the marine medaka , oryzias dancena , is a truly euryhaline teleost fish , having a great 
 capacity for hypo and hyper - osmoregulation . most of its physiological attributes are similar across a 
 wide spectrum of salinities , ranging from fresh water to normal seawater ( inoue & takei , 2003 ; kang et 
 al . , 2008 ; cho et al . , 
 2010 ) . therefore , much attention has been directed at extending the utility of functional 
 transgenic marine medaka strains for ornamental purposes , because they can be used at most naturally 
 occurring salinities ( cho et al . , 2011 ) . in 
 addition , in a recent study of transgenic marine medaka containing the myosin light chain-2 
 ( mlc2f ) promoter , the expression of a vivid red fluorescent color in their fast 
 skeletal muscles suggested great potential for these as novel ornamental fish for both freshwater and 
 seawater aquaria ( cho et al . , 2012 ) . triploidization is a technique used to generate sterile aquatic animals by taking advantage of the 
 incompatibility in pairing the three homologous chromosomes during meiosis i ( don & avtalion , 1986 ) . this technique has also been used to enhance the 
 productivity of several fish species because of its assumed ability to increase yield by channeling the 
 energy required from gonadal development to somatic growth ( tave , 
 1993 ) . more importantly , it generates fish that are unable to breed and contribute to the 
 local gene pool if they were to accidentally escape from confinement . by conferring sterility of exotic 
 fish for a limited purpose , triploidy can serve as an effective method for reducing or eliminating the 
 environmental risks of genetically modified organisms ( kim et al . , 
 1994 ) . growth rates tend to 
 slow or cease in maturing fish , and it is during the later stages of sexual maturation that a growth 
 advantage of triploid fish over diploids is most likely to be observed ( hatanaka et al . , 1991 ; kobayashi , 1992 ; benfey , 1999 ) . in species that survive spawning , diploids frequently 
 exhibit compensatory growth and therefore , a growth advantage of triploids is most likely to be seen in species where 
 the diploids have high or complete mortality associated with sexual maturation and spawning , as in ayu 
 ( plecoglossus altivelis ) and twicespawned rainbow trout ( oncorhynchus 
 mykiss ) ( hatanaka et al . , 1991 ; thyroid stimulating hormone ( tsh ) and thyroxine 
 ( t4 ) play indispensable roles during the embryonic and larval periods of fish development ( khalil et al . , because of the potentiating 
 effects of tsh and t4 on fish larval growth and survival , it is important to investigate whether 
 application of exogenous thyroid stimulating hormone and thyroxine to female brood fish results in 
 better growth and survival of larvae ( khalil et al . , 
 2011 ) . no previous study of the marine medaka has included a comparative analysis of the diploid and triploid 
 fish . therefore , we undertook a comparative analysis of the comparative study of growth and gonad 
 maturation in diploid and triploid marine medaka , oryzias dancena . so , the objectives 
 of this study were to evaluate the reproductive characteristics of the triploid form of this 
 species . the specimens of marine medaka , oryzias dancena used in this study were from a 
 laboratory stock maintained at the institute of marine living modified organisms 
 ( imlmo ) , pukyong national university , busan , korea . the general maintenance of the 
 experimental fish was according to the method of song et al . breeding occurred in brackish water ( 5 psu ) , as described by cho et al . the breeding conditions included a temperature of 25  1c and a 
 16 h light:8 h dark cycle . triploid and diploid specimens were fed brine shrimp ( artemia 
 nauplii ; inve , salt lake city , utah , usa ) and micro - particle feed ( 150500 m diameter ; 
 ewha oil co. , busan , korea ) . fertilized eggs were obtained each day by mating male and female broodfish in a glass tank containing 30 
 l of well - aerated water . the eggs were collected from females immediately following fertilization . at 2 
 min after fertilization the fertilized eggs were subjected to a cold shock treatment at 0c for 45 min . 
