Fish Physiol Biochem DOI 10.1007/s10695-013-9829-z
Comparative study of dietary soy phytoestrogens genistein and equol effects on growth parameters and ovarian development in farmed female beluga sturgeon, Huso huso A. Yousefi Jourdehi • M. Sudagar • M. Bahmani • S. A. Hosseini • A. A. Dehghani • M. A. Yazdani
Received: 21 February 2013 / Accepted: 24 June 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Oocyte maturation in fish is a hormonally regulated process. In the light of long-term oocyte maturation in beluga, the aim of this research was to study the estrogenic effects of different concentrations of soy dietary genistein (GE) and equol (EQ) on the growth performance and ovary development in farmed female Huso huso. Fish were fed with concentrations 0 (control), 0.2, 0.4, 0.8 and 1.6 g of EQ and GE per kg of isoproteic (CP 45 %) and isoenergetic (19.5 MJ/kg) diets during a year. Blood samples and ovary biopsies were collected from each fish seasonally. The main results of the present experimentation are that growth performance was not affected significantly both in GE and EQ (P [ 0.05). EQ at concentration 0.4 g/kg had more estrogenic effects than other concentrations of EQ and GE in beluga so that 64 % of fish were matured sexually. Some reproductive indices such as oocyte diameter, testosterone (T) and 17b-estradiol (E2)
increased significantly at EQ 0.4 g/kg at the end of experiment (P \ 0.05), while 17a-hydroxy progesterone level (17a-OHP) showed no significant changes at all concentrations. Biochemical indices such as calcium, phosphorous and cholesterol increased at GE concentrations, but decreased at EQ concentrations similarly at the end of experiment. There was a negative relationship between plasma phosphorous and alkaline phosphatase enzyme levels. Based on results, EQ at concentration 0.4 g/kg improved oocyte development more than the other concentrations of GE and EQ, and therefore, it can be used as an additive to diets for inducing ovary development in this species.
A. Yousefi Jourdehi (&) M. Sudagar The University of Gorgan Agricultural Sciences and Natural Resources, Gorgan, Iran e-mail: [email protected]
S. A. Hosseini Ecology, The University of Gorgan Agricultural Sciences and Natural Resources, Gorgan, Iran
Keywords Phytoestrogens Oocyte maturation stages Sex steroid hormones Biochemical indices Huso huso
A. Yousefi Jourdehi International Sturgeon Research Institute of the Caspian Sea, Rasht, Iran
A. A. Dehghani Water Engineering, The University of Gorgan Agricultural Sciences and Natural Resources, Gorgan, Iran
M. Bahmani Department of Fish Physiology and Biochemistry and Rearing and Propagation, International Sturgeon Research Institute of the Caspian Sea, Rasht, Iran
M. A. Yazdani Department of Sturgeon Fish Rearing and Propagation, International Sturgeon Research Institute of the Caspian Sea, Rasht, Iran
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Introduction Phytoestrogens are by definition plant-derived substances that are able to activate the estrogen receptors (ER). Most of these plant substances are to some extent similar to the estrogen either in structure or function (Cassidy et al. 2000). Isoflavones are a group of phytoestrogens and can be found in many plants, particularly in the legume family. The major constituents of soy isoflavones, genistein (GE) and equol (EQ) interact with the ER in several tissues including gonads. GE and daidzein are two of the major metabolites from isoflavones, but can be further metabolized by intestinal microorganisms (Schoefer et al. 2002; Wang et al. 2005). Daidzein can be metabolized to EQ (King and Bursill 1998). Steroid hormones are the most important hormones for improving reproduction in fishes (So et al. 1985). Changes in the concentration of serum steroid hormones have been used as indicators of reproductive response in fish. Vitellogenin (VTG), the major constituent of yolk, is synthesized in the liver under estrogenic induction (Wallace 1985). VTG is then carried in blood to the ovaries, where it is sequestered by the developing oocytes by binding to specific receptors associated with endocytotic vesicles (Le Menn and Nunez Rodriguez 1991). In fish, only reproductively active females normally synthesize VTG. Estradiol is the main inducer during the reproductive cycle (Fostier et al. 1983). A large number of natural or synthetic chemicals present either in food or in the environment. The estrogenic effects of phytoestrogens are well documented in fish (Pelissero et al. 2001). Phytoestrogens can bind to steroid-binding proteins (Dechaud et al. 1999) and to ER of target cells (Casanova et al. 1999). Moreover, they exhibit endocrine-disturbing activity interfering with enzymatic reactions either on steroid metabolism, i.e., aromatization (Chen et al. 1997) or on the mechanism of action of estrogens, i.e., tyrosine kinase activity (Huang et al. 1999). As a result, phytoestrogens can induce reproductive disorders (Maclatchy and Van Der Kraak 1995). Phytoestrogens are able to stimulate VTG synthesis in vivo in Siberian sturgeon (Pelissero et al. 1991) and in vitro in trout hepatocyte (Pelissero et al. 1993). There have been very few
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studies on the effects of GE and EQ in teleosts. Intraperitoneal injections of GE and EQ to yearling Siberian sturgeon (Acipenser baerii) induced vitellogenesis, a physiological response associated with estrogenic activity (Pelissero et al. 1991). GEenriched diets were fed to rainbow trout (Oncorhynchus mykiss) for a year, and gametogenesis and reproductive efficiency of the trout were reduced (Pelissero et al. 2001). The beluga sturgeon (Huso huso) is the largest growing sturgeon species, one of the largest fish found in freshwater. The beluga is now extensively farmed to produce caviar. Due to the long reproductive cycle (18-year-old in females) in beluga of the Caspian Sea in nature, many countries such as Iran like to farm this species in fresh waters. Considering the last maturity of beluga, this study was carried out with the aims of using dietary soy phytoestrogens GE and EQ for increase oocyte development in females of beluga.
Materials and methods Fishes and experimental design Experiments were carried out on 54 female H. huso 5-year-old with average body weight 13.25 ± 0.3 kg and total length 140.3 ± 1.2 cm that had been farmed in terrestrial ponds of the Caspian Sea International Sturgeon Research Organization during a year (from summer 2011 to summer 2012). Ovary of all fish was at sage II of sexual maturation at the start of the experiment. Fish were anesthetized using a Clove powder 250 ppm for 10 min and selected by biopsy and laparoscopy methods (using a Stema Company, model M-CAM1700, 30 degree telescope with 4 mm in size and with length 17.5 cm and with the cold light source a halogen 250 W, Germany) and were stocked randomly in 18 concrete ponds (with dimensions 3 9 2.5 9 1.2 m3) equipped with aeration system and hole mixed the Sepidrood River water together. Fishes were treated with a practical beluga diet was formulated to contain at concentrations of 0, 0.2, 0.4, 0.8 and 1.6 g/kg of purified GE and EQ (Xi, and Keen— Source Biotechnology Co., Ltd). GE and EQ were
Fish Physiol Biochem
dissolved in 75 ml ethanol and added to the diet. Diets were mixed by a mixer in 2,000-g portions for 7–10 min after adding 15 % of water. Food pellets were prepared in diameter 12 mm with a standard meat grinder. Before preparing each diet, 0.5 kg was run through the extruder and discarded to remove any residue from the previous diets. The diets were mixed from low to high levels of GE to minimize the overlap in GE and EQ concentrations. They were air-dried 24 h. Each treatment includes 6 fish in two replicates with 3 fish per each concrete pond. One percent of biomass and twice daily (0800 and 1600 h), fishes received a diet based on fish meal 58 %, wheat meal 20 %, meat powder 15 %, fish oil 2.5 %, mineral supplements 1 %, vitamin supplements 1 %, vitamin E 1 % and calcium phosphate 1 % consisted of crude protein (CP 45 %), fat 14 %, carbohydrate 20 %, ash 9 %, moisture \10 % and energy (19.5 MJ of dry matter). Physicochemical parameters were measured by using Oxi-pHmeter40i (WTW, Germany) daily. Oocyte diameter was measured using a stereomicroscope. For histological studies, ovaries samples were fixed in Bouin’s fluid during 48 h and were dehydrated, paraffinated, mounted, sectioned (5–7 lm) by Microtome set (Leitz 1512, Germany), stained by hematoxylin and eosin (H&E) and studied by light microscope. Blood samples (5 ml) were taken from a caudal vein of fish seasonally.
