In order to avoid the invasiveness of arousing associated with biopsy in hausen ( Huso huso ) , we explored the possibilities of utilizing sex steroid degrees to sex farmed immature fish. Beluga were selected indiscriminately from 2, 4 and 5-year-old stocks, cultured under the same environmental and feeding governments. Conventional methods of arousing by biopsy and histological observation were used to find the sex and gonadal phase, while enzyme-linked immunosorbent check was employed to mensurate the degrees of testosterone ( T ) and 17i??-estradiol ( E2 ) in serum.
All fish had differentiated sex glands and males and females could be distinguished microscopically in all age groups. T degrees differed significantly between males and females, but made for hapless forecasters of sex due to the significant convergence in scopes of T degrees in both sexes. There was no important difference in E2 degrees between males and females. When ciphering the Thymine: E2 ratios ( TER ) , a clear separation was obtained for 5-year-old fish, ratios over 40 foretelling male, and those below 40 foretelling a female phenotype.
The survey demonstrated that the TER can be used as an effectual index to divide males and females in civilized immature 5-year-old hausen.
In sturgeon aquaculture the industry, it is of import to place the sex of single fish at an early age in order to work 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 can non be determined externally until the pre-spawning stage, and hence, sturgeon have been sexed by biopsy during minor surgery ( Doroshov et al. , 1994 ) . In Iran, such surgical trials have likewise been used for arousing of farmed sturgeon ( Bahmani & A ; Kazemi, 1998 ) . While this method is straight-forward and utile for unambiguous designation of the sex of single fish, the process is clip devouring. Furthermore, the invasiveness of the process is likely to bring on significant emphasis for the fish, non in the least due to the possibility of infections, doing productiveness losingss.
Biochemical trials for solutes with sex-specific profiles, notably sex steroids, may be more rapid, cause less emphasis and be less invasive to the fish ( Semenkova et al. , 2006 ; Mojazi Amiri et al. , 1996a, B ) . Concentrations of testosterone ( T ) normally increase at the oncoming of miosis in males and during oocyte growing in females ( Cuisset et al. , 1995 ) and remain high boulder clay spermiation or ovarian ripening ( Cuisset et al. , 1995 ; Mojazi Amiri et al. , 1996a, B ; Webb et al. , 2002a, B ) . Serum 17i??-estradiol ( E2 ) degrees increases during vitellogenesis in females, but remain low in the males at any phase 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 survey, the suitableness of sex steroid profiling ( T and E2 ) of farmed immature hausen, Huso huso, as a arousing tool was evaluated ( Webb et al. , 2002a ; Fiest et al. , 2004 ; Malekzadeh Viayeh et al. , 2006 ) in order to supply a tool for sturgeon culturists.
Materials and Methods
The fish used in this survey were two, four and five-years-old farmed hausen reared in the Propagation and Culture Center of Shahid Marjani near Gorgan metropolis, North-Eastern Iran. These fish were kept in fresh H2O in separate pools ( 1500 M2, depth 1.2m ; lading denseness 2kg/m2 ) depending on age. Thirty fish were selected indiscriminately from each age category. Environmental steps were changeless during the civilization period ( H2O temperature: 9.7A°C, DO: 6.5 mg/l, salt: 4 ppt, nitrate: 0.02 mg/l, phosphate: 0.03 mg/l, pH: 7.9 ) .
Blood sampling process
Immediately after taking fish from pools and anaesthetized with clove pulverization ( 150 ppm ) , biometrical factors were recorded ( Fish at age 2 year: n=30, organic structure weight ( BW ) 4.32A±0.78 kilogram, organic structure length ( BL ) 94A±6.13 centimeter ; at age 4 year: n=30, BW 9.25A±1.99 kilogram, BL 118.63A±74 centimeter ; and at age 5 year: n=30, BW 15.48A±1.98 kilogram, BL 137.47A±5.94 centimeter ) . Thereafter, blood samples were collected from the caudal vena utilizing a 5 milliliter syringe ( inserted behind the anal five ) . Blood was centrifuged ( Behdad type: BH-1200, Iran ) at 3000 revolutions per minute for 20 proceedingss, and serum collected and stored frozen at -20°C until assay for steroids.