 following treatment the hatching success 
 and the incidence of abnormal larvae were assessed based on stereoscopic microscope ( c - ds ; nikon co. , 
 tokyo , japan ) examination of at least 23 eggs per group . the values for these parameters in the 
 experimental treatments were expressed as percentages of treated eggs . after 60 days , triploid marine 
 medaka were distinguished by flowcytometry and chromosome analysis . over 12 months the standard length , body weight , condition factor , and gonadosomatic index ( gsi ) for each 
 individual 120 were measured to investigate the growth and maturation of diploid and triploid fish . the 
 condition factor was determined using the equation : condition factor = ( body weight  100)/(body 
 length ) , and the gsi was determined using the equation : gsi = ( gonad weight / body weight ) 
  100 . sex hormone and growth hormone were analyzed through the plasma of each ploidy group at 120 days 
 after hatching . the estradiol-17 and testosterone concentrations were measured using fluorophotometry 
 method while 48 hrs ( i - chroma , sun kyung medical , korea ) . to measure the concentrations of estradiol-17 
 and testosterone , next , mix a specimen with 
 buffer by voltex mixer and leave to the specimen at room temperature for 15 min , finally , insert it to 
 the i - chroma reader .   to investigate changes in growth hormone levels , the concentrations of thyroid stimulating hormone ( tsh ) 
 and thyroxine ( t4 ) in samples of each group were measured using fluorophotometry during the 12 months 
 following hatching . to measure the concentrations of tsh and t4 , next , mix a specimen with buffer by voltex mixer and leave to the specimen at room 
 temperature for 10 min , finally , insert it to the i - chroma reader . the gonad morphology was observed in one - year - old diploid and triploid males and females . the gonads were 
 surgically removed and fixed in buffered 10% formaldehyde solution for histological analysis , following 
 morphological analysis . conventional histological techniques were used to assess gonad development , 
 including embedding in paraffin wax , sectioning to 6 m thickness , and staining using mayer s 
 hematoxylin and eosin . the standard length , body weight , condition factor , and gsi were assessed using anova followed by 
 duncan s multiple range test at the significance level p=0.05 . the von bertalanffy growth parameters for diploid and triploid marine medaka , oryzias dancena 
 estimated using the non - linear regression method , are shown in fig . the von bertalanffy growth equations were 
 lt=30.2(1e ) 
 and lt=30.9(1e ) 
 for the diploid and triploid females , respectively . the growth coefficients ( k ) for the 
 diploid and triploid females were estimated to be 3.22/year and 3.08/year , respectively , their 
 asymptotic maximum length ( l ) was estimated to be 30.2 mm and 30.9 mm , 
 respectively , and the theoretical age at zero length ( t0 ) was estimated to 
 be 0.03 and 0.04 , respectively . the von bertalanffy growth equations were 
 lt=30.2(1e ) 
 and lt=30.9(1e ) 
 for the diploid and triploid males , respectively . the k and l values for the diploid and 
 triploid males were similar to those of the females . the t0 values differed between the 
 diploid and triploid males ( 0.01 and 0.02 , respectively ) , and was greater than that for the females . 
 for each measured parameter , significant differences were found between the diploids and triploids , and 
 the males and females ( p<0.05 ) . the change in body weight for diploid and triploid marine medaka is shown in fig . 2 . during the experimental period the growth patterns for the diploids and 
 triploids ( female and male groups ) were similar ; all fish grew rapidly from 3 to 4 months of age , and 
 grew slowly from 8 to 12 months of age . for both males and females the triploids were larger than their 
 diploid counterparts during the experimental period . among both diploid and triploid fish the body 
 weight of males was greater than that of females . fish condition was affected by ploidy , but not by 
 gender v ( table 1 ; p<0.05 ) , and the condition 
 of all groups decreased rapidly from 1 to 2 months of age . up to 4 months of age the condition factor 
 for both the male and female diploids was significantly higher than for their triploid counterparts . 