Blood analyses Blood serum was separated by using a centrifuge (Labofuge 200, Heraeus Sepatech, Germany) with 3,000 rpm per 10 min. Samples were stored at -20 °C (Pottinger and Carrick 2001). Sex steroid hormone levels (including testosterone (T), 17a-hydroxy progesterone and 17b-estradiol) and alkaline phosphatase enzyme (ALP) level were measured using radio-immune assay (RIA) with Immunotech kit and I125 (France) and gamma counter LKB (Finland) based on ng/ml in the Dr. Fadaie laboratory (Rasht, Iran). Total calcium and phosphorous were analyzed using a photometric technique (Roche Modular automated clinical chemistry analyzer). Plasma cholesterol was measured by standard enzymatic method (Pars Azmoon kit, Pars Azmoon Inc., Tehran, Iran) using a spectrophotometer with wave length 570 nm at the hematology laboratory.
Growth data calculation CF ¼ BWf = FL3 100 BWf ¼ final body weight and FL ¼ final length BWI ¼ 100 ðBWf BWi Þ=BWi BWf ¼ final body weight and BWi ¼ initial body weight SGR ¼ ðln Wf ln Wi Þ=t 100 Wf ¼ final fish weight and Wi ¼ initial fish weight; and t ¼ day FCR ¼ F=ðWf Wi ÞF ¼ food; Wi ¼ final fish weight and Wi ¼ initial fish weight PER ¼ ðBWf BWi Þ=protein intake BWf ¼ final body weight and BWi ¼ initial body weight Statistical analysis Minimum, maximum, variance and standard error were used to describe biochemical indices. One-way ANOVA was used for comparison between 2 groups, and Duncan’s test was used for differential studies. Levene’s test was used to test for equality of variance. An independent-sampled t test was used to detect differences between hormonal and biochemical indices. Excel and SPSS17 software were used for data analysis and drawing diagrams. Data are presented as mean ± SEM. Results Physicochemical and growth performance results The minimum and maximum of temperature were 11.2 ± 1.5 °C in winter and 22.5 ± 1.6 °C in summer, respectively. Dissolved oxygen (O2) was decreased with increase in temperature and reached to a minimum in summer (Table 1). Growth parameters results showed that the mean initial weight and length of H. huso were 13.25 ± 0.3 kg and 140.3 ± 1.2 cm and reached to 29.26 ± 0.6 kg and 163.4 ± 1.3 cm at the end of experiment, respectively (P \ 0.05), but showed no significant differences between GE and EQ concentrations and controls (P [ 0.05). There were no significant
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Fish Physiol Biochem Table 1 Water physicochemical parameters change during different seasons (mean ± SEM) Seasons
Physiochemical parameters Temperature (°C)
O2 (mg/l)
pH
Spring
17.4 ± 1.9
7 ± 0.2
7.8 ± 0.3
Summer
22.5 ± 1.6
6.2 ± 0.5
7.8 ± 0.3
Autumn
16.9 ± 2
8.2 ± 0.2
7.7 ± 0.3
Winter
11.2 ± 1.5
8.8 ± 0.2
7.9 ± 0.3
differences in growth rate (GR), food conversion ratio (FCR), specific growth ratio (SGR), condition factor (CF), feed efficiency (FE) and protein conversion ratio (PER) between the treatments and corresponding controls (P [ 0.05) (Table 2). Laparoscopic and Histological Observations Stage II Oocytes are not round but orthogonal. Yolk resembles a narrow strip. The protoplasmic growth and increase in oocyte diameter are detectable in this stage. Nucleoli are completely stuck to the membrane of nucleus. A chromatin mass is observed in the center of nucleus. Number of nucleolus close to the membrane of nucleus is raised, and vacuoles are observed around nucleus inside the cytoplasm. Animal pole is not differentiated from vegetable pole. Appearance of pigments in lateral layer of oocyte cytoplasm is the most obvious index of transition from stage II. Stage III In this stage, growth of oocytes is associated with reduction in fat layer. Along with thickening of oocytes, pigments and fat tissue are observed in the middle and lateral parts of gonads; however, do not completely fill up the body cavity. Pigments are dark gray in color and formed beneath the cell membrane. Oocytes firmly adhere to the fat layer of ovary. As a result, they do not pass through special caviar processing sieves. The ovary is fully grown in volume, oocytes are visible with naked eyes but yet indistinguishable from each other and blood vessels are clearly dispersed. A thick layer of fat is present in the middle part of ovary while oocyte is found in lateral area of ovary. In comparison with the previous stage, oocytes abundance and volume are
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higher, fat is reduced, and oocytes pass through the sieve in this stage. Stage IV Yolk granules almost fill the whole space around nucleus, and only a tiny cytoplasm is spread out near the nucleolus and oocyte membranes. Small yolk granules and nucleus are located in animal pole while larger yolk granules and fat droplets in vegetal pole. Nucleus is positioned from cell center toward animal pole at micropyle site. Nucleoli are mostly situated at the center although few are distributed in different areas of the cell. Based on histological study on the ovary of H. huso, 9 major layers (from outside to inside) are discernible at stage IV of maturity: follicle epithelial layer, jelly coat, external zona radiata, internal zona radiata, fat layer, pigments, cytoplasm, nucleus, nucleoli and micropyle (Fig. 1A–F). Oocyte diameter Oocyte diameter increased significantly at all concentrations of GE and EQ except GE 1.6 g/kg than control. At the end of experiment, it increased significantly at EQ 0.4 g/kg (reached to a maximum 3.1 ± 0.2 mm) and 1.6 g/kg than others (P \ 0.05) (Fig. 2). Sex steroid hormones Testosterone level increased significantly at the end of experiment than start at all concentrations. At the end of experiments, testosterone level reached to a maximum at EQ 0.4 g/kg (P \ 0.05) (Fig. 3). 17b-estradiol (E2) levels were more at all treatments at the end than start of experiment and showed significant increase at EQ 0.4 g/kg at the end of experiment (Fig. 4). 17a-hydroxy progesterone levels were more at the end of experiment than start but showed no significant difference between the start and the end at all concentrations of GE and EQ (P [ 0.05) (Fig. 5). Biochemical indices results Alkaline phosphates enzyme (ALP) levels decreased significantly at all concentrations of GE and EQ at the end than the start and was maximum at EQ 0.4 g/kg (P \ 0.05) (Fig. 6). Calcium ion levels were increased
2.9 ± 0.2
0.16 ± 0.01
PER
0.16 ± 0.01
36.8 ± 2.4
69.2 ± 11.8
0.93 ± 0.2
0.74 ± 0.02
13.3 ± 1.1
0.18 ± 0.02
40.3 ± 3.5
84.3 ± 18.5
0.99 ± 0.2
2.6 ± 0.3
0.05 ± 0.004
0.6 ± 0.01
170.1 ± 2.6
141.2 ± 3.4
29.1 ± 2
13.2 ± 0.7
0.19 ± 0.02
40.3 ± 4.2
82.3 ± 11.6
1 ± 0.2
2.6 ± 0.4
0.05 ± 0.005
0.64 ± 0.01
167.7 ± 5.8
144.2 ± 4.4
30.4 ± 1.3
13.2 ± 0.7
0.17 ± 0.02
41.9 ± 4.6
82.9 ± 13.6
1 ± 0.2
2.8 ± 0.3
0.056 ± 0.006
0.71 ± 0.02
162.1 ± 3.9
143.1 ± 4.5
30.4 ± 1.3
13.3 ± 1.1
0.16 ± 0.01
35.4 ± 2.4
79.4 ± 12.6
0.98 ± 0.2
2.7 ± 0.3
0.05 ± 0.005
0.71 ± 0.02
160.3 ± 2
136.2 ± 4.3
29.6 ± 1.9
13.2 ± 1.1
0.15 ± 0.02
38.4 ± 3.8
76.1 ± 13.8
0.98 ± 0.2
2.9 ± 0.2
0.04 ± 0.003
0.67 ± 0.01
163.5 ± 4.2
140.9 ± 2.8
29.6 ± 2.6
0.16 ± 0.01
35.9 ± 2.3
76.3 ± 11.9
0.98 ± 0.2
2.8 ± 0.2
0.047 ± 0.003
0.64 ± 0.01
162.2 ± 3.2
137.2 ± 3.2
27.5 ± 1.8
13.3 ± 0.7
0.8
0.16 ± 0.01
36.9 ± 1.2
80.9 ± 15.1
0.97 ± 0.2
2.7 ± 0.1
0.05 ± 0.002
0.69 ± 0.02
163.6 ± 2.3
138.1 ± 4.6
30.3 ± 1.7
13.3 ± 1.1
1.6
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
Significant
IBW initial body weight, FBW final body weight, IL initial length, FL final length, CF condition factor, GR growth rate, FCR feed conversion ratio, SGR specific growth rate, BW body weight, FER feed efficiency ratio, PER protein efficiency rate
A total of 54 fish including 6 fish per each group were sampled in each season
69.4 ± 11.5
35.2 ± 3.9
BWI (%)
FE
3.1 ± 0.3
0.96 ± 0.2
FCR
0.04 ± 0.0
GR (kg/day)
SGR (%/day)
0.05 ± 0.002
0.68 ± 0.01
CF (%)
140.3 ± 2.7
160.1 ± 2.6
141.5 ± 3.1
160.2 ± 5.6
I L (cm)
FL (cm)
30.6 ± 1.5
0.4
13.2 ± 0.5
13.2 ± 1.2
28.2 ± 2.3
IBW (kg)
1.6
0.2
0.8
0.2
0
0.4
Equol levels (g/kg diet)
Genistein levels (g/kg diet)
FBW (kg)
Dietary treatments Growth parameter
Table 2 Growth parameters performances in H. huso fed with different concentrations of genistein and equol (mean ± SEM, n = 54)
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A
D
B
E
C
F
Fig. 1 The comparison between laparoscopy and histological studies at the same and different stages of maturation of the ovaries. A, D Oocytes at stage II, previtellogenesis, B, E oocytes
