in teleosts , the sex differentiation pattern may be classified as synchronous hermaphroditism 
 ( protandrous and protogynous ) or gonochorism ( undifferentiated and differrentiated ) ( yamamoto , 1969 ) .
both genetic and environmental factors can 
 significantly impact the sex determination of fish ( devlin & 
 nagahama , 2002 ) .
the physicochemical factors relevant to the sex determination of fish 
 include the water temperature , salinity , and ph ( baroiller et al . , 
 1999 ) , while the related chemical factors include polychorinated biphenyls ( psbs ) , 
 nonylphenol , bisphenol , and dioxin by endocrine disruptor ( gray & 
 metcalf , 1997 ) .
to improve the economics of aquaculture , these parameters may be used to 
 render fish populations artificially mono - sexual ( e.g. , by adjusting the water temperature , controlling 
 the ph , or applying hormone treatments ) . the korean rose bitterling , rhodeus uyekii , is an indigenous species in korea . falling 
 within the cypriniformes , cyprinidae , and rhodeus ,
these fish live on the muddy floor 
 at the edges of reservoirs , plant - filled and/or slow - moving streams , and ponds .
they are distributed in 
 the watershed of the nakdong river and other streams in the southern part of korea ( jeon , 1982 ) .
the korean rose bitterling breeds between the end of 
 april and mid - june , and shows special features in breeding in the fresh water bivalves of the family 
 unionidae by extending the ovipositor in the female ( uchida , 
 1939 ; nakamura , 1969 ) .
seen from the side , the body of the 
 korean rose bitterling is relatively compressed from nose to tail and relatively tall from the caudal to 
 dorsal aspect , with the male generally being taller than the female . in color , the body is light brown 
 on the back , darker on the front back of the dorsal fin and the caudal peduncle , and reddish white 
 silver on the flank .
the male is black on the outside of its rear fin and exhibits a small white spot 
 inside .
given its excellent visual effect , the 
 male is far preferred over the female as an aquarium fish , besides the taxonomic group with urgent 
 preservation due to easy exposure to the recent biological and environmental pollution ( kang et al .
, researchers have studied : the early life history of laboratory - reared 
 fish ; egg and larval development ; the seasonal sex ratio in the field ; the reproductive cycle of the 
 spring - spawning bitterling ; osteology ; elongation of the ovipositor ; the temperaturedependent index of 
 the mitotic interval ( 0 ) for chromosome manipulation ; morphometric traits ; cytogenetics of 
 induced crosses and reciprocal hybrids between korean rose bitterling and r. notatus ; 
 and the use of lidocaine hydrochloride and clove oil as an anesthetic ( for both korean rose bitterling 
 and oily bitterling , acheilognathus koreensis ) ( kim 
 & han , 1990 ; park & kim , 1990 ; an , 1995 ; kim , 1997 ; chae , 2001 ; kim et al . , 2011 , 
 2012 ) .
studies have also examined trends in the early 
 development of the gonad and the potential for sex change , but the process of sex differentiation in the 
 korean rose bitterling is not yet fully understood .  
here , we investigated the sex differentiation and gonadal development of the korean rose bitterling , and 
 their dependence on the initial growth and integral water temperature at the constant breeding water 
 temperature for the hatching fry through the artificial insemination as a part of production of the 
 monosexual population and the seed production of the korean rose bitterling .
brood stock of the korean rose bitterling , rhodeus uyekii , were obtained from the 
 inland aquaculture research center , national institute of fisheries science ( nifs ; 
 changwon , korea ) and housed in a mariculture facility at the fishery genetics and breeding sciences 
 laboratory of the korea maritime and ocean university in busan , korea .
the fish were maintained in a 
 glass tank ( w 120 cm  l 30 cm  h 50 cm ) at a water temperature of 201c until the 
 experiments were initiated . during the 2013 spawning season ( late april to midjune ) , densely colored males and 
 ovipositor - extended females were selected and used as brood stock .
eggs were harvested by abdominal 
 compression of ovipositor - extended females , semen was harvested by abdominal compression of males 
 and inseminated within 2 minutes .
plastic tanks ( w 30 cm  l 20 cm  h 15 cm ; fresh plus , mirae 
 chemical , korea ) were loaded with 200 eggs each and maintained at a water depth of 10 cm and a l : d 
 ratio of 13:11 until hatching .
the water was replaced twice daily with aerated bottled water 
 ( natural mineral water , pulmuone , korea ) , and the water temperature was maintained at 220.5 
 oc .
hatched larvae were divided into groups of 10 and distributed to transparent plastic containers ( w 10 
 cm  l 5 cm  h 3 cm ; coolrara , easyfilm , korea ) .
the larvae began to feed once the yolk was completely 
 absorbed , at 21 days post - hatching ( dph ) .