 however , after 4 months of age the condition factor for both male and female groups was not 
 significantly different between the diploid and triploid fish . during the experimental period , the 
 condition factor within the diploid and triploid groups did not differ significantly between females and 
 males . the different superscripts of each value are 
 significantly different between ploid and sex ( p<0.05 ) changes in the gsi for diploid and triploid marine medaka are shown in fig . the gsi for triploid females and males was lower than that for their diploid counterparts , 
 and within each ploidy group the gsi for females was higher than that for males . the gsi of triploid females increased from 3.17% at 1 month 
 following hatching to 20.45% at 4 months ( p<0.05 ) , and decreased to 15.43% at 12 
 months . the gsi of triploid males increased from 1.56% at 1 month following hatching to 9.05% at 4 
 months ( p<0.05 ) , and decreased to 7.18% at 12 months . for all groups the gsi was 
 highest at 4 months , and decreased from 4 to 12 months . morphological and histological analyses of the 
 gonads of one - year - old fish showed that the triploid genotype caused significant depression of gonad 
 development in marine medaka . the ovaries of the diploid females were well developed , with fully 
 yolk - laden eggs , whereas those of the triploid females were poorly developed and significantly smaller 
 than in the diploid fish ( fig . 4a and 4b ) . in contrast , there was 
 no clear visual difference in the testicular morphology of diploid and triploid males , although the 
 testes of the triploid fish were slightly smaller , suggesting that the gsi of the triploid males 
 differed from that of their diploid counterparts ( fig . 4c and 
 4d ) . ( a ) : diploid ovary ; ( b ) : triploid ovary ; ( c ) : diploid testis ; ( d ) : triploid testis . histological analysis of the gonads showed clear differences between the diploid and triploid females . unlike the diploid fish , which had highly developed ovaries filled with mature yolk - containing oocytes , 
 the ovaries of the triploid females contained a considerable number of oogonia , with very few oocytes at 
 the chromatin in addition , the triploid males showed depressed gonad development compared with their 
 diploid counterparts , even though the gross morphology of the diploid and triploid testes was not 
 significantly different . normal spermatids and sperm were present in the testes of the diploid fish , 
 whereas the testes of the triploid males had few spermatids or no sperm ( fig . 5c and 5d ) . gonads : ( a ) diploid ovary ; ( b ) triploid ovary ; ( c ) diploid testis ; ( d ) triploid testis . imo : 
 immature oocyte ; oyg : oocyte in yolk granule stage ; oyv : oocyte in yolk vesicle stage ; sg : 
 spermatogonium ; smc : spermatocyte . the concentrations of thyroid stimulating hormone and thyroxine were affected by the ploidy of fish , but 
 not by their gender ( table 2 ; p<0.05 ) . the 
 concentration of thyroid stimulating hormone and thyroxine in all groups increased slowly during the 
 experimental period . in both the diploid and triploid groups the concentrations of these two hormones 
 were lower in the females than in the males , but for both genders were higher in triploid fish than in 
 the diploids . 6 , the testosterone concentration in 
 the diploid fish gradually decreased after the experiment . at 24 h , values of testosterone restored 
 initial value , and at 48 h the testosterone concentration was similar to that at 0 and 24 h. however , 
 the triploids had lower testosterone concentrations than the diploids because of their lower gsi 
 ( p<0.05 ; fig . 6 ) . the estradiol-17 
 concentration in the diploid fish showed a tendency to gradually decrease from 6 to 12 h ( fig . however , at 24 and 48 h the estradiol-17 concentration had 
 returned to the level at 0 h. in the triploids the estradiol-17 concentration did not change , and the 
 concentrations of testosterone and estradiol-17 in the induced triploids was significantly less than in 
 the diploids ( p<0.05 ; fig . differences between ploid 
 and sex / stage are significant at this each values are meanss.d . of triplicate 
 experiment . differences between ploid and sex / stage are significant at this level 
 ( p<0.05 ) . different letters on error bars are 
 significantly different for each group ( p<0.05 ) . different letters on error bars are 
 significantly different for each group ( p<0.05 ) . the triploid marine medaka , oryzias dancena grew more rapidly than their diploid 
 counterparts ( p<0.05 ) , which is consistent with the results of 
 previous studies . ( 2001 ) reported 
 that triploid mud loach had a growth rate 2225 times that of diploid fish . in this study the triploid 
 marine medaka were larger than the diploid form , but giantism of the triploid was not responsible . kim et al . ( 2001 ) & seol et al . 