at stage III, vitellogenesis, C, F: matured oocyte at stage IV, arrows indicated the developmental changes of vitellogenin and oocytes at stages II, III and IV (H&E, 40X)
Fig. 2 Mean ± SEM of oocytes diameter at different concentrations of GE and EQ (n = 54, 6 fish per each group); A, B indicate the significant comparison between treatments at the
end of experiment. *Significant comparison between the start and the end of experiment
Fig. 3 Mean ± SEM of testosterone at different concentrations of GE and EQ (n = 54, 6 fish per each group); A, B indicate the significant comparison between treatments at the end of
experiment; *significant comparison between the start and the end of experiment
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Fig. 4 Mean ± SEM of 17b-estradiol at different concentrations of GE and EQ (n = 54, 6 fish per each group); A, B indicate the significant comparison between treatments at the end of
experiment; *significant comparison between the start and the end of experiment
Fig. 5 Mean ± SEM of 17a-hydroxy progesterone at concentrations of GE and EQ (n = 54, 6 fish per each group)
Fig. 6 Mean ± SEM of alkaline phosphatase at different concentrations of GE and EQ (n = 54, 6 fish per each group); ab indicate the significant comparison between treatments at the
start of experiment; A, B indicate the significant comparison between treatments at the end of experiment; *significant comparison between the start and the end of experiment
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Fig. 7 Mean ± SEM of calcium ion at different concentrations of GE and EQ (n = 54, 6 fish per each group); A, B indicate the significant comparison between treatments at the end of
experiment; *significant comparison between the start and the end of experiment
Fig. 8 Mean ± SEM of phosphorous at different concentrations of GE and EQ (n = 54, 6 fish per each group); ab indicate the significant comparison between treatments at the start of
experiment; A, B significant comparison between treatments at the end of experiment; *significant comparison between the start and the end of experiment
Fig. 9 Mean ± SEM of cholesterol at different concentrations of GE and EQ (n = 54, 6 fish per each group); abc indicate the significant comparison between treatments at the start of
experiment; A, B indicate the significant comparison between treatments at the end of experiment; *significant comparison between the start and the end of experiment
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at all concentrations of GE at end than the start and reached to a maximum at GE 1.6 g/kg, but in EQ was lower at the end than the start and decreased significantly at EQ 0.4 g/kg (P \ 0.05) (Fig. 7). Phosphorous ion levels change similar to calcium ion levels (Fig. 8). Cholesterol levels increased significantly at GE 1.6 g/kg and decreased significantly at EQ 0.4 at the end of experiment (Fig. 9) (P \ 0.05).
Discussion Growth performance The fish diets could be considered equivalent from the point of view of composition; the only relevant difference is phytoestrogenic activity. Growth parameters results showed that soy dietary isoflavonic phytoestrogens GE and EQ concentrations induced a growth increase about twice at the end than the start of experiment, but there was no significant difference among different concentrations. The final body weight increased significantly at all treatments at the end of experiments than start, but showed no significant effects on growth performances that indicating good balance of diet for ovary development. It appears that the effectiveness of phytoestrogens on growth is more evident for male animals, which may not be surprising when considering that they have estrogenic activity. This may also remind us that animal gender should be considered for experimental design. With conventional weaning of piglets, soy daidzein or soy GE did not have significant effects on weight gain and feed intake (Greiner et al. 2001), though daidzein could slightly improve the gain/feed ratio. Reproductive performance This is the first study to report on the estrogenic effects of soy phytoestrogens consumption on sturgeon in Iran. In this research, estrogenic effects of isoflavonic phytoestrogens GE and EQ have been investigated. Results showed that EQ supplemented to the diets at 0.4 g/kg improved significantly ovary maturation than GE. This result was confirmed by measuring reproductive indices changes such as histological and morphological changes in oocytes, increase in their diameter, increase in testosterone (T) and estradiol (E2) levels and decrease in progesterone, calcium and ALP levels that indicated
inducing vitellogenesis and oocyte development. Phytoestrogens have been reported to have many biological activities in fertilization, immunostimulation, anabolism and balancing hormones (Leusch et al. 2006). Although their structure is not related to vertebrate steroids, they are able to produce estrogen-like effects. The biological activities of phytoestrogens are usually studied on the level of ER binding and typically induce biological responses through interaction with ER, either agonist or antagonist (Sicuro et al. 2010). Phytoestrogens effects have been investigated in fish (Ng et al. 2006). Ko et al. (1999) reported that in yellow perch, GE induced VTG synthesis by the liver and had a growth promoting effect similar to that of 17b-estradiol. Phytoestrogens are able to stimulate VTG synthesis in vivo in Siberian sturgeon (Pelissero et al. 1991) and in vitro in trout hepatocyte (Pelissero et al. 1993). Latonelle et al. (2002) reported that the estrogenic potency of the isoflavones ranged differently between the two species in the following order: biochanin A \ daidzein = formononetin \ GE \ EQ in trout and biochanin A \ GE \ daidzein \ formononetin \ EQ in Siberian sturgeon (A. baerii) that was according to present study results about GE and EQ. The levels of GE utilized in striped bass (Morone saxatilis) affect the hepatic, gonadal or normal somatic growth (Pollack et al. 2003). In present study, a significant effect of soy isoflavone consumption on testosterone was observed. Doses of GE and EQ produce significant effects on some serum steroid hormones levels. Testosterone levels were elevated significantly at EQ 0.4 and 1.6 g/kg than other treatments. The blood 17b-estradiol level of Huso huso significantly increased at EQ 0.4 g/kg compared with the control and others. In Japanese medaka (Oryzias latipes) exposed intraperitoneally to GE, VTG was not induced in males and females, but plasma 17b-estradiol (E2) was increased in exposed females and plasma testosterone (T) levels were reduced in exposed males (Zhang et al. 2011). Daidzein at 3 mg/kg significantly increased progesterone levels of 330-day-old laying hens (Meng et al. 2001). The increases in testosterone and estradiol at the final stages of maturity in teleost fish (Nagahama 1994) and in different species of sturgeon (Frederiek et al. 2007) are due to decreases in aromatase enzyme activity; reductions in testosterone are controlled by gonadotropins, but time-dependent fluctuations of hormones can occur in various species (Bahmani et al. 2009). The concentration of testosterone increase in females during gametogenesis (Barannikova et al. 2004). Steroid hormones of theca