the hatchlings ( n = 20 ) were sampled daily from hatching to 5 dph , at 2-day 
 intervals from 7 to 21 dph , at 4-day intervals from 25 to 41 dph , at 6-day intervals from 47 to 83 
 dph , and at 30-day intervals from 110 to 170 dph .
the fish were sampled at different stages and 
 anesthetized with 300 ppm lidocaine - hcl / nahco3 at 25c according to method of 
 kang et al .
body weight ( bw ) was measured to the 
 nearest 0.01 g using an electric balance ( ax 200 , shimadzu corp . ,
japan ) , the total length ( tl ) was 
 measured to the nearest 0.01 cm using a vernier caliper ( cd-20 cp , mitutoyo , japan ) , and growth was 
 estimated using an exponential growth curve and the calculation of a condition factor . a mimetic diagram was produced according to the growth for indicate morphological changes .
each 
 sample of whole fish was fixed in bouin s solution for 24 h , washed thoroughly with water , exposed 
 to decalcification solution for 24 h , washed again , and dehydrated through a graded alcohol series 
 ( 70% , 80% , 90% , and 100% ethanol 1 h each ) . the samples were then cleared with xylene , impregnated 
 with soft paraffin , impregnated with hard paraffin , embedded , trimmed , and cut ( 6 m ) .
eosin , mounted with canadian balsam , and 
 examined / photographed under optical microscopy ( axiocam mr , carl zeiss , germany ) .
the korean rose bitterling , rhodeus uyekii , hatched at two days post - fertilization with 
 a post - fertilization integral water temperature ( iwt ) of 43c .
fig . 1 , fig . 2 , and 3 present the changes in total length ( tl ) , body weight ( bw ) , condition factor , and 
 morphology from
just - hatched ( iwt post - hatching 0c ) to 170 days post - hatching ( dph ; iwt 
 post - hatching 3,740c ) . at hatching ,
the average tl and bw were 6.10.09 ( sd ) mm and 4.90.07 
 mg , respectively , while at 83 dph , these values were 18.90.28 mm and 48.20.72 mg , respectively ( fig . 1 and fig .
3 ) . tl increased 
 continuously from hatching to 83 dph , and could be represented by the growth equation : 
 tl=4.914e ( r = 0.961 ; t , time ) ( fig . 
 1 , upper panel ) .
bw showed a slight reduction between hatching and the completion of yolk 
 absorption ( 21 dph , see below ) , but increased rapidly once the hatchling began to feed ; it could be 
 represented by the growth equation : bw=2.200e ( r=0.725 ) ( fig .
the condition factor decreased from just - hatching to 21 dph , 
 but remained steady thereafter ( fig .
yolk absorption was complete at 21 dph ( 9.20.14 mm tl , 
 4.80.07 mg bw ; 462c iwt post hat ching ) ( fig .
abbreviations : tl , total length ; bw , body weight ; and t , time .
condition factor = 
 ( a / b)1,000 where a is bw and 
 b is tl .
bars = 3 mm . a : at 17 dph , 374c iwt ; inset shows a high - power view of a primordial germ cell ( pgc ; inset bar 
 = 10 m ) .
abbreviations : ab , air bladder ; cc , condensed chromatin ; ec , 
 endoovarian canal ; g , gut ; gr , genital ridge ; m , mesentery ; mf , me - iotic figure ; mp , meiotic 
 prophase ; mpgc , mitotic primordial germ cell ; pgc , primordial germ cell ; sc , somatic cell ; sd , 
 sperm duct ; sg , spermatogonia ; and y , yolk .
bars = 20 m . at 17 dph ( 7.90.12 mm tl , 3.70.06 mg bw , 374c iwt ) ( fig .
2-g ) when the first feeding 
 commenced following complete absorption of the yolk , gonads became apparent on both sides , running 
 beneath the air bladder mesentery to the genital ridge in the abdominal cavity ( fig .
the primordial germ cells ( pgcs ) were oval , approximately 7.2 m in 
 diameter , and enclosed by loose connective tissue .
their cytoplasm was weakly stained by hematoxylin , 
 and they contained round nuclei of 2 m in diameter ( fig .
3a 
 insert ) . by 19 dph ( 8.20.12 mm tl , 3.60.05 mg bw , 418c iwt ) ( fig .
2-h ) the number of pgcs was increased and numerous mitotic pgcs and condensed chromatin 
 were apparent ( fig .
3b ) . at 21 dph ( fig . 2-i : 9.20.14 mm tl , 4.80.07 mg bw , 
 462c iwt ) , some of the pgcs contained condensed chromatin ; oocytes were observed in meiotic 
 prophase ; and the pgcs and oocytes were enclosed by somatic cells ( fig . 

