Studies on Improvement of Seed Production Techniques in Salmonids and Osmerids

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1 Aqu-BioScience Monogrphs, Vol. 5, No. 4, pp (2012) Studies on Improvement of Seed Production Techniques in Slmonids nd Osmerids Shiny Mizuno Slmon nd Freshwter Fisheries Reserch Institute Fisheries Reserch Deprtment Locl Independent Administrtive Agency Hokkido Reserch Orgniztion 3-373, Kitkshiwgi, Eniw, Hokkido , Jpn e-mil: Astrct Andromous slmonids nd osmerids re rtificilly propgted in Jpn y relese of their seeds. However, there re mny unsolved prolems in the techniques of their propgtion. In the present monogrph, studies on the improvement of seed production techniques in 4 fishes (msu slmon Oncorhynchus msou, chum slmon O. ket, shishmo smelt Spirinchus lnceoltus nd Jpnese smelt Hypomesus nipponensis) were outlined. Techniques to evlute dorsl fin pigmenttion during smoltifiction s n externl seed qulity, nd to improve seed qulity of htchery-rered fish, nd the discovery of metolic prolems in htchery-rered fish were descried in yerling msu slmon. In underyerling msu slmon, techniques to evlute nutritionl conditions using kidney melno-mcrophge density ws developed, nd pplied to the evlution of the nutritionl condition in htchery-rered fish fter relese. In chum slmon fry, the development of techniques to monitor the physicl condition nd to find its pproprite culture conditions ws reviewed. In egg cultures of shishmo nd Jpnese smelt, techniques to eliminte egg dhesiveness with tretments of kolin or scllop shell powder suspension were estlished in order to improve htching rtes. In ddition, the pproprite emryogenetic stge for the relese of shishmo smelt emryos ws demonstrted. Consequently, this monogrph revels tht these techniques contriute directly to the development of rtificil propgtion in some slmonids nd osmerids. Received on Jnury 10, 2012 Accepted on June 21, 2012 Online pulished on Decemer 21, 2012 Keywords culture condition htchery-rered fish htching rte Hypomesus nipponensis kolin Oncorhynchus ket Oncorhynchus msou physicl condition scllop shell powder seed production seed qulity Spirinchus lnceoltus 1. Generl introduction Andromous slmonids nd osmerids including msu slmon Oncorhynchus msou, chum slmon O. ket, shishmo smelt Spirinchus lnceoltus nd Jpnese smelt Hypomesus nipponensis re importnt species for costl nd freshwter commercil fisheries in Hokkido, the northern-most prefecture in Jpn. These fish re not lwys undnt under nturl reproduction systems (Ngt nd Keriym 2004; Toro 2005, ; Miykoshi 2006). Therefore, these fish re rtificilly propgted y relesing their seeds in order to increse their stock in vrious plces in Hokkido (Kusud nd Ternishi 1996; Keriym 1999; Torisw 1999). Msu slmon, which inhit oth Pcific Ocen nd the Jpn Se costs of Hokkido, spwn in upstrem regions in utumn nd htch the winter of the sme yer (Kto 1991). The fish re seprted into two types: ndromous nd strem-resident types. The ndromous type migrtes to the ocen in spring fter spending 1 or 2 yers in the strems. Before the sewrd migrtion, they undergo smoltifiction, series of ehviorl, morphologicl, physiologicl nd iochemicl chnges controlled y multiple hormonl pthwys (Hor 1988) tht trnsform the freshwter prr into sewrd-migrtory smolt. After 1 yer of life in the ocen, the fish return to their ntl strem in spring nd spwn in utumn (Kuo 1980; Mchidori nd Kto 1984; Kto 1991). For the rtificil propgtion of msu slmon, htchery-rered (htchery) juveniles, originting from rtificil insemintion nd fed commercil food in htcheries, re relesed into strems (Mym 1992). Effects of msu slmon propgtion, which re expressed s the recovery rte of relesed htchery seed, re of 0.41 to 2.12% (Miykoshi 2006), while the recovery rte of chum slmon rtificil propgtion ws >10% (Ty 1989). In other 2012 TERRAPUB, Tokyo. All rights reserved. doi: /sm