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cells in ovary (testosterone) can incorporate to granulosa cells and induce aromatase enzyme (P450) gene expression which converts testosterone for promotes ova that lead to decrease testosterone and increase estradiol hormone during gametogenesis because of increase in aromatase activity of ova and temporal changes (Zhang et al. 2011). In immature fish, sex steroid hormone levels were low but increase with maturity. Although 17bestradiol hormone is one of the most important sex steroids in females, there are also significant levels of testosterone and progesterone in blood plasma, because these hormones are precursors of estradiol (Barannikova et al. 2004). In fact, testosterone is the main hormone controlling the reproductive process in sturgeon under natural and cultural conditions (Bukovskaya et al. 1997). There is a continued and positive relationship among steroid hormones of blood plasma (Barannikova et al. 2006). In this case, cholesterol converts to pregnenolone, progesterone and finally androstenedione. It converts to 17b-estradiol after changing with testosterone in the granulosa layer (Nagahama 1994). Therefore, 17bestradiol production increases at vitellogenesis with increasing gonad development. In teleost fish, progesterone concentrations decreased with development of the ovary and gonad cells especially at vitellogenesis because ovary promotion is similar to that in sturgeon (Dahle et al. 2003). Decreases in estradiol level after vitellogenesis were may be because aromatse activity ceased resulting from completion of egg development and a sharp reduction in feedback from steroid hormones. It seems that cessation of ovary aromatic activates and no requirement for testosterone and using it for spawning is the major reason to increase testosterone hormone levels at this time. Higher levels of progesterone in more mature female breeders compared with immature breeders was because of the effectiveness of progestin (especially progesterone) in promoting oocytes in some adult teleost fish reaches a maximum at spawning time. Dietary GE and EQ could also affect content of cholesterol. So that, it decreased significantly at EQ 0.4 g/kg might be because of more consumption for supporting required energy of gonadic activities and using for steroid hormone synthesis. Dietary daidzein could affect egg content of cholesterol. Daidzein supplemented at 40 mg/kg could significantly decrease egg cholesterol content by 19 % egg yolk cholesterol concentration by 11 % as compared to control (Yin et al. 2004). In present study, ALP enzyme
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level decreased significantly in all concentrations of GE and EQ at the end than the start because of using for vitellogenesis. ALP is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. Calcium and phosphorus ion levels sowed similar changes and increased at GE concentrations but decreased at EQ concentrations at the end of experiment because of using for vitellogenesis. Phosphorous formed major ingredients of egg VTG in fish. The increase in calcium ion concentration in females was at vitellogenesis, but it decreases after completing vitellogenesis. They are in accordance with previous studies, demonstrating that the calcium ion concentration was increased with the increase in estradiol hormone in blood plasma at vitellogenesis (Stahl 2008). Results of the present study also showed the relationship between calcium ion and reproductive cycle development. Calcium ion levels of plasma increased until before spawning as a result of an increase in stannius glands in females during ovary development cycle. This increase is due to calcium ion linking to proteins (vitelline cells) of the egg (Urasa and Wendelaar Bonga 1985).
Conclusion Isoflavonic phytoestrogens can exhibit estrogenic activity on reproduction and exert ovary development at proper dosage, and improve reproductive performance of female H. huso. These effects were usually coupled with their influence on some steroid hormones especially in testosterone level, calcium and phosphorous ions and ALP enzyme. The observed effects of isoflavonic phytoestrogens may be mediated by their modulation of endocrine. EQ added to the diet increased oocyte development at certain concentrations, whereas the phytoestrogens had no significant effect on growth parameters. Equol can be added to the diet to advance gonadal development which would be beneficial to the production of caviar by aquaculture. Acknowledgments Experiments were performed in the International Sturgeon Research Organization (ISRO) of the Caspian Sea (Physiology and Biochemistry and Rearing and Propagation Depts.) and were funded by ISRO and the Gorgan University of Agricultural Sciences and Natural Resources. The authors thank all staff.
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References Bahmani M, Yousefi Jourdehi A, Kazemi R, Pourdehghani M, Hallajian A, Dejhandian S, Jalilpoor J (2009) Seasonal fluctuations of testosterone (T), 17a-hydroxy progesterone (17a-OHP), 17b-estradiol (E2) during sexual maturation in Acipenser stellatus. Iran J Fish 4:7–16 Barannikova IA, Bayounova LV, Semenkova TB (2004) Serum levels of testosterone, 11-ketotestosterone and 17b-estradiol in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment. J Fish Biol 64(5):1330–1338 Barannikova IA, Bayounova LV, Semenkova TB (2006) Serum sex steroids and their specific cytosol binding in the pituitary and gonads of Russian sturgeon (Acipenser gueldenstaetdii Brandt) during final maturation. J Appl Ichthyol 22:331–333 Bukovskaya OS, Lambert JGD, Kime DE (1997) In vitro steroidogenesis by gonads of the Russian sturgeon, Acipenser gueldenstaedti Brandt. Fish Physiol Biochem 16:345–353 Casanova M, You L, Gaido KW, Archibeque-Engle S, Janszen DB, Heck HA (1999) Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro. Toxicol Sci 51:236–244 Cassidy A, Hanley B, Lamuela-Raventos RM (2000) Isoflavones, lignans and stilbenes-origins, metabolism and potential importance to human health. J Sci Food Agric 80:1044–1062 Chen S, Kao YC, Laughton CA (1997) Binding characteristics of aromatase inhibitors and phytoestrogens to human aromatase. J Steroid Biochem Mol Biol 61:107–115 Dahle R, Taranger GL, Karlsen Ø, Kjesbu OS, Norberg B (2003) Gonadal development and associated changes in liver size and sexual steroids during the reproductive cycle of captive male and female Atlantic cod (Gadus morhua L.). Comp Biochem Physiol A Mol Integr Physiol 136(3):641–653 Dechaud H, Ravard C, Claustrat F, Perriere AB, Pugeat M (1999) Xenoestrogen interaction with human sex hormonebinding globulin (hSHBG). Steroids 64:328–334 Fostier A, Jalabert B, Billard R, Breton B, Zoar Y (1983) The gonadal steroids. In: Hoar WS, Randall DJ, Donaldson EM (eds) Fish physiology, vol IX. Academic Press, New York, pp 277–372 Frederiek J, Munday L, Westcott A, Hobbs V, Robin Liley N (2007) Aromatase pathway mediates sex change in each direction. Biol Sci 272:1399–1405 Greiner LL, Stahly TS, Stabel TJ (2001) The effect of dietary soy genistein on pig growth and viral replication during a viral challenge. J Anim Sci 79:1272–1279 Huang RQ, Fang MJ, Dillon GH (1999) The tyrosine kinase inhibitor genistein directly inhibits GABAA receptors. Brain Res Mol Brain Res 67:177–183 King RA, Bursill DB (1998) Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans. Am J Clin Nutr 67:867–872 Ko K, Malison JA, Reed JR (1999) Effect of genistein on growth and reproductive function of male and female yellow perch Perca flavescens. J World Aquac Soc 30:73–77 Latonelle K, Lemenn F, Kaushik SJ, Plessiero B (2002) Effects of dietary phytoestrogens in vivo and in vitro in rainbow
trout and Siberian sturgeon: interests and limits of the in vitro studies of interspecies differences general comparative. Endocrinology 126:39–51 Le Menn F, Nunez Rodriguez J (1991) Receptors mediate endocytosis of VTG in fish follicle. In: Proceedings of the 4th international symposium on reproductive physiology of fish, Norwich, UK, pp 300–302 Leusch FD, van den Heuvel MR, Chapman HF, Gooneratne SR, Eriksson AM, Tremblay LA (2006) Development of methods for extraction and in vitro quantification of estrogenic and androgenic activity of wastewater samples. Comp Biochem Physiol C Toxicol Pharmacol 143(1):117–126 Maclatchy DL, Van Der Kraak GJ (1995) The phytoestrogen beta-sitosterol alters the reproductive endocrine status of goldfish. Toxicol Appl Pharmacol 134:305–312 Meng T, Han ZK, Wang GJ (2001) Effect of diet supplemented with daidzein on laying chicken performance and serum estradiol levels. In: The 7th national academic conference of the animal physio-biochemistry, Zhengzhou, pp 197–198 (abstract) Nagahama Y (1994) Endocrine regulation of gametogenesis in fish. Int J Dev Biol 38:217–229 Ng Y, Hanson S, Malison JA, Wentworth B, Barry TP (2006) Genistein and other isoflavones found in soybeans inhibit estrogen metabolism in salmonid fish. Aquaculture 254:658–665 Pelissero C, Bennetau B, Babin P, Le Menn F, Dunogues J (1991) The estrogenic activity of certain phytoestrogens in the Siberian sturgeon Acipenser baeri. J Steroid Biochem Mol Biol 38:292–299 Pelissero C, Flouriot G, Foucher JL, Bennetau B, Dunogues J, Le Gac F, Sumpter JP (1993) Vitellogenin synthesis in cultured hepatocytes: an in vitro test for the estrogenic potency of chemicals. J Steroid Biochem Mol Biol 44:263–272 Pelissero C, Breton B, Bennetau B, Corraze G, Le Menn F, Cuisset B, Helou C, Kaushik SJ (2001) Effect of genistein enriched diets on the endocrine process of gametogenesis and on reproduction efficiency of the rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol 121:173–187 Pollack SJ, Mary AO, Sullivan VC, Woods LC (2003) The effects of the soy isoflavone genistein on the reproductive development of striped bass. N Am J Aquac 65:226–234 Pottinger TG, Carrick TR (2001) ACTH does not mediate divergent stress responsiveness in rainbow trout. Comp Biochem Physiol 129:399–404 Schoefer L, Mohan R, Braune A, Birringer M, Blaut M (2002) Anaerobic C-ring cleavage of genistein and daidzein by Eubacterium ramulus. FEMS Microbiol Lett 208:197–202 Sicuro B, Badino P, Dapra F, Gai F, Galloni M, Odore R, Palmegiano GB, Macchi E (2010) Physiological effects of natural olive oil antioxidants utilization in rain bow trout (Oncorhynchus mykiss). Aquacult Int 18:415–431 So Y, Idler DR, Truscott B, Walsh JM (1985) Progesterone and androgens and their glucuronides in the terminal stages of oocyte maturation in Atlantic salmon. Steroid Biochem 23:583–891 Stahl MT (2008) Reproductive physiology of Shovelnose sturgeon from the middle Mississippi River in relation to seasonal variation in plasma sex steroids, vitellogenin, calcium, and oocyte diameters, chap 2. Use of sex hormone