2-k : 11.20.17 mm tl , 10.5 0.16 mg 
 bw , 726c iwt ) , numerous oocytes were observed in meiotic prophase , meiotic figures were 
 present , and the oocytes were in the chromatin - nucleolus stage , with blood vessels and endo - ovarian 
 canals beginning to fill the ovary ( fig .
13.60.20 mg bw , 1,166c iwt ) , 
 the oocytes were in the perinucleolus stage and had increased to 50 m in diameter ( fig .
perinucleolus - stage oocytes increased in number and size to 83 dph . from 
 110 to 170 dph ,
abbrevia - tions : ec , endoovarian canal ; pno , oocyte in 
 the perinucleolus stage ; sc , somatic cell ; sg , spermatogonia ; smc , spermatocyte .
bars : in a and b = 30 m ; in c = 500 m ; and in d = 20 m .
the korean rose bitterling appeared to begin its sex differentiation when yolk absorption was completed 
 and the first feeding began ( 21 dph ) . at 25 dph ( fig .
2-j : 
 10.10.15 mm tl , 5.40.08 mg bw , 550c iwt ) , spermatogonia could be distinguished from pgcs 
 ( fig .
3d ) . at 33 dph ( fig . 
 2-k : 11.20.17 mm tl , 10.50.16 mg bw , 726c iwt ) , the spermatogonia had increased 
 in number ( fig .
no significant development of the testes was 
 seen up to 83 dph ( 18.9 mm tl , 48.2 mg bw , 1,826c iwt ) .
spermatocytes and sperm cells were 
 first observed at 140 dph ( 31.2 mm tl , 265.8 mg bw , 3,080c iwt ) and continued to mature 
 through 170 dph ( fig .
these observations indicate that the 
 korean rose bitterling belongs to the differentiated type of gonochoristic teleosts ( fig .
the present study shows that although the tl of the korean rose bitterling , rhodeus 
 uyekii , increased continuously post - hatching , the bw decreased somewhat until yolk 
 absorption was complete and feeding began ( at 21 dph ) .
this likely reflects the dependence of the 
 metabolism on energy from the yolk rather than the intake of exogenous food .
the sex differentiation of teleostei varies and can show irregular features , and many gonochoric fishes , 
 including some hermaphroditic fishes , easily change sex ( devlin & 
 nagahama , 2002 ) . in aquaculture , this has been used to produce monosexual populations with 
 improved economics .
thus , we need to fundamentally understand the sex differentiation and sex change of 
 fishes ( park et al . , 2004 ) . here , the sex differentiation of the 
 korean rose bitterling was analyzed with respect to tl and bw at various dph .
result of gonadogenesis 
 and sex differentiation in european eel , anguilla anguilla and bagrid catfish , 
 pseudobagrus fulvidraco .
these results relatively correct better than pre - vious 
 studies based on simply dph ( colombo & grandi , 1996 ; park et al . , 2004 ) .
moreover , as include the integral water 
 temperature for check factor in this study , it will be more correctly standard than previous 
 studies .
those of the tilapia , 
 tilapia zillii , appear at hatching ; those of the rainbow trout , salmo 
 gairdneri , are first seen at 36 dph under the mesonephric duct ; those of the ussurian 
 bullhead , leiocassis ussuriensis , and black bullhead , p. koreanus , 
 appear at 1 dph ; those of the ko - rean bullhead appear at 3 dph ; and those of the chinese minnow , 
 moroco oxycephalus , appear at 8 dph under the air bladder mesentery ( yoshikawa & oguri , 1978 ; takashima et al .
, 1980 ; park et al . , 1998 , 2001 , 2004 , 2008 ) .
the present study shows that the pgcs of the korean rose 
 bitterling first appear at 17 dph under the air bladder mesentery , and formation genital ridge thus 
 confirmed early gonad .
undifferentiated gonads of fish during gonadal differentiation in the ovary or testis differentiation 
 into different duct can be determined by forming patterns .
ovarian differentiation in the korean rose 
 bitterling began at 21 dph ( 9.20.14 mm tl , 4.80.07 mg bw , 462c iwt ) , as indicated by the 
 appearance of meiotic prophase oocytes .
the timing of sex differentiation differs by species , beginning 
 at 40 dph ( 326c iwt ) in the cherry salmon , oncorhynchus masou , but at 
 ( 270c iwt ) in the coho salmon , o. kisutch ( pifferrer & donaldson , 1989 ; park et al . , 

however , there seems to be some consistency in that the sex differentiation of various 
 fish species begins at the time the yolk sac is completely absorbed and the first feeding occurs , such 
 as in the rainbow trout , the cherry salmon ( 40 dph ) ( park et al .