2 104 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 words, msu slmon propgtion costs more money compred to chum slmon propgtion. It is essentil for the sustinle rtificil propgtion of msu slmon to reduce the cost of seed production y incresing the survivl rte of seed. Htchery msu slmon re relesed in two wys. One is clled the smolt relese method where yerling (1+) smoltified juveniles re relesed into downstrem regions in spring, nd the other is clled the spring juvenile relese method, where underyerling (0+) juveniles re relesed into upstrem regions in spring (Mym 1992). One of the keys to incresing survivl rte of seed is stocking htchery juveniles with high seed qulities determined from ethologicl, physiologicl nd iochemicl chrcteristics. However, seed qulity of htchery juveniles hs not een perfectly evluted in msu slmon. In ddition, there hs een no ttempt to improve the seed qulity of htchery juveniles. Chum slmon, which inhit the whole region of Hokkido, htch in rivers in winter, nd migrte to the ocen the following spring. All of the fish re ndromous. After 2 to 6 yers of mrine life, dults return to spwn in their ntl rivers in utumn (Slo 1991). In the progrm, 0+ fry, which were produced in htcheries in the sme wy s the msu slmon, re relesed into the river in spring (Koyshi 1980). However, the incresed stock due to the propgtionprogrm hs cused downsizing nd ging (Keriym nd Edpline 2004). This phenomenon is elieved to e due to popultion density-dependent effect: the growth of chum slmon fry is restrined y crrying cpcity, such s the mount of food or hitt size, in the North Pcific (Keriym et l. 2007). Htchery fish hve to coexist with wild fish in nrrow crrying cpcity. Therefore, iologicl interctions etween htchery popultions nd wild popultions re eing studied (Keriym nd Edpline 2004). Hilorn (1992) hs wrned tht the excessive relese of htchery fish decreses the genetic diversity of Pcific slmon, including chum slmon. Accordingly, rtificil propgtion nd the preservtion of wild popultions must e jointly considered in order to preserve the nturl lnce. Keriym (2002) ws concerned out the infection of wild fish y htchery fish diseses. Considering this ckground, the relile production of htchery fry with good physicl conditions is more importnt thn n incresed numer of relesed htchery fry for the sustinle rtificil propgtion of chum slmon. Chum slmon fry re intensively cultured in high density conditions in htchery ponds. In generl, intensively cultured fish re commonly under high physiologicl stress resulting from deteriorted rering conditions (Ptinõ et l. 1986; Ppoutsoglou et l. 1987). On the other hnd, it hs een impossile to monitor the physicl conditions of htchery fry cultured in high density conditions, nd prcticl environmentl conditions for the culture of fry in good physicl conditions hve not yet een found. Shishmo smelt migrte to their ntl river on the Pcific Ocen side of Hokkido in erly winter to spwn (Hikit 1930, 1958). Adults spwn 1.4 mm sized eggs in freshwter environments out 3 to 9 km upstrem from the estury (Ito 1959, 1963, 1964; Okd nd Sski 1960; Okd et l. 1975, 1976; Omi 1978). The lrve migrte to the estury immeditely fter htching crried y the snow-melt wter during the following spring, nd mture fter 2 or 3 yers of life in the ocen (Omi 1978). All of the fish re ndromous (Miyji et l. 1976). On the other hnd, Jpnese smelt, which re distriuted round the whole of Hokkido, consist of ndromous nd lke-resident types (Hmd 1961; Ktym et l. 1999). Andromous-type fish migrte to their ntl strem nd spwn 0.8 mm sized eggs in shllow strems from April to June (Shirishi 1961; Torisw 1999). The generl lifespn of the fish is 1 to 2 yers (Utoh nd Skzki 1983, 1984, 1987; Torisw 1999). For the rtificil propgtion of shishmo nd Jpnese smelt, lrve, otined y rtificil insemintion, re relesed into the rivers or lkes through the dringe of htchery wters (Iwi nd Osm 1986; Kusud nd Ternishi 1996; Izuk 2003; Kitsukw et l. 2003). However, the efficiency of the propgtion hs not yet een ccurtely evluted. It is necessry to first produce lot of seed to perform pilot relese, in order to estlish effective seed-relese methods nd to elucidte the effects of relesing htchery seed. Some shishmo nd Jpnese smelt htcheries intensively culture eggs using upwelling flow-type crylic nd cylindricl jr incutors (Kusud nd Ternishi 1996; Kitsukw et l. 2006). However, it ws found tht eggs cultured in the jr incutors showed