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Fish Physiol Biochem profiles to determine sexual maturation in Shovelnose sturgeon. Southern Illinois University Carbondale, p 28 Urasa FM, Wendelaar Bonga SE (1985) Stannius corpuscles and plasma calcium levels during the reproductive cycle in the cichlid teleost fish, Oreochromis mossambicus. Cell Tiss Res 241:219–227 Wallace RA (1985) Oocyte growth in non mammalian vertebrates. In: Jones RE (ed) The vertebrate ovary. Plenum, New York, pp 469–502 Wang XL, Hur HG, Lee JH, Kim KT, Kim SI (2005) Enantioselective synthesis of S-equol from dihydrodaidzein by a
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newly isolated anaerobic human intestinal bacterium. Appl Environ Microbiol 71:214–219 Yin JD, Qi GH, Zheng JJ, Huo QG (2003) Influence of dietary isoflavonoids on stability of egg yolk lipids. Sci Agricultura Sinica 36:438–442 Zhang Y, Doroshov S, Famula T, Conte F, Kueltz D, LinartesCasenave J, Van Eenennaam J, Struffenegger P, Beer K, Murata K (2011) Egg quality and plasma testosterone and estradiol-17b in white sturgeon (Acipenser transmontanus) farmed for caviar. J Appl Ichthyol 27:558–564
Comparative study of dietary soy phytoestrogens genistein and equol effects on growth parameters and ovarian development in farmed female beluga sturgeon, Huso huso A. Yousefi Jourdehi • M. Sudagar • M. Bahmani • S. A. Hosseini • A. A. Dehghani • M. A. Yazdani
Received: 21 February 2013 / Accepted: 24 June 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Oocyte maturation in fish is a hormonally regulated process. In the light of long-term oocyte maturation in beluga, the aim of this research was to study the estrogenic effects of different concentrations of soy dietary genistein (GE) and equol (EQ) on the growth performance and ovary development in farmed female Huso huso. Fish were fed with concentrations 0 (control), 0.2, 0.4, 0.8 and 1.6 g of EQ and GE per kg of isoproteic (CP 45 %) and isoenergetic (19.5 MJ/kg) diets during a year. Blood samples and ovary biopsies were collected from each fish seasonally. The main results of the present experimentation are that growth performance was not affected significantly both in GE and EQ (P [ 0.05). EQ at concentration 0.4 g/kg had more estrogenic effects than other concentrations of EQ and GE in beluga so that 64 % of fish were matured sexually. Some reproductive indices such as oocyte diameter, testosterone (T) and 17b-estradiol (E2)
increased significantly at EQ 0.4 g/kg at the end of experiment (P \ 0.05), while 17a-hydroxy progesterone level (17a-OHP) showed no significant changes at all concentrations. Biochemical indices such as calcium, phosphorous and cholesterol increased at GE concentrations, but decreased at EQ concentrations similarly at the end of experiment. There was a negative relationship between plasma phosphorous and alkaline phosphatase enzyme levels. Based on results, EQ at concentration 0.4 g/kg improved oocyte development more than the other concentrations of GE and EQ, and therefore, it can be used as an additive to diets for inducing ovary development in this species.
A. Yousefi Jourdehi (&) M. Sudagar The University of Gorgan Agricultural Sciences and Natural Resources, Gorgan, Iran e-mail: [email protected]
S. A. Hosseini Ecology, The University of Gorgan Agricultural Sciences and Natural Resources, Gorgan, Iran
Keywords Phytoestrogens Oocyte maturation stages Sex steroid hormones Biochemical indices Huso huso
A. Yousefi Jourdehi International Sturgeon Research Institute of the Caspian Sea, Rasht, Iran
A. A. Dehghani Water Engineering, The University of Gorgan Agricultural Sciences and Natural Resources, Gorgan, Iran
M. Bahmani Department of Fish Physiology and Biochemistry and Rearing and Propagation, International Sturgeon Research Institute of the Caspian Sea, Rasht, Iran
M. A. Yazdani Department of Sturgeon Fish Rearing and Propagation, International Sturgeon Research Institute of the Caspian Sea, Rasht, Iran
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Introduction Phytoestrogens are by definition plant-derived substances that are able to activate the estrogen receptors (ER). Most of these plant substances are to some extent similar to the estrogen either in structure or function (Cassidy et al. 2000). Isoflavones are a group of phytoestrogens and can be found in many plants, particularly in the legume family. The major constituents of soy isoflavones, genistein (GE) and equol (EQ) interact with the ER in several tissues including gonads. GE and daidzein are two of the major metabolites from isoflavones, but can be further metabolized by intestinal microorganisms (Schoefer et al. 2002; Wang et al. 2005). Daidzein can be metabolized to EQ (King and Bursill 1998). Steroid hormones are the most important hormones for improving reproduction in fishes (So et al. 1985). Changes in the concentration of serum steroid hormones have been used as indicators of reproductive response in fish. Vitellogenin (VTG), the major constituent of yolk, is synthesized in the liver under estrogenic induction (Wallace 1985). VTG is then carried in blood to the ovaries, where it is sequestered by the developing oocytes by binding to specific receptors associated with endocytotic vesicles (Le Menn and Nunez Rodriguez 1991). In fish, only reproductively active females normally synthesize VTG. Estradiol is the main inducer during the reproductive cycle (Fostier et al. 1983). A large number of natural or synthetic chemicals present either in food or in the environment. The estrogenic effects of phytoestrogens are well documented in fish (Pelissero et al. 2001). Phytoestrogens can bind to steroid-binding proteins (Dechaud et al. 1999) and to ER of target cells (Casanova et al. 1999). Moreover, they exhibit endocrine-disturbing activity interfering with enzymatic reactions either on steroid metabolism, i.e., aromatization (Chen et al. 1997) or on the mechanism of action of estrogens, i.e., tyrosine kinase activity (Huang et al. 1999). As a result, phytoestrogens can induce reproductive disorders (Maclatchy and Van Der Kraak 1995). Phytoestrogens are able to stimulate VTG synthesis in vivo in Siberian sturgeon (Pelissero et al. 1991) and in vitro in trout hepatocyte (Pelissero et al. 1993). There have been very few
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studies on the effects of GE and EQ in teleosts. Intraperitoneal injections of GE and EQ to yearling Siberian sturgeon (Acipenser baerii) induced vitellogenesis, a physiological response associated with estrogenic activity (Pelissero et al. 1991). GEenriched diets were fed to rainbow trout (Oncorhynchus mykiss) for a year, and gametogenesis and reproductive efficiency of the trout were reduced (Pelissero et al. 2001). The beluga sturgeon (Huso huso) is the largest growing sturgeon species, one of the largest fish found in freshwater. The beluga is now extensively farmed to produce caviar. Due to the long reproductive cycle (18-year-old in females) in beluga of the Caspian Sea in nature, many countries such as Iran like to farm this species in fresh waters. Considering the last maturity of beluga, this study was carried out with the aims of using dietary soy phytoestrogens GE and EQ for increase oocyte development in females of beluga.