Sera Testosterone and Estradiol concentrations ( ng/ml ) were determined, utilizing Enzyme Linked Immuno Sorbent Assay ( ELISA ) method ( The EiAsy Way Estradiol and The EiAsy Way Testosterone, kits, Diagnostic Biochem Canada Inc, Ontario, Canada ) harmonizing to Bayunova et Al. ( 2002 ) and Semenkova et Al. ( 2002 ) , severally. The assay sensitiveness for Testosterone was 0.022ng/mL and for Estradiol 10pg/mL. The intra assay coefficient of fluctuation for Testosterone and Estradiol were 9.1 % and 7.7 % severally. Harmonizing the kit insert for Testosterone, the high cross responsiveness 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.
A little portion of one sex gland was removed from each anaesthetized fish by biopsy and preserved in Bouin ‘s solution for histological scrutiny to find the sex and gonadal phase. Conventional histological methods were used to fix gonadal tissues for observation and phase was determined harmonizing to Mojazi Amiri et Al. ( 1996a, B ) . Briefly, the undermentioned phases were identified in males: Phase I™ = increasing Numberss of spermatogonia by mitosis ( spermatogonial proliferation ) ; Stage I™I™ = seminal lobules filled with primary and secondary spermatocytes and low Numberss of spermatids ( early spermatogenesis ) ; and Stage I™I™I™ = the presence of many spermatids and little populations of sperm cell in the cardinal portion of lobules ( mid-spermatogenesis ) . In females, ovaries were characterized as Stage I™ = smallest oocytes about 10-60 Aµm in diameter with a big karyon ( chromatin nucleus phase ) ; Stage I™I™ = oocytes about 60-200 Aµm in diameter with distribution of nucleole around the interior portion of the atomic envelope ( perinucleolus phase ) ; and Stage I™I™I™ = oocytes about 200-480 Aµm in diameter with the visual aspect of oil droplets at the fringe of the karyon in a cytol ( oil droplet phase ) .
After proving for normalcy, steroid informations were subjected to independent samples t trial utilizing the package bundle SPSS16. Furthermore, the T to E2 ratio ( TER ) was calculated for each fish. Steroid and TER information for both sexes were presented as scatter secret plans against age to measure to efficaciousness of each variable as a sex identifier.
Based on histological observations of sex glands from 2-year old fish, 89 % of males were in phase I and 11 % in phase II, while in females, 18 % were in phase I, 73 % in phase II and 9 % in phase III. In 4-year olds, 11 % of male fish were in phase I, 78 % in phase II and 11 % in phase VI ( atretic testicles ) . Among the females, phase II predominated ( 78 % ) , whilst staying females had ovaries in phase III. By age 5 old ages, 22 % of males were in phase II and 78 % in phase III, whereas all females had progressed to present III ( Figs 1, 2 ) .
Serum T degrees were significantly higher in males than in females, most notably for the 5-year old fish. In this age category, male T degrees averaged 248.88A±188.51 ng/ml, well higher than degrees found in females ( 42.93A±30.77 ng/ml ) . However, there was noteworthy convergence in T degrees between sexes ( Fig 3a ) and this convergence was more marked at younger ages than in the oldest age category. Indeed, T degrees were utile as forecaster of sex at high ( & gt ; 30 ng/ml ) or low ( & lt ; 20 ng/ml ) degrees, but non at intermediate values ( Ta‰? 4 and Ta‰¤ 494 ) .
Serum E2 degrees averaged 0.51-1.42 ng/ml for the different experimental groups and did non differ between males and females ( informations non shown ) . Scatter secret plans indicated a high grade of convergence between males and females, particularly in the younger age categories ( Fig 5b ) .