 ( 2008 ) suggested that the absence of giantism in triploids is because of their smaller cell 
 number . in the present study the male marine medaka ( diploid and triploid ) were larger than the females 
 ( p<0.05 ) . female fish are typically larger than male fish of 
 the same age , although in some species the reverse is true ; examples include the gudgeon , gobio 
 gobio ( mann , 1980 ) & the filefish , 
 brachaluteres ulvarum ( akagawa et al . , 1995 ) . it has been suggested in several studies that the evolution of a larger body size in males 
 probably results from male  male competition associated with a polygynous mating systems ( katano , 1998 ; kim et al . , therefore , exploring the nature and extent of sexual dimorphism can extend our 
 understanding of social structure and adaptation , as well as species identification . over 1 month we 
 found that a rapid increase in length caused a reduction in condition , as measured using the condition 
 factor , and the condition factor for triploids was lower than that for the diploids . unfortunately , no 
 previous studies have reported such a rapid decrease in condition , or differences in the condition 
 factor between diploid and triploid marine medaka or other fish species , so our observations remain 
 unexplained . the 
 odd chromosome number induced during triploidization precludes homologous chromosomal pairing during 
 meiosis i , which leads to inefficient gamete differentiation and consequently confers sterility on the 
 triploid fish ( zhang et al . , 2005 ) . overall , most of our 
 observations of the gonads in this study were similar to those previously reported for other triploid 
 fish : smaller gonads , significantly delayed gonadal development , and more pronounced sterility in 
 females ( kim et al . , 1994 ; felip 
 et al . , 1999 ; tiwary et al . , 2000 ; feindel et al . , 2011 ) . in a recent study cal et al . ( 2010 ) reported that the induction of triploidy lowered the estradiol-17 
 concentration and changed its internal secretion , resulting in oogenesis disorders . however , numerous 
 previous studies have also claimed that triploid fish , especially old triploid males , may have a 
 functional but reduced capacity to reproduce , despite their sterile - like gonadal development ( benfey , 1999 ) . the artificial insemination of normal haploid eggs of 
 tench ( tinca tinca ) using sperm from triploid fish has been reported to produce some 
 abnormal larvae ( linhart et al . , 2006 ) . ( 2011 ) reported that treatment of triploid african 
 catfish ( clarias gariepinus ) with the ovulation/ spermiating agent ovaprim induced 
 advanced vitellogenin sequestration in triploid oocytes , and promoted the fertilization capacity of milt 
 from triploid males . sex hormones , including testosterone and estradiol-17 , are commonly found in induced triploids because 
 their testes and ovaries do n't mature ( kim et al . , 1994 ; park & kim , 1994 ) . unlike the normal gonadal maturation of 
 diploids , in induced triploids the gonads are immature on formal and histological ( lincoln & scott , 1984 ; kim et al . , 
 1994 ) , and induced triploids showed lower concentrations of sex hormones because of decreased 
 hormone secretion resulting from immaturity ( lincoln , 1981 ; lincoln & scott , 1984 ) . the testes of diploids had normal 
 spermatids and spermatozoa , while few were seen in the induced triploids , and the ovaries of diploids 
 had many well developed oocytes , while those of the induced triploids exhibited oogonia ( kim et al . , 1994 ; park & kim , 
 1994 ; kim et al . , 2001 ) . the concentrations of thyroid stimulating hormone ( tsh ) and thyroxine ( t4 ) were found to be higher in the 
 induced triploid fish over 1 year ( p<0.05 ) . in both the diploid and 
 triploid groups in previous 
 studies the initial survival of freshwater fish , transformation , and the effects of tsh and t4 on 
 development and growth have been observed ( lam & sharma , 
 1985 ; weatherley & gill , 1987 ) . administration of 
 growth hormone caused a reduction in the weight to length ratio ( condition factor ) in diploids but not 
 in triploids , and caused triploids to deplete lipid energy stores more rapidly ( mclean et al . the induced triploids were expected to show 
 better growth compared with the diploids , and the condition factor for the latter was expected to 
 decrease faster . as no comparable studies have investigated differences in tsh and t4 between diploid 
 and triploid in marine medaka or other fish species , no explanation for our observations is available . 
 although induced triploids are infertile , their production is economically feasible because they grow 
 faster than their diploid counterparts . in particular , longer - term observations of the potential for maturation in older triploid 
 marine medaka should extend our understanding of their reproductive capacity .
abstractthe marine medaka , oryzias dancena is a suitable sample as a laboratory animal 
 because it has a small size and clearly distinguishes between female and male . 
 data on the growth 
 and maturity of the diploid and triploid sea cucurbit species suitable for laboratory animals are 
 very useful for studying other species . 
 triploidy was induced in the marine medaka by cold shock 
 treatment ( 0c ) of fertilized eggs for 45 min , applied two minutes after fertilization . 
 the diploid 
 and triploid male fish were larger than their female counterparts 
 ( p<0.05 ) , and the concentrations of thyroid stimulating 
 hormone ( tsh ) and thyroxine ( t4 ) were higher in the induced triploids over 1 year 
 ( p<0.05 ) . in both the diploid and tri - ploid groups 
 the 
 concentrations of tsh and t4 were higher in the male fish than in the females 
 ( p<0.05 ) , while the testo - sterone and estradiol-17 
 concentrations in the induced triploids were lower than in the diploids 
 ( p<0.05 ) . the gonadosomatic index ( gsi ) of 
 the triploid 
 fish was lower than that for the diploids , and the gsi for females in each ploidy group were higher 
 than that for the males . 
 for both groups the gsi was highest at 4 months of age , and decreased 
 thereafter to 12 months . 
 analysis of the gonads of one - year - old triploid fish suggested that the 
 induction of triploidy probably causes sterility in this species ; this effect was more apparent in 
 females than in males .