Materials and methods Fishes and experimental design Experiments were carried out on 54 female H. huso 5-year-old with average body weight 13.25 ± 0.3 kg and total length 140.3 ± 1.2 cm that had been farmed in terrestrial ponds of the Caspian Sea International Sturgeon Research Organization during a year (from summer 2011 to summer 2012). Ovary of all fish was at sage II of sexual maturation at the start of the experiment. Fish were anesthetized using a Clove powder 250 ppm for 10 min and selected by biopsy and laparoscopy methods (using a Stema Company, model M-CAM1700, 30 degree telescope with 4 mm in size and with length 17.5 cm and with the cold light source a halogen 250 W, Germany) and were stocked randomly in 18 concrete ponds (with dimensions 3 9 2.5 9 1.2 m3) equipped with aeration system and hole mixed the Sepidrood River water together. Fishes were treated with a practical beluga diet was formulated to contain at concentrations of 0, 0.2, 0.4, 0.8 and 1.6 g/kg of purified GE and EQ (Xi, and Keen— Source Biotechnology Co., Ltd). GE and EQ were
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dissolved in 75 ml ethanol and added to the diet. Diets were mixed by a mixer in 2,000-g portions for 7–10 min after adding 15 % of water. Food pellets were prepared in diameter 12 mm with a standard meat grinder. Before preparing each diet, 0.5 kg was run through the extruder and discarded to remove any residue from the previous diets. The diets were mixed from low to high levels of GE to minimize the overlap in GE and EQ concentrations. They were air-dried 24 h. Each treatment includes 6 fish in two replicates with 3 fish per each concrete pond. One percent of biomass and twice daily (0800 and 1600 h), fishes received a diet based on fish meal 58 %, wheat meal 20 %, meat powder 15 %, fish oil 2.5 %, mineral supplements 1 %, vitamin supplements 1 %, vitamin E 1 % and calcium phosphate 1 % consisted of crude protein (CP 45 %), fat 14 %, carbohydrate 20 %, ash 9 %, moisture \10 % and energy (19.5 MJ of dry matter). Physicochemical parameters were measured by using Oxi-pHmeter40i (WTW, Germany) daily. Oocyte diameter was measured using a stereomicroscope. For histological studies, ovaries samples were fixed in Bouin’s fluid during 48 h and were dehydrated, paraffinated, mounted, sectioned (5–7 lm) by Microtome set (Leitz 1512, Germany), stained by hematoxylin and eosin (H&E) and studied by light microscope. Blood samples (5 ml) were taken from a caudal vein of fish seasonally.
Blood analyses Blood serum was separated by using a centrifuge (Labofuge 200, Heraeus Sepatech, Germany) with 3,000 rpm per 10 min. Samples were stored at -20 °C (Pottinger and Carrick 2001). Sex steroid hormone levels (including testosterone (T), 17a-hydroxy progesterone and 17b-estradiol) and alkaline phosphatase enzyme (ALP) level were measured using radio-immune assay (RIA) with Immunotech kit and I125 (France) and gamma counter LKB (Finland) based on ng/ml in the Dr. Fadaie laboratory (Rasht, Iran). Total calcium and phosphorous were analyzed using a photometric technique (Roche Modular automated clinical chemistry analyzer). Plasma cholesterol was measured by standard enzymatic method (Pars Azmoon kit, Pars Azmoon Inc., Tehran, Iran) using a spectrophotometer with wave length 570 nm at the hematology laboratory.
Growth data calculation CF ¼ BWf = FL3 100 BWf ¼ final body weight and FL ¼ final length BWI ¼ 100 ðBWf BWi Þ=BWi BWf ¼ final body weight and BWi ¼ initial body weight SGR ¼ ðln Wf ln Wi Þ=t 100 Wf ¼ final fish weight and Wi ¼ initial fish weight; and t ¼ day FCR ¼ F=ðWf Wi ÞF ¼ food; Wi ¼ final fish weight and Wi ¼ initial fish weight PER ¼ ðBWf BWi Þ=protein intake BWf ¼ final body weight and BWi ¼ initial body weight Statistical analysis Minimum, maximum, variance and standard error were used to describe biochemical indices. One-way ANOVA was used for comparison between 2 groups, and Duncan’s test was used for differential studies. Levene’s test was used to test for equality of variance. An independent-sampled t test was used to detect differences between hormonal and biochemical indices. Excel and SPSS17 software were used for data analysis and drawing diagrams. Data are presented as mean ± SEM. Results Physicochemical and growth performance results The minimum and maximum of temperature were 11.2 ± 1.5 °C in winter and 22.5 ± 1.6 °C in summer, respectively. Dissolved oxygen (O2) was decreased with increase in temperature and reached to a minimum in summer (Table 1). Growth parameters results showed that the mean initial weight and length of H. huso were 13.25 ± 0.3 kg and 140.3 ± 1.2 cm and reached to 29.26 ± 0.6 kg and 163.4 ± 1.3 cm at the end of experiment, respectively (P \ 0.05), but showed no significant differences between GE and EQ concentrations and controls (P [ 0.05). There were no significant
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Fish Physiol Biochem Table 1 Water physicochemical parameters change during different seasons (mean ± SEM) Seasons
Physiochemical parameters Temperature (°C)
O2 (mg/l)
pH
Spring
17.4 ± 1.9
7 ± 0.2
7.8 ± 0.3
Summer
22.5 ± 1.6
6.2 ± 0.5
7.8 ± 0.3
Autumn
16.9 ± 2
8.2 ± 0.2
7.7 ± 0.3
Winter
11.2 ± 1.5
8.8 ± 0.2
7.9 ± 0.3
differences in growth rate (GR), food conversion ratio (FCR), specific growth ratio (SGR), condition factor (CF), feed efficiency (FE) and protein conversion ratio (PER) between the treatments and corresponding controls (P [ 0.05) (Table 2). Laparoscopic and Histological Observations Stage II Oocytes are not round but orthogonal. Yolk resembles a narrow strip. The protoplasmic growth and increase in oocyte diameter are detectable in this stage. Nucleoli are completely stuck to the membrane of nucleus. A chromatin mass is observed in the center of nucleus. Number of nucleolus close to the membrane of nucleus is raised, and vacuoles are observed around nucleus inside the cytoplasm. Animal pole is not differentiated from vegetable pole. Appearance of pigments in lateral layer of oocyte cytoplasm is the most obvious index of transition from stage II. Stage III In this stage, growth of oocytes is associated with reduction in fat layer. Along with thickening of oocytes, pigments and fat tissue are observed in the middle and lateral parts of gonads; however, do not completely fill up the body cavity. Pigments are dark gray in color and formed beneath the cell membrane. Oocytes firmly adhere to the fat layer of ovary. As a result, they do not pass through special caviar processing sieves. The ovary is fully grown in volume, oocytes are visible with naked eyes but yet indistinguishable from each other and blood vessels are clearly dispersed. A thick layer of fat is present in the middle part of ovary while oocyte is found in lateral area of ovary. In comparison with the previous stage, oocytes abundance and volume are
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higher, fat is reduced, and oocytes pass through the sieve in this stage. Stage IV Yolk granules almost fill the whole space around nucleus, and only a tiny cytoplasm is spread out near the nucleolus and oocyte membranes. Small yolk granules and nucleus are located in animal pole while larger yolk granules and fat droplets in vegetal pole. Nucleus is positioned from cell center toward animal pole at micropyle site. Nucleoli are mostly situated at the center although few are distributed in different areas of the cell. Based on histological study on the ovary of H. huso, 9 major layers (from outside to inside) are discernible at stage IV of maturity: follicle epithelial layer, jelly coat, external zona radiata, internal zona radiata, fat layer, pigments, cytoplasm, nucleus, nucleoli and micropyle (Fig. 1A–F). Oocyte diameter Oocyte diameter increased significantly at all concentrations of GE and EQ except GE 1.6 g/kg than control. At the end of experiment, it increased significantly at EQ 0.4 g/kg (reached to a maximum 3.1 ± 0.2 mm) and 1.6 g/kg than others (P \ 0.05) (Fig. 2). Sex steroid hormones Testosterone level increased significantly at the end of experiment than start at all concentrations. At the end of experiments, testosterone level reached to a maximum at EQ 0.4 g/kg (P \ 0.05) (Fig. 3). 17b-estradiol (E2) levels were more at all treatments at the end than start of experiment and showed significant increase at EQ 0.4 g/kg at the end of experiment (Fig. 4). 17a-hydroxy progesterone levels were more at the end of experiment than start but showed no significant difference between the start and the end at all concentrations of GE and EQ (P [ 0.05) (Fig. 5). Biochemical indices results Alkaline phosphates enzyme (ALP) levels decreased significantly at all concentrations of GE and EQ at the end than the start and was maximum at EQ 0.4 g/kg (P \ 0.05) (Fig. 6). Calcium ion levels were increased
2.9 ± 0.2
0.16 ± 0.01
PER
0.16 ± 0.01
36.8 ± 2.4
69.2 ± 11.8
0.93 ± 0.2
0.74 ± 0.02
13.3 ± 1.1
0.18 ± 0.02
40.3 ± 3.5
84.3 ± 18.5
0.99 ± 0.2
2.6 ± 0.3
0.05 ± 0.004
0.6 ± 0.01
170.1 ± 2.6
141.2 ± 3.4
29.1 ± 2
13.2 ± 0.7
0.19 ± 0.02
40.3 ± 4.2
82.3 ± 11.6
1 ± 0.2
2.6 ± 0.4
0.05 ± 0.005
0.64 ± 0.01
167.7 ± 5.8
144.2 ± 4.4
30.4 ± 1.3
13.2 ± 0.7
0.17 ± 0.02
41.9 ± 4.6
82.9 ± 13.6
1 ± 0.2
2.8 ± 0.3
0.056 ± 0.006
0.71 ± 0.02
162.1 ± 3.9
143.1 ± 4.5
30.4 ± 1.3
13.3 ± 1.1
0.16 ± 0.01
35.4 ± 2.4
79.4 ± 12.6
0.98 ± 0.2
2.7 ± 0.3
0.05 ± 0.005
0.71 ± 0.02
160.3 ± 2
136.2 ± 4.3
29.6 ± 1.9
13.2 ± 1.1
0.15 ± 0.02
38.4 ± 3.8
76.1 ± 13.8
0.98 ± 0.2
2.9 ± 0.2
0.04 ± 0.003
0.67 ± 0.01
163.5 ± 4.2
140.9 ± 2.8
29.6 ± 2.6
0.16 ± 0.01
35.9 ± 2.3
76.3 ± 11.9
0.98 ± 0.2
2.8 ± 0.2
0.047 ± 0.003
0.64 ± 0.01
162.2 ± 3.2
137.2 ± 3.2
27.5 ± 1.8
13.3 ± 0.7
0.8
0.16 ± 0.01
36.9 ± 1.2
80.9 ± 15.1
0.97 ± 0.2
2.7 ± 0.1
0.05 ± 0.002
0.69 ± 0.02
163.6 ± 2.3
138.1 ± 4.6
30.3 ± 1.7
13.3 ± 1.1
1.6
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
Significant
IBW initial body weight, FBW final body weight, IL initial length, FL final length, CF condition factor, GR growth rate, FCR feed conversion ratio, SGR specific growth rate, BW body weight, FER feed efficiency ratio, PER protein efficiency rate
A total of 54 fish including 6 fish per each group were sampled in each season
69.4 ± 11.5
35.2 ± 3.9
BWI (%)
FE
3.1 ± 0.3
0.96 ± 0.2
FCR
0.04 ± 0.0
GR (kg/day)
SGR (%/day)
0.05 ± 0.002
0.68 ± 0.01
CF (%)
140.3 ± 2.7
160.1 ± 2.6
141.5 ± 3.1
160.2 ± 5.6
I L (cm)
FL (cm)
30.6 ± 1.5
0.4
13.2 ± 0.5
13.2 ± 1.2
28.2 ± 2.3
IBW (kg)
1.6
0.2
0.8
0.2
0
0.4
Equol levels (g/kg diet)
Genistein levels (g/kg diet)
FBW (kg)
Dietary treatments Growth parameter
Table 2 Growth parameters performances in H. huso fed with different concentrations of genistein and equol (mean ± SEM, n = 54)
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A
D
B
E
C
F
Fig. 1 The comparison between laparoscopy and histological studies at the same and different stages of maturation of the ovaries. A, D Oocytes at stage II, previtellogenesis, B, E oocytes
at stage III, vitellogenesis, C, F: matured oocyte at stage IV, arrows indicated the developmental changes of vitellogenin and oocytes at stages II, III and IV (H&E, 40X)
Fig. 2 Mean ± SEM of oocytes diameter at different concentrations of GE and EQ (n = 54, 6 fish per each group); A, B indicate the significant comparison between treatments at the
end of experiment. *Significant comparison between the start and the end of experiment
Fig. 3 Mean ± SEM of testosterone at different concentrations of GE and EQ (n = 54, 6 fish per each group); A, B indicate the significant comparison between treatments at the end of
experiment; *significant comparison between the start and the end of experiment
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Fig. 4 Mean ± SEM of 17b-estradiol at different concentrations of GE and EQ (n = 54, 6 fish per each group); A, B indicate the significant comparison between treatments at the end of
experiment; *significant comparison between the start and the end of experiment
Fig. 5 Mean ± SEM of 17a-hydroxy progesterone at concentrations of GE and EQ (n = 54, 6 fish per each group)
Fig. 6 Mean ± SEM of alkaline phosphatase at different concentrations of GE and EQ (n = 54, 6 fish per each group); ab indicate the significant comparison between treatments at the
start of experiment; A, B indicate the significant comparison between treatments at the end of experiment; *significant comparison between the start and the end of experiment
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Fig. 7 Mean ± SEM of calcium ion at different concentrations of GE and EQ (n = 54, 6 fish per each group); A, B indicate the significant comparison between treatments at the end of
experiment; *significant comparison between the start and the end of experiment
Fig. 8 Mean ± SEM of phosphorous at different concentrations of GE and EQ (n = 54, 6 fish per each group); ab indicate the significant comparison between treatments at the start of
experiment; A, B significant comparison between treatments at the end of experiment; *significant comparison between the start and the end of experiment
Fig. 9 Mean ± SEM of cholesterol at different concentrations of GE and EQ (n = 54, 6 fish per each group); abc indicate the significant comparison between treatments at the start of
experiment; A, B indicate the significant comparison between treatments at the end of experiment; *significant comparison between the start and the end of experiment
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at all concentrations of GE at end than the start and reached to a maximum at GE 1.6 g/kg, but in EQ was lower at the end than the start and decreased significantly at EQ 0.4 g/kg (P \ 0.05) (Fig. 7). Phosphorous ion levels change similar to calcium ion levels (Fig. 8). Cholesterol levels increased significantly at GE 1.6 g/kg and decreased significantly at EQ 0.4 at the end of experiment (Fig. 9) (P \ 0.05).
Discussion Growth performance The fish diets could be considered equivalent from the point of view of composition; the only relevant difference is phytoestrogenic activity. Growth parameters results showed that soy dietary isoflavonic phytoestrogens GE and EQ concentrations induced a growth increase about twice at the end than the start of experiment, but there was no significant difference among different concentrations. The final body weight increased significantly at all treatments at the end of experiments than start, but showed no significant effects on growth performances that indicating good balance of diet for ovary development. It appears that the effectiveness of phytoestrogens on growth is more evident for male animals, which may not be surprising when considering that they have estrogenic activity. This may also remind us that animal gender should be considered for experimental design. With conventional weaning of piglets, soy daidzein or soy GE did not have significant effects on weight gain and feed intake (Greiner et al. 2001), though daidzein could slightly improve the gain/feed ratio. Reproductive performance This is the first study to report on the estrogenic effects of soy phytoestrogens consumption on sturgeon in Iran. In this research, estrogenic effects of isoflavonic phytoestrogens GE and EQ have been investigated. Results showed that EQ supplemented to the diets at 0.4 g/kg improved significantly ovary maturation than GE. This result was confirmed by measuring reproductive indices changes such as histological and morphological changes in oocytes, increase in their diameter, increase in testosterone (T) and estradiol (E2) levels and decrease in progesterone, calcium and ALP levels that indicated
inducing vitellogenesis and oocyte development. Phytoestrogens have been reported to have many biological activities in fertilization, immunostimulation, anabolism and balancing hormones (Leusch et al. 2006). Although their structure is not related to vertebrate steroids, they are able to produce estrogen-like effects. The biological activities of phytoestrogens are usually studied on the level of ER binding and typically induce biological responses through interaction with ER, either agonist or antagonist (Sicuro et al. 2010). Phytoestrogens effects have been investigated in fish (Ng et al. 2006). Ko et al. (1999) reported that in yellow perch, GE induced VTG synthesis by the liver and had a growth promoting effect similar to that of 17b-estradiol. Phytoestrogens are able to stimulate VTG synthesis in vivo in Siberian sturgeon (Pelissero et al. 1991) and in vitro in trout hepatocyte (Pelissero et al. 1993). Latonelle et al. (2002) reported that the estrogenic potency of the isoflavones ranged differently between the two species in the following order: biochanin A \ daidzein = formononetin \ GE \ EQ in trout and biochanin A \ GE \ daidzein \ formononetin \ EQ in Siberian sturgeon (A. baerii) that was according to present study results about GE and EQ. The levels of GE utilized in striped bass (Morone saxatilis) affect the hepatic, gonadal or normal somatic growth (Pollack et al. 2003). In present study, a significant effect of soy isoflavone consumption on testosterone was observed. Doses of GE and EQ produce significant effects on some serum steroid hormones levels. Testosterone levels were elevated significantly at EQ 0.4 and 1.6 g/kg than other treatments. The blood 17b-estradiol level of Huso huso significantly increased at EQ 0.4 g/kg compared with the control and others. In Japanese medaka (Oryzias latipes) exposed intraperitoneally to GE, VTG was not induced in males and females, but plasma 17b-estradiol (E2) was increased in exposed females and plasma testosterone (T) levels were reduced in exposed males (Zhang et al. 2011). Daidzein at 3 mg/kg significantly increased progesterone levels of 330-day-old laying hens (Meng et al. 2001). The increases in testosterone and estradiol at the final stages of maturity in teleost fish (Nagahama 1994) and in different species of sturgeon (Frederiek et al. 2007) are due to decreases in aromatase enzyme activity; reductions in testosterone are controlled by gonadotropins, but time-dependent fluctuations of hormones can occur in various species (Bahmani et al. 2009). The concentration of testosterone increase in females during gametogenesis (Barannikova et al. 2004). Steroid hormones of theca
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cells in ovary (testosterone) can incorporate to granulosa cells and induce aromatase enzyme (P450) gene expression which converts testosterone for promotes ova that lead to decrease testosterone and increase estradiol hormone during gametogenesis because of increase in aromatase activity of ova and temporal changes (Zhang et al. 2011). In immature fish, sex steroid hormone levels were low but increase with maturity. Although 17bestradiol hormone is one of the most important sex steroids in females, there are also significant levels of testosterone and progesterone in blood plasma, because these hormones are precursors of estradiol (Barannikova et al. 2004). In fact, testosterone is the main hormone controlling the reproductive process in sturgeon under natural and cultural conditions (Bukovskaya et al. 1997). There is a continued and positive relationship among steroid hormones of blood plasma (Barannikova et al. 2006). In this case, cholesterol converts to pregnenolone, progesterone and finally androstenedione. It converts to 17b-estradiol after changing with testosterone in the granulosa layer (Nagahama 1994). Therefore, 17bestradiol production increases at vitellogenesis with increasing gonad development. In teleost fish, progesterone concentrations decreased with development of the ovary and gonad cells especially at vitellogenesis because ovary promotion is similar to that in sturgeon (Dahle et al. 2003). Decreases in estradiol level after vitellogenesis were may be because aromatse activity ceased resulting from completion of egg development and a sharp reduction in feedback from steroid hormones. It seems that cessation of ovary aromatic activates and no requirement for testosterone and using it for spawning is the major reason to increase testosterone hormone levels at this time. Higher levels of progesterone in more mature female breeders compared with immature breeders was because of the effectiveness of progestin (especially progesterone) in promoting oocytes in some adult teleost fish reaches a maximum at spawning time. Dietary GE and EQ could also affect content of cholesterol. So that, it decreased significantly at EQ 0.4 g/kg might be because of more consumption for supporting required energy of gonadic activities and using for steroid hormone synthesis. Dietary daidzein could affect egg content of cholesterol. Daidzein supplemented at 40 mg/kg could significantly decrease egg cholesterol content by 19 % egg yolk cholesterol concentration by 11 % as compared to control (Yin et al. 2004). In present study, ALP enzyme
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level decreased significantly in all concentrations of GE and EQ at the end than the start because of using for vitellogenesis. ALP is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. Calcium and phosphorus ion levels sowed similar changes and increased at GE concentrations but decreased at EQ concentrations at the end of experiment because of using for vitellogenesis. Phosphorous formed major ingredients of egg VTG in fish. The increase in calcium ion concentration in females was at vitellogenesis, but it decreases after completing vitellogenesis. They are in accordance with previous studies, demonstrating that the calcium ion concentration was increased with the increase in estradiol hormone in blood plasma at vitellogenesis (Stahl 2008). Results of the present study also showed the relationship between calcium ion and reproductive cycle development. Calcium ion levels of plasma increased until before spawning as a result of an increase in stannius glands in females during ovary development cycle. This increase is due to calcium ion linking to proteins (vitelline cells) of the egg (Urasa and Wendelaar Bonga 1985).
Conclusion Isoflavonic phytoestrogens can exhibit estrogenic activity on reproduction and exert ovary development at proper dosage, and improve reproductive performance of female H. huso. These effects were usually coupled with their influence on some steroid hormones especially in testosterone level, calcium and phosphorous ions and ALP enzyme. The observed effects of isoflavonic phytoestrogens may be mediated by their modulation of endocrine. EQ added to the diet increased oocyte development at certain concentrations, whereas the phytoestrogens had no significant effect on growth parameters. Equol can be added to the diet to advance gonadal development which would be beneficial to the production of caviar by aquaculture. Acknowledgments Experiments were performed in the International Sturgeon Research Organization (ISRO) of the Caspian Sea (Physiology and Biochemistry and Rearing and Propagation Depts.) and were funded by ISRO and the Gorgan University of Agricultural Sciences and Natural Resources. The authors thank all staff.
Fish Physiol Biochem
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