decreases during late winter and early spring in stage IV and V females (Figures 1 and 2). There was no significant difference in levels of E2 between stages IV and V (P>0.05).
Mean calcium concentrations had a similar pattern to mean E2 concentrations (Figure 1). The highest concentration of calcium was observed in stage III females (6.67 ± 1.15 mg/dl). Lowest serum levels of calcium were observed in V and stage II females and these values did not differ significantly between stage II and V (P>0.05).
There was no significant correlation between E2 and calcium in stages ΙΙ, IV and V (data not shown), but generally, the results show that there was a positive relationship between concentration of E2 and total calcium of serum (Figure 3, r2=0.6789, P<0.01).
In the present study, correlation was found between serum calcium and E2 levels during ovary development in female Persian sturgeon. Seasonal variation in serum calcium and E2 levels was observed, associated with ovarian stage and in agreement with earlier observations [11,18-21]. Serum E2 does not increase during the spawning (Figure 2). It increases during stage III (vitellogeniesis). Serum concentration of estrogen increase during phase III and varies consistently with the natural reproductive cycle [19,22-24]. The increase of estrogen secretion results in a rise of serum calcium during ovarian maturation. GTH-I circulate in the blood and upon reaching the ovaries, stimulates the follicle cells to synthesize E2, which is then released into the blood. E2 diffuses across the membrane of liver cells and binds to the estrogen receptors, resulting in initiation of transcription and translation of VTG . Increasing E2 concentrations during oogenesis indirectly caused an increase in calcium levels in female shovelnose sturgeon because E2 simulates the liver to produce VTG .
Fleming et al. , Ho and Vanstone  and Woodhead  detected increased serum calcium levels in female fish after the administration of E2. Enhanced secretion of estrogen during the sexual maturation of females increases the serum calcium level . Collectively, these results are in agreement with our work, as increased secretion of E2 during stage III is correlated with increased serum calcium concentration.
Several authors have correlated the increased blood calcium content with ovarian maturation [28,29]. E2 has been shown to stimulate hepatic formation of VTG, increase serum VTG levels, and increase protein-bound fraction of plasma calcium levels .
Female bester Huso huso×Acipneser ruthenus, stellate, and Atlantic sturgeon exhibit peak E2 concentrations when gravid and E2 concentrations drop significantly following ovulation [5,16,25]. Concomitant increases in plasma calcium and VTG concentration following estrogenic stimulation have been reported previously in salmonid fish .
In the present study, relationship was observed between the total calcium level in blood serum and ovarian maturation of Persian sturgeon. These results indicate that the gonads play an important role in the calcium regulatory system of females. The seasonal peaks in serum E2 and calcium concentrations occur during stage III, not at ovulation. Additionally calcium appears to be at its lowest level in stage V (Figure 1). Therefore, seasonal fluctuations in serum E2 and calcium concentrations in female Persian sturgeon may be indicative of ovulation timing.
Also, there was significantly correlation between serum calcium and E2 during stage III, therefore both of these values can be utilized to identify vitellogenic females in sturgeon broodstocks.
The authors are grateful to staffs of Shahid Marjani sturgeon Propagation Center in Gorgan, Iran, for helping our study. And we would like to thank Mr. Seyed Mostafa Aghilinejhad for the help.