low htching rtes (Tked et l. 2002). In few shishmo smelt htcheries, eyed-stge emryos re relesed into the river (Mizuno et l. 2004, 2005) efore they htch. However, the most pproprite time for relesing eyedstge emryos into the river is unknown. In this monogrph, I review reserch performed on the improvement of seed production techniques in slmonids nd osmerids, with the im to solve some of the prolems mentioned ove. In Section 2, three pproches to evluting nd improving the seed qulity of htchery msu slmon smolt re descried. Section 3 reviews 2 studies on the development nd ppliction of techniques evluting seed qulity in htchery msu slmon prr for spring juvenile relese. Section 4 dels with 2 studies deling with the estlishment of techniques to monitor physicl conditions nd the elucidtion of pproprite culture conditions in chum slmon fry. In Section 5, three ttempts to develop techniques to improve survivl rte in rtificil propgtion of shishmo nd Jpnese smelt were de-

3 S. Mizuno / Aqu-BioSci. Monogr. 5: , scried. The finl chpter descries the implictions nd perspective studies of the improvement of seed production techniques in slmonids nd osmerids. 2. Evlution nd improvement of seed qulities in 1+ htchery msu slmon smolts 2-1. Introduction Externl smoltifiction is minly chrcterized y the lck pigmenttion of dorsl nd cudl fin mrgins due to the diffusion of melnin grnules in melnophores nd y ody silvering due to the deposition of gunine on the skin (Ur et l. 1994). A quntittive nlysis of the ody silvering hve een developed in msu slmon (Chid nd Kijim 1994; Ando et l. 2005), mgo slmon O. rhodurus (Kuwd et l. 2000) nd Atlntic slmon Slmo slr (Duston 1995; Hner et l. 1995). In contrst, quntifiction of the dorsl fin pigmenttion hs not yet een estlished in slmonids. The most well-known internl chnges occurring during smoltifiction is the development of sewter dptility, which results from physiologicl nd iochemicl chnges in the osmoregultory orgns such s the gill, kidney, intestine nd esophgus (Clrke nd Hirno 1995). Sewter dptility, which possily reflects the survivl of juveniles fter their entry into sewter, is often rised s relile indictor to judge the internl qulity of htchery smolt seed. A sewter chllenge test, which exmines the chnge in serum N + levels during trnsfer from freshwter to sewter (Clrke nd Blckurn 1977), nd gill N +,K + -ATPse ctivity (Zugg nd McLin 1971; Folmer nd Dickhoff 1980; Boeuf nd Hrche 1982; Boeuf nd Prunet 1985; McCormick 1993) re used to determine sewter dptility. Until now, however, only the survivl rte fter the trnsfer from freshwter to sewter hs een exmined in the smolt seeds of msu slmon from htcheries (Misk et l. 1998). Some previous studies hve pointed out the prolems of htchery smolt: sewter dptility is low (Shrimpton et l. 1994), development of sewter dptility is not synchronized with externl smoltifiction (Wedemeyer et l. 1980), nd hormonl regultion relted to the development of sewter dptility is incomplete (Shrimpton et l. 1994; McCormick nd Björnsson 1994). Chnges in metolism re lso generlly ccepted s n effect of internl smoltifiction. Metolic chnges during smoltifiction hve een reported through metolic enzyme ctivity, mounts of metolites, nd metolic rtes in Atlntic nd coho slmon (O. kisutch). In Atlntic slmon, stndrd nd ctive metolic rte is higher in smolts compred to prr (Mxime et l. 1989). Atlntic slmon smolts hve lower heptic nd muscle glycogen nd incresed lood glucose levels, which reflects n ctivtion of glycolysis, sent in prr (Wendt nd Sunders 1973). Sheridn et l. (1985) demonstrted tht the low heptic glycogen content ws cused y comintion of reduced glycogen synthesis nd incresed glycogenolysis in coho slmon smolts. Muscle phosphofructokinse ctivity, glycolytic enzyme, ws high during smoltifiction in Atlntic slmon (Leonrd nd McCormick 2001). Citrte synthse ctivity, citric cid cycle enzyme of liver, gill nd kidney ws enhnced during smoltifiction in Atlntic slmon (McCormick et l. 1989). In the gill nd liver, the ctivity of respirtory enzymes incresed during smoltifiction in Atlntic slmon (Lngdon nd Thorpe 1985; McCormick nd Sunders 1987). These previous reports demonstrte tht the ctivtion of glycolysis, nd of the citric cid cycle, nd the increse in respirtion re notle metolic chnges which occur during smoltifiction. However, there ws little informtion on chnges in metolism nd metolic enzymes during smoltifiction in msu slmon. Consequently, seed qulity relted to sewter dptility nd metolism hs not yet een sufficiently evluted in msu slmon smolt. Andromous slmonid juveniles migrting from freshwter to sewter environment re prone to high mortlity (Prker 1968; Heley 1982; Bx 1983). One of the principl cuses for this is predtion y seirds nd lrge fish (Bemish nd Neville 1995; Ngsw 1998; Kwmur nd Kudo 2000). Individul survivl, then, is dependent on swimming ility, in order to escpe from predtors (Jyne nd Luder 1993). This swimming ility is more closely relted to urst swimming, which expresses the sprint swimming in fish (Jyne nd Luder 1993), rther thn to criticl swimming, which reflects sustined swimming. Burst swimming is powered y white glycolytic fires tht constitute the ulk of the myotoml mss nd tht depend on n neroic metolism (Domenici nd Blke 1997; Frnklin nd Johnston 1997), wheres criticl swimming principlly relies upon eroic red oxidtive fires (Bone et l. 1978; Gllugher et l. 1995). Burst swimming results in the production of lctte nd H + y the muscle, which cuse mrked reduction in plsm ph, s well s respirtory nd metolic cidosis (Turner et l. 1983; Millign nd Wood 1986). In the rinow trout O. mykiss, increses in lood hemogloin (H) concentrtions following urst swimming hve een oserved (Millign nd Wood 1986, 1987; Person nd Stevens 1991). The incresed H concentrtions hve, in turn, een relted to recovery from cidosis-induced ftigue (Millign nd Wood 1987; Wng et l. 1994). It is plusile, therefore, tht there is reltionship etween concentrtions of H nd the urst swimming cpcity in slmonids, ut this hypothesis hs not yet een tested. Woodwrd nd Smith (1985) noted tht htchery

4 106 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 slmon displyed plcid ehvior nd poor swimming ility due to the rering stress of high densities nd low wter flow. The swimming ility cn e improved y chronic swimming exercises, resulting in incresed H concentrtions (Hochchk 1961; Burrows 1969; Znyszek nd Smith 1984). Furthermore, incresed wter flow results in improved swimming performnce in htchery msu slmon in circulr tnk (Azum et l. 2002). However, it is often difficult or unprcticl to extrct enough wter to generte fst wter flow or to mke htchery fish exercise in circulr tnks, given the limited mount of utilizle wter, nd the fct tht rectngulr ponds re used y mny Hokkido Prefecture htcheries. Accordingly, other methods must e developed to improve the swimming ility of htchery fish. Incresing dietry iron is known to increse H concentrtions in fish (Kwtsu 1972; Iked et l. 1973; Skmoto nd Yone 1978; Crriquiriorde et l. 2004), ut its effect on urst swimming cpcity hs not yet een studied. Susection 2-2 focuses on the estlishment of quntifying system for the lck pigmenttion of the dorsl fin mrgin during smoltifiction using imge nlysis nd on the elucidtion of the reltionships etween the dorsl fin pigmenttion nd gill N +,K + - ATPse ctivity. Susection 2-3 dels with chnges in the ctivities nd trnscription levels of severl metolic enzymes during smoltifiction, in oth wild nd htchery fish, in order to show physiologicl qulities prticulr to htchery smolt. In Susection 2-4, differences in urst swimming cpcity nd physiologicl prmeters other thn those shown in Susections 2-2 nd 2-3 of oth etween wild nd htchery smolts, nd the effects of diets supplemented with iron on these prmeters in the htchery smolt were investigted Development of techniques to evlute dorsl fin pigmenttion during smoltifiction Smolting htchery fish of the Donn Reserch Center of Slmon nd Freshwter Fisheries Reserch Institute (SFRI) nd smolting wild fish cptured in the two rivers in Hokkido were studied. One of the two rivers, the Ken-ichi River, ws utilized for rering the SFRIhtchery fish. After lethl nesthesi of the fish, the depressed portion of the dorsl fin memrne etween the fourth nd fifth soft rys (Fig. 1) ws oserved with light microscope. The gryscle digitl picture of the portion ws divided into lck nd white prts include ll melnophores in the lck prt using n imge nlysis softwre (Fig. 2). The dorsl fin pigmenttion level ws expressed s the percentge of the lck prt over the whole picture size. The level of dorsl fin pigmenttion incresed from Jnury to My during smoltifiction in oth stocks of wild fish nd the stock of htchery fish (P < 0.05, One-wy ANOVA) (Fig. 3), reflecting the progress of exterior IV V Fig. 1. Oserved portion of dorsl fin pigmenttion. Pnel A indictes the entire dorsl fin of wild msu slmon smolt in the Ken-ichi River. Pnel B is mgnified from the smll rectngulr re indicted in pnel A. In pnel B, the white squre (100 µm 100 µm) shows the portion of the dorsl fin oservtion. IV: fourth soft ry, V: fifth soft ry, VI: sixth soft ry. Scle rs show 1.00 mm. Reprinted from Aquculture, 229, Mizuno et l., Quntittive chnges of lck pigmenttion in the dorsl fin mrgin during smoltifiction in msu slmon, Oncorhynchus msou, , 2004, with permission from Elsevier. smoltifiction. The levels converged t round 83 84% in My, which is the pek time of downstrem migrtion to the se in wild msu slmon. Moreover, there ws no significnt difference in the pek level etween wild nd htchery fish (P > 0.05, One-wy ANOVA). These findings revel the estlishment of quntittive system for dorsl fin pigmenttion with level of 83 84% eing n pproprite indictor for externl seed qulity in htchery smolt. SFRI-htchery smolt is regrded s excellent seed sed on dorsl fin pigmenttion. Gill N +,K + -ATPse ctivity incresed from Jnury to My during smoltifiction in ll wild nd htchery fish (P < 0.05, One-wy ANOVA) (Fig. 4). There were no significnt differences in the ctivity etween wild nd htchery fish t ny given period (P > 0.05, Onewy ANOVA). These results suggest tht the SFRIhtchery smolts hve s high sewter dptility s wild fish. Figure 5 shows the reltionship etween dorsl fin pigmenttion nd gill N +,K + -ATPse ctivity in ll fish. A positive nonliner correltion ws found etween the level of pigmenttion of the dorsl fin nd gill ATPse ctivity in oth the wild nd htchery fish (P < , Spermn s rnk correltion coefficient) (Figs. 3, 4). The period of incresed dorsl fin pigmenttion preceded tht of enhnced gill N +,K + - ATPse ctivity in ll fish. However, it hs een generlly ccepted tht externl smoltifiction coincided with typicl internl smoltifiction, such s the development of sewter dptility, in other slmonids (Hor 1988). The gp etween the increse of dorsl VI

5 S. Mizuno / Aqu-BioSci. Monogr. 5: , Fig. 2. Dorsl fin pigmenttion during smoltifiction of wild msu slmon in the Utetsu River. Upper pictures in gryscle, lower pictures fter lck nd white trnsformtion y n imging softwre. The lines from to e indictes dorsl fins in Jnury, Ferury, Mrch, April nd My, respectively. Scle r shows 100 µm. Reprinted from Aquculture, 229, Mizuno et l., Quntittive chnges of lck pigmenttion in the dorsl fin mrgin during smoltifiction in msu slmon, Oncorhynchus msou, , 2004, with permission from Elsevier. Level of dorsl fin pigmenttion (%) Wild (Ken-ichi) Wild (Utetsu) Htchery Jn. Fe. Mr. Apr. My Smpling time Fig. 3. Quntittive chnges in dorsl fin pigmenttion during smoltifiction in wild nd htchery-rered msu slmon. The letter djcent to symol indictes significnt difference with the initil vlue of ech respective group (P < 0.05; One wy ANOVA). Reprinted from Aquculture, 229, Mizuno et l., Quntittive chnges of lck pigmenttion in the dorsl fin mrgin during smoltifiction in msu slmon, Oncorhynchus msou, , 2004, with permission from Elsevier. Gill N +,K + -ATPse ctivity ( mol Pi/ mg protein hour) Wild (Ken-ichi) Wild (Utetsu) Htchery Jn. Fe. Mr. Apr. My Smpling time Fig. 4. Chnges in gill N +,K + -ATPse ctivity during smoltifiction in wild nd htchery-rered msu slmon. The letter djcent to symol indictes significnt difference with the initil vlue of ech respective group (P < 0.05; One wy ANOVA). Reprinted from Aquculture, 229, Mizuno et l., Quntittive chnges of lck pigmenttion in the dorsl fin mrgin during smoltifiction in msu slmon, Oncorhynchus msou, , 2004, with permission from Elsevier. fin pigmenttion nd gill N +,K + -ATPse ctivity is possily prticulr to msu slmon. However, the pek of dorsl fin pigmenttion coincided with tht of gill N +,K + -ATPse ctivity. Therefore, we cn use the level of dorsl fin pigmenttion s n indictor in order to find the pek of smoltifiction in SFRIhtchery msu slmon Differences in metolism etween 1+ wild nd htchery smolts Wild nd SFRI-htchery smolting fish were smpled t the sme time. The river where the wild fish were cught, ws lso utilized for rering the htchery fish. Serum, gill nd liver of the fish were smpled. Serum

6 108 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Level of dorsl fin pigmenttion (%) Wild (Ken-ichi) Wild (Utetsu) Htchery Gill N +,K + -ATPse ctivity ( mol Pi/ mg protein hour) Fig. 5. Correltion of gill N +,K + -ATPse ctivity with dorsl fin pigmenttion level, in wild nd htchery-rered smolting msu slmon. Spermn s rnk correltion coefficient y rnk test ws used s sttisticl nlysis. The norml, old nd dotted line show the nonliner correltions [Pigment] = ln [ATPse] (r 2 = 0.752, P < ) in the Ken-ichi River, [Pigment] = ln [ATPse] (r 2 = 0.879, P < ) in the Utetsu River nd [Pigment] = ln [ATPse] (r 2 = 0.806, P < ) in the htchery-rered groups, respectively. Reprinted from Aquculture, 229, Mizuno et l., Quntittive chnges of lck pigmenttion in the dorsl fin mrgin during smoltifiction in msu slmon, Oncorhynchus msou, , 2004, with permission from Elsevier. glucose concentrtion nd heptic glycogen content decresed during smoltifiction in wild fish (P < 0.05, One-wy ANOVA), while the decrese ws not found in htchery fish (P > 0.05, One-wy ANOVA) (Fig. 6). Heptic glycogen content of htchery fish ws significntly lower thn in wild fish in Mrch (P < 0.05, Student s t-test) (Fig. 6). These findings reflect low glycogenolysis during smoltifiction nd low use of glycogen s n energy source in htchery fish. Moreover, decrese in heptic pyruvte kinse (PRK) ctivity ws oserved from April to My in htchery fish (P < 0.05, One-wy ANOVA), while there ws no decrese in the ctivity t the sme stge in wild fish (P > 0.05, One-wy ANOVA) (Fig. 7). An increse in gill PRK ctivity during smoltifiction ws reveled in wild fish (P < 0.05, One-wy ANOVA), while it did not increse in htchery fish (P > 0.05, One-wy ANOVA) (Fig. 7). Fig. 6. Chnges in serum glucose concentrtion (GL) nd liver glycogen content (GC) during smoltifiction in wild nd htchery-rered msu slmon. Different smll lpheticl letters indicte significnt differences etween wild nd htchery-rered fish t ech smpling time (P < 0.05; Student s t-test). Asterisks revel significnt differences compred to the vlue of the sme prmeter in Mrch within ech group of fish (P < 0.05; One-wy ANOVA). Cross mrks indicte significnt chnge from the vlue of the sme prmeter one month efore for the sme group of fish (P < 0.05; Student s t-test). Modified from Aquculture, , Mizuno et l., Chnges in ctivity nd trnscript level of liver nd gill metolic enzymes during smoltifiction in wild nd htchery-rered msu slmon (Oncorhynchus msou), , 2010, with permission from Elsevier. These results suggest low glycolytic chnges during smoltifiction nd low use of glycolysis t the smolt stge in htchery fish. Furthermore, this study discovered temporl difference in incresed heptic citrte synthse (CS) ctivity etween htchery nd wild fish, nd the sence of n incresed gill CS ctivity during smoltifiction in htchery fish (P > 0.05, One-wy ANOVA) (Fig. 8), which revels tht there is little chnge of the citric cid cycle t the smolt stge in htchery fish. Htchery smolt hd lower liver cytochrome c oxidse (COX) ctivity compred to wild smolt in My (P < 0.05, Student s t-test), which possily mens tht htchery smolt cnnot prepre for the ctivtion of the heptic respirtory chin efore their sewrd migrtion (Fig. 9). Liver nd gill trnscrip-

7 S. Mizuno / Aqu-BioSci. Monogr. 5: , Fig. 7. Chnges in liver nd gill pyruvte kinse (PRK) ctivity during smoltifiction in wild nd htchery-rered msu slmon. Different smll lpheticl letters indicte significnt differences etween wild nd htchery-rered fish t ech smpling time (P < 0.05; Student s t-test). Asterisks revel significnt differences compred to the vlue of the sme prmeter in Mrch within ech group of fish (P < 0.05; One-wy ANOVA). Cross mrks indicte significnt chnge from the vlue of the sme prmeter one month efore for the sme group of fish (P < 0.05; Student s t-test). Modified from Aquculture, , Mizuno et l., Chnges in ctivity nd trnscript level of liver nd gill metolic enzymes during smoltifiction in wild nd htchery-rered msu slmon (Oncorhynchus msou), , 2010, with permission from Elsevier. Fig. 8. Chnges in liver nd gill citrte synthse (CS) ctivities during smoltifiction in wild nd htchery-rered msu slmon. Different smll lpheticl letters indicte significnt differences etween wild nd htchery-rered fish t ech smpling time (P < 0.05; Student s t-test). Asterisks revel significnt differences compred to the vlue of the sme prmeter in Mrch within ech group of fish (P < 0.05; One-wy ANOVA). Cross mrks indicte significnt chnge from the vlue of the sme prmeter one month efore for the sme group of fish (P < 0.05; Student s t-test). Modified from Aquculture, , Mizuno et l., Chnges in ctivity nd trnscript level of liver nd gill metolic enzymes during smoltifiction in wild nd htchery-rered msu slmon (Oncorhynchus msou), , 2010, with permission from Elsevier. tion levels of ATP synthse suunit 8 (AST) showed opposite chnges etween htchery fish (decrese) nd wild fish (increse) from April to My (P < 0.05, Onewy ANOVA) (Fig. 10). Another notle point is tht there re temporl differences in the increse in liver nd gill AST trnscription levels etween htchery nd wild fish. Htchery smolt indicted lower AST trnscription levels in the liver nd gill in My compred to the wild smolt (P < 0.05; Student s t-test) (Fig. 10). These findings suggest tht htchery fish cnnot produce mny respirtory chin enzymes efore their sewrd migrtion. The present study found tht SFRI-htchery msu slmon hd some prolems in the crohydrte metolism, citric cid cycle, nd respirtory chin, which suggests tht the htchery fish my not e le to djust to mrine environments fter migrting from the river to the se leding to high mortlity rtes. Wlton (1986) reported tht low crohydrte nd high protein food intke induced low ctivities of glycolytic enzymes nd the citric cid cycle in rinow trout. Bruge et l. (1994) demonstrted tht elevted digestile crohydrte intke resulted in glycemi in rinow trout. These findings imply tht the prolem of SFRI-htchery fish is cused y low crohydrte diet. Menwhile, Leonrd nd McCormick (2001) hve found tht there were significnt fluctutions nd temporl differences etween htchery fish nd wild Atlntic slmon of the liver β-hydroxycyl-coenzyme A dehydrogense, hert phosphofructokinse nd white

8 110 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Fig. 9. Chnges in liver cytochrome c oxidse (COX) ctivity during smoltifiction in wild nd htchery-rered msu slmon. Different smll lpheticl letters indicte significnt differences in the vlue etween wild nd htcheryrered fish t ech smpling time (P < 0.05; Student s t-test). Asterisks revel significnt differences compred to the vlue of the sme prmeter in Mrch within ech group of fish (P < 0.05; One-wy ANOVA). Cross mrks indicte significnt chnge from the vlue of the sme prmeter one month efore for the sme group of fish (P < 0.05; Student s t-test). Modified from Aquculture, , Mizuno et l., Chnges in ctivity nd trnscript level of liver nd gill metolic enzymes during smoltifiction in wild nd htchery-rered msu slmon (Oncorhynchus msou), , 2010, with permission from Elsevier. muscle lctte dehydrogense during smoltifiction, which indicted other metolic prolems in htchery fish. McCormick nd Björnsson (1994) hve found higher levels of plsm cortisol, stress-relted hormone involved in stimulting or mintining incresed plsm glucose nd gluconeogenesis, in htchery smolt thn in migrting htchery smolt relesed into strem, which my reflect tht htchery fish experience greter stress thn wild fish. This considertion my demonstrte tht metolic prolems of htchery fish result from the stress of the rtificil rering environment such s feeding, rtificil hndling nd high rering densities. In ddition, Misk et l. (2004) elucidted tht fsting induced rpid decrese in heptic glycogen in msu slmon, which suggests tht the mount of food supplied impcts the heptic glycogen content. Therefore, the smll heptic glycogen content of htchery fish is possily cused y stress relted to low food supply in the present study. Acute hndling stress, which often occurs during fish trnsporttion in htchery fish, induces temporry increse in plsm cortisol nd glucose levels (Crey nd McCormick 1998). These erlier results suggest the considerle increse in plsm cortisol levels relted to hndling stress disturs the crohydrte metolism in the SFRIhtchery fish. Furthermore, high rering densities resulted in reduced plsm levels of thyroid hormones nd cortisol relted to smoltifiction in smolting coho Fig. 10. Chnges in trnscription levels of liver nd gill ATP synthse suunit 8 (AST) during smoltifiction in wild nd htchery-rered msu slmon. Different smll lpheticl letters indicte significnt differences in the vlue etween wild nd htchery-rered fish t ech smpling time (P < 0.05; Student s t-test). Asterisks reveled significnt differences compred to the vlue of the sme prmeter in Mrch within ech groups of fish (P < 0.05; One-wy ANOVA). Cross mrks indicte significnt chnge from the vlue of the sme prmeter one month efore for the sme group of fish (P < 0.05; Student s t-test). Modified from Aquculture, , Mizuno et l., Chnges in ctivity nd trnscript level of liver nd gill metolic enzymes during smoltifiction in wild nd htchery-rered msu slmon (Oncorhynchus msou), , 2010, with permission from Elsevier. slmon (Ptinõ et l. 1986) nd in incresed plsm glucose levels nd low growth rte in rinow trout (Procrione et l. 1999). Crohydrte metolism is directly or indirectly regulted y not only cortisol ut lso insulin, glucgon nd epinephrine in fish (Plisetsky et l. 1988; Plisetsky 1990; Vijyn et l. 1993). Therefore, this considertion my revel tht predictle socil stress relted to high rering densities disturs the endocrine control of the crohydrte metolism in SFRI-htchery fish. The est wy to solve the metolic prolems of the htchery msu slmon smolt is possily to chieve n improvement of dietry qulity, repletion, reduce hndling nd/or develop low density-rering conditions.

9 S. Mizuno / Aqu-BioSci. Monogr. 5: , CF Wild Control Iron-1 Iron-2 cd Jn. Fe. Apr. My Smpling time d Wild Control Iron-1 Jn. Fe. Apr. My Smpling time c Fig. 11. Condition fctor (CF) in wild nd htchery-rered msu slmon t ech smpling time of the 2003 nd 2004 experiments. The letters,, c nd d indicte significnt differences etween the wild, htchery control, iron-1 nd iron-2 groups t given smpling time, respectively (P < 0.05; One-wy ANOVA). Asterisks show significnt differences compred to the vlue of the previous smpling time within the sme group (P < 0.05; Student s t-test). Modified from Aquculture, 273, Mizuno et l., Effects of diets supplemented with iron citrte on some physiologicl prmeters nd on urst swimming velocity in smoltifying htchery-rered msu slmon (Oncorhynchus msou), , 2007, with permission from Elsevier Development of techniques to improve the seed qulity of 1+ htchery smolts In the 2003 experiment, 1+ SFRI-htchery fish were divided into 3 groups of 40 fish in Jnury. Ech group ws housed in seprte tnk nd fed to stition either the sme diet s tht supplied efore Jnury (control), feely iron-enriched diet (iron-1) or highly iron-enriched diet (iron-2), respectively. The iron-1 nd iron-2 diets contined 2.50 nd 7.50 g iron citrte per kg dry weight of control food, resulting in men totl iron levels in the control, iron-1 nd iron-2 diets of , nd mg/kg dry weight, respectively. Ech of the 3 groups nd smolting wild fish, which were cptured in the river of SFRI, were smpled from Jnury to My on the sme dys. In the 2004 experiment, forty thousnd 1+ SFRI-htchery fish were divided into 2 equl groups in Ferury. The control diet ws ssigned to one group of fish, while the other group ws fed the iron-1 diet. Ech of the two groups nd smolting wild fish were smpled s in the 2003 experiment. The smpled fish were used to clculte the condition fctor (CF) (Bgenl nd Tesch 1978), mesure urst swimming cpcity nd for the smpling of stomch remnnts, lood, liver nd white muscle. A decrese of the CF