, 
 1997 ; van den hurk & solf , 1981 ) , and the korean 
 rose bitterling ( 21 dph ; this work ) .  
the sex differentiation of teleosts can be grouped at the level of species or family , with the process 
 occurring at 10 ~40 dph in cichlids , 10~30 dph in cyprinodontids , and 3~40 dph in anabantids ( yamazaki , 1976 ; shelton & 
 jensen , 1979 ; pandian & sheela , 1995 ) . the sex 
 differentiation of the ussurian bullhead occurs at 15~20 dph and that of the korean bullhead occurs in a 
 similar range of 12 - 20 dph , while those of the mandarin fish , siniperca scherzeri , and 
 cherry salmon begin somewhat later , at 40 dph ( park et al . , 1997 , 
 2001 , 2004 ) .
the 
 present study shows that the sex differentiation of the korean rose bitterling occurs at 21~25 dph , with 
 ovaries first seen at 21 dph and testes observed at 25 dph . in some fishes , such as the ocean perch
, 
 diterma temmincki , testicular differentiation is seen first ; however , most fish 
 show initial differentiation of the ovary ( takashima et al . , 
 1980 ;
lee & lee , 1996 ; park et al . , 2004 ) , as observed here for the korean rose bitterling .
the entovarian sac forms at the center of the ovarian 
 cavity , while the parovarian sac forms on the edge of ovary ( lee & 
 lee , 1996 ) . the sweet fish , plecoglossus altivelis , and the threespine 
 stickleback , gasterosteus aculeatus , reportedly form parovarian sacs ( bang et al . , 2000 ) , while the white spotted char , salvelinus 
 leucomaenis , the rainbow trout , and the ussurian bullhead reportedly form entovarian sacs . 

the present study shows that the korean rose bitterling forms its sacs in the middle of the ovary 
 pattern , indicating that this species forms entovarian sacs ( takashima 
 et al . , 1980 ; park et al . , 2001
the observation that the korean rose bitterling began ovarian differentiation at 21 dph and testicular 
 differentiation at 25 dph indicates that this species exhibits gonochorism . in this , the korean rose 
 bitterling resembles the rainbow trout , the chum salmon , o. keta , the medaka , 
 oryzias latipes , the ussurian bullhead , the chinese minnow , and the ocean perch 
 ( robertson , 1953 ; tuzuki et 
 al .
, 1966 ; yoshikawa & oguri , 1978 ; takashima et al . , 1980 ;
lee & 
 lee , 1996 ; park et al . , 1998 , 2001 ) .
the korean rose bitterling is endemic species to korea and the importance in the resources enforcement 
 has been emerged ( jeon , 1982 ) .
therefore , it is important that we 
 understand the timing of sex differentiation timing and the sex ratio in this species .
future studies 
 should focus on tracking the differentiation and development of the gonad in the korean rose bitterling 
 to see how the sex ratio and timing of sex differentiation can be altered by artificial factors ( e.g. , 
 hormones and environmental factors ) .
the results of the present study could be utilized as fundamental 
 data for the resources proliferation and the ecological changes in this species .
 abstractthis report describes the sex differentiation of the korean rose bitterling , rhodeus 
 uyekii , from hatching to 170 days post - hatch ( dph ) in relation to total length ( tl ) , 
 body weight ( bw ) , and integral water temperature ( iwt ) . 
 the growth curve of tl from just hatching to 
 83 dph was 5.144e0.045 t ( r = 0.961 ; t , time ) , and that of bw was 
 2.398e0.086 t ( r = 0.725 ) . 
 primordial germ cells ( pgcs ) were 
 observed at 17 dph ( 7.9 mm tl , 3.74 mg bw , 374c iwt ) , and thereafter began to protrude into the 
 peritoneal cavity . at 21 dph ( 9.20.14 mm tl , 4.80.07 mg bw , 462c iwt ) , 
 some pgcs contained 
 condensed chromatin and oocyte were observed in meiotic prophase . 
 in contrast to the ovaries , which 
 grew gradually after sexual differentiation , testes began multiplying at 25 dph ( 10.1 mm tl , 5.42 mg 
 bw , 550c iwt ) , when testicular differentiation was first identified , and multiplied continuously 
 thereafter . at 33 dph ( 11.2 mm tl , 10.5 mg bw , 726c iwt ) , 
 the developing testes contained 
 spermatogonia that exhibited mitotic activity . 
 no spermatocyte or sperm cell was observed until 83 
 dph ( 18.9 tl , 48.2 mg bw , 1,826c iwt ) . at 170 dph ( 32.5 mm tl , 270.1 mg bw , 3,740c iwt ) , which was 
 the end point of this study , the mature ovaries showed germinal vesicle breakdown , while the mature 
 testes contained observable spermatocytes and sperm cells . 
 these results allow us to identify the 
 sex differentiation type of the korean rose bitterling as differentiated gonochoristic . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION