In order to avoid the invasiveness of sexing associated with biopsy in beluga (Huso huso), we explored the possibilities of using sex steroid levels to sex farmed immature fish. Beluga were selected randomly from 2, 4 and 5-year-old stocks, cultured under the same environmental and feeding regimes. Conventional methods of sexing by biopsy and histological observation were used to determine the sex and gonadal stage, while enzyme-linked immunosorbent assay was employed to measure the levels of testosterone (T) and 17ï¢-estradiol (E2) in serum. All fish had differentiated gonads and males and females could be distinguished microscopically in all age groups. T levels differed significantly between males and females, but made for poor predictors of sex due to the substantial overlap in ranges of T levels in both sexes. There was no significant difference in E2 levels between males and females. When calculating the T:E2 ratios (TER), a clear separation was obtained for 5-year-old fish, ratios over 40 predicting male, and those below 40 predicting a female phenotype. The study demonstrated that the TER can be used as an effective indicator to separate males and females in cultured immature 5-year-old beluga.
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In sturgeon aquaculture the industry, it is important to identify the sex of individual fish at an early age in order to exploit males for meat production and females for caviar production or as broodstock (Williot et al., 2001; Fiest et al., 2004). The sex of these fish cannot be determined externally until the pre-spawning phase, and therefore, sturgeon have been sexed by biopsy during minor surgery (Doroshov et al., 1994). In Iran, such surgical tests have similarly been used for sexing of farmed sturgeon (Bahmani & Kazemi, 1998). While this method is straight-forward and useful for unambiguous identification of the sex of individual fish, the procedure is time consuming. Furthermore, the invasiveness of the procedure is likely to induce substantial stress for the fish, not in the least due to the possibility of infections, causing productivity losses.
Biochemical tests for solutes with sex-specific profiles, notably sex steroids, may be more rapid, cause less stress and be less invasive to the fish (Semenkova et al., 2006; Mojazi Amiri et al., 1996a,b). Concentrations of testosterone (T) usually increase at the onset of meiosis in males and during oocyte growth in females (Cuisset et al., 1995) and remain high till spermiation or ovarian maturation (Cuisset et al., 1995; Mojazi Amiri et al., 1996a, b; Webb et al., 2002a, b). Serum 17ï¢-estradiol (E2) levels increases during vitellogenesis in females, but remain low in the males at any stage of testicular development (Webb et al., 2002a, b; Semenkova et al., 2006). Differences in sex steroid profiles between the sexes have been used in several acipenserids (e.g., Feist et al., 2004).
In this study, the suitability of sex steroid profiling (T and E2) of farmed immature beluga, Huso huso, as a sexing tool was evaluated (Webb et al., 2002a; Fiest et al., 2004; Malekzadeh Viayeh et al., 2006) in order to provide a tool for sturgeon culturists.
Materials and Methods
The fish used in this study were two, four and five-years-old farmed beluga reared in the Propagation and Culture Center of Shahid Marjani near Gorgan city, North-Eastern Iran. These fish were kept in fresh water in separate ponds (1500 m2, depth 1.2m; loading density 2kg/m2) depending on age. Thirty fish were selected randomly from each age class. Environmental measures were constant during the culture period (water temperature: 9.7Â°C, DO: 6.5 mg/l, salinity: 4 ppt, nitrate: 0.02 mg/l, phosphate: 0.03 mg/l, pH: 7.9).
Blood sampling procedure
Immediately after removing fish from ponds and anaesthetized with clove powder (150 ppm), biometrical factors were recorded (Fish at age 2 yr: n=30, body weight (BW) 4.32Â±0.78 kg, body length (BL) 94Â±6.13 cm; at age 4 yr: n=30, BW 9.25Â±1.99 kg, BL 118.63Â±74 cm; and at age 5 yr: n=30, BW 15.48Â±1.98 kg, BL 137.47Â±5.94 cm). Thereafter, blood samples were collected from the caudal vein using a 5 ml syringe (inserted behind the anal fin). Blood was centrifuged (Behdad type: BH-1200, Iran) at 3000 rpm for 20 minutes, and serum collected and stored frozen at -20°C until assay for steroids.
Sera Testosterone and Estradiol concentrations (ng/ml) were determined, using Enzyme Linked Immuno Sorbent Assay (ELISA) method (The EiAsy Way Estradiol and The EiAsy Way Testosterone, kits, Diagnostic Biochem Canada Inc, Ontario, Canada) according to Bayunova et al. (2002) and Semenkova et al. (2002), respectively. The assay sensitivity for Testosterone was 0.022ng/mL and for Estradiol 10pg/mL. The intra assay coefficient of variation for Testosterone and Estradiol were 9.1% and 7.7% respectively. According the kit insert for Testosterone, the high cross reactivity was related to 5-a dihydro testosterone (5.2%) and other androgen have less than 1% crosses reaction and about Estradiol, estriol has 1.6% and other estrogens less than 1% cross reaction.
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A small part of one gonad was removed from each anaesthetized fish by biopsy and preserved in Bouin's solution for histological examination to determine the sex and gonadal stage. Conventional histological methods were used to prepare gonadal tissues for observation and stage was determined according to Mojazi Amiri et al. (1996a, b). Briefly, the following stages were identified in males: Stage Î™ = increasing numbers of spermatogonia by mitosis (spermatogonial proliferation); Stage Î™Î™ = seminal lobules filled with primary and secondary spermatocytes and low numbers of spermatids (early spermatogenesis); and Stage Î™Î™Î™ = the presence of many spermatids and small populations of spermatozoa in the central part of lobules (mid-spermatogenesis). In females, ovaries were characterized as Stage Î™ = smallest oocytes about 10-60 Âµm in diameter with a large nucleus (chromatin nucleus stage); Stage Î™Î™ = oocytes about 60-200 Âµm in diameter with distribution of nucleoli around the inner part of the nuclear envelope (perinucleolus stage); and Stage Î™Î™Î™ = oocytes about 200-480 Âµm in diameter with the appearance of oil droplets at the periphery of the nucleus in a cytoplasm (oil droplet stage).
After testing for normality, steroid data were subjected to independent samples t test using the software package SPSS16. Furthermore, the T to E2 ratio (TER) was calculated for each fish. Steroid and TER data for both sexes were presented as scatter plots against age to assess to efficacy of each variable as a sex identifier.
Based on histological observations of gonads from 2-year old fish, 89% of males were in stage I and 11% in stage II, while in females, 18% were in stage I, 73% in stage II and 9% in stage III. In 4-year olds, 11% of male fish were in stage I, 78% in stage II and 11% in stage VI (atretic testes). Among the females, stage II predominated (78%), whilst remaining females had ovaries in stage III. By age 5 years, 22% of males were in stage II and 78% in stage III, whereas all females had progressed to stage III (Figs 1, 2).
Serum T levels were significantly higher in males than in females, most notably for the 5-year old fish. In this age class, male T levels averaged 248.88Â±188.51 ng/ml, substantially higher than levels found in females (42.93Â±30.77 ng/ml). However, there was notable overlap in T levels between sexes (Fig 3a) and this overlap was more pronounced at younger ages than in the oldest age class. Indeed, T levels were useful as predictor of sex at high (> 30 ng/ml) or low (< 20 ng/ml) levels, but not at intermediate values ( Tâ‰¥ 4 and Tâ‰¤ 494).
Serum E2 levels averaged 0.51-1.42 ng/ml for the different experimental groups and did not differ between males and females (data not shown). Scatter plots indicated a high degree of overlap between males and females, especially in the younger age classes (Fig 5b).
The ratio of T: E2 was around 50 for females, regardless of age. In males, however, TER gradually increased with age (Fig 3c), such that 4-year old fish started to show a pattern (TERmale > TERfemale) that became highly predictive by the time fish had become 5 years of age (Fig 3c). Indeed, in all but one out of seventeen 5-year-old fish (94% of cases), sex could be correctly predicted on the basis of the TER, such that TER-values over 40 were indicative of the male phenotype, and those below 40 of the female phenotype.
This study was conducted in order to identify biochemical tools that may be applicable to sexing of immature sturgeon under culture conditions. Histological analysis confirmed that all sampled fish were immature, varying between stages I-III of gonadogenesis. Our findings further indicated that Huso huso can be reliably sexed from an early age by biopsy, and that sex differentiation occurs earlier in females than in males, a finding that is in accord with that of Yousefian (2006). Moreover, our histological studies demonstrated that early stages of spermatogenesis in males and the previtellogenic period in females lasts at least 2-3 years, indicating that the speed of germ cell development in sturgeon is slow compared to that in many teleosts (Bahmani and Kazemi, 1998).
In captive beluga, significant increases of T levels in males may be used as an "indicator" for separation of sexes at early stages of gonadal development under aquaculture conditions (Yousefian, 2006). In the present study, serum T levels were only marginally useful as a predictor of sex, and only in the oldest age class. Indeed, only about half of the fish (Tâ‰¥4 ng/ml or Tâ‰¤494 ng/ml) could be sexed on the basis of serum T levels, despite clear statistically significant differences between the sexes in 5-year olds. In an earlier study by Feist et al. (2004), significant differences in androgen levels (T and 11-ketotestosterone) between sexes of Acipenser transmontanus were similarly reported (Feist et al., 2004), but notable differences between populations limited the use of plasma androgens levels as sexing tools to specific localities.
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In our study the levels of E2 did not separate males and females. Likewise, there were no significant differences in serum E2 levels between males and females of Sciaenops ocellatus L in previtellogenesis stage (Kucherka et al, 2006), nor in female A. transmontanus (Feist et al., 2004). Pottinger et al (2005) came to the same conclusion in salmonids. Eï€² thus appears to be a good indicator to separate males and females only at the time of vitellogenesis.
In our endeavour to identify a useful non- or less-invasive sexing tool for immature beluga, we also evaluated the T:E2 ratio. To our delight, the TER was highly predictive of sex in 5-year old fish, with a clear separation of the sexes; only a single individual was miss-assigned to be female on the basis of the TER. On the other hand, the E2:T ratio is measured in plasma and chorioallantoic/amniotic fluid (CAF) of hatchling loggerhead turtles, Caretta caretta so that the ratio were significantly lower in males than those in females (Gross et al., 1995). The 11KT to E2 ratio was higher in males than females during embryogenesis and sexual differentiation in eurasian perch, Perca fluviatilis (Rougeot et al., 2007). Nevertheless, in many species testosterone seems to play an important role in sex differentiation and is closely related to gonadal differentiation (Nakamura et al., 1989 and Chang et al., 1995) and also it plays an intermediate role as precursor of 11-ketotestosterone and 17Î² estradiol (Baroiller et al., 1999).
In conclusion, we have identified a procedure for the sexing of farmed immature beluga that is considerably less invasive than surgical methods used to date. Indeed, beluga can be reliably sexed on the basis of the T: E2 ratio (values over 40 represent males), but only in 5-year-old fish. The method has limited applicability to 4-year olds and is not useful for fish younger than 4 years of age; rather, other biochemical indices may need to be explored to sex younger fish. It is anticipated that the TER will be used in future for the selection of early maturing males that can be used for breeding with older females from different lines.