The ratio of T: E2 was about 50 for females, irrespective of age. In males, nevertheless, TER bit by bit increased with age ( Fig 3c ) , such that 4-year old fish started to demo a form ( TERmale & gt ; TERfemale ) that became extremely prognostic by the clip fish had become 5 old ages of age ( Fig 3c ) . Indeed, in all but one out of 17 5-year-old fish ( 94 % of instances ) , sex could be right predicted on the footing of the TER, such that TER-values over 40 were declarative of the male phenotype, and those below 40 of the female phenotype.
This survey was conducted in order to place biochemical tools that may be applicable to arousing of immature sturgeon under civilization conditions. Histological analysis confirmed that all sampled fish were immature, changing between phases I-III of gonadogenesis. Our findings further indicated that Huso huso can be faithfully sexed from an early age by biopsy, and that sex distinction occurs earlier in females than in males, a determination that is in agreement with that of Yousefian ( 2006 ) . Furthermore, our histological surveies demonstrated that early phases of spermatogenesis in males and the previtellogenic period in females stopping points at least 2-3 old ages, bespeaking that the velocity of source cell development in sturgeon is slow compared to that in many teleosts ( Bahmani and Kazemi, 1998 ) .
In confined hausen, important additions of T degrees in males may be used as an “ index ” for separation of sexes at early phases of gonadal development under aquaculture conditions ( Yousefian, 2006 ) . In the present survey, serum T degrees were merely marginally utile as a forecaster of sex, and merely in the oldest age category. Indeed, merely about half of the fish ( Ta‰?4 ng/ml or Ta‰¤494 ng/ml ) could be sexed on the footing of serum T degrees, despite clear statistically important differences between the sexes in 5-year olds. In an earlier survey by Feist et Al. ( 2004 ) , important differences in androgen degrees ( T and 11-ketotestosterone ) between sexes of Acipenser transmontanus were likewise reported ( Feist et al. , 2004 ) , but noteworthy differences between populations limited the usage of plasma androgens degrees as sexing tools to specific vicinities.
In our survey the degrees of E2 did non separate males and females. Likewise, there were no important differences in serum E2 degrees between males and females of Sciaenops ocellatus L in previtellogenesis phase ( Kucherka et al, 2006 ) , nor in female A. transmontanus ( Feist et al. , 2004 ) . Pottinger et Al ( 2005 ) came to the same decision in salmonids. Eiˆ? therefore appears to be a good index to divide males and females merely at the clip of vitellogenesis.
In our enterprise to place a utile non- or less-invasive sexing tool for immature hausen, we besides evaluated the Thymine: E2 ratio. To our delectation, the TER was extremely prognostic of sex in 5-year old fish, with a clear separation of the sexes ; merely a individual person was miss-assigned to be female on the footing of the TER. On the other manus, the E2: T ratio is measured in plasma and chorioallantoic/amniotic fluid ( CAF ) of hatchling loggerhead polo-necks, 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 distinction in Eurasian perch, Perca fluviatilis ( Rougeot et al. , 2007 ) . Nevertheless, in many species testosterone seems to play an of import function in sex distinction and is closely related to gonadal distinction ( Nakamura et al. , 1989 and Chang et al. , 1995 ) and besides it plays an intermediate function as precursor of 11-ketotestosterone and 17I? estradiol ( Baroiller et al. , 1999 ) .
In decision, we have identified a process for the sexing of farmed immature hausen that is well less invasive than surgical methods used to day of the month. Indeed, hausens can be faithfully sexed on the footing of the T: E2 ratio ( values over 40 represent males ) , but merely in 5-year-old fish. The method has limited pertinence to 4-year olds and is non utile for fish younger than 4 old ages of age ; instead, other biochemical indices may necessitate to be explored to arouse younger fish. It is anticipated that the TER will be used in future for the choice of early maturing males that can be used for engendering with older females from different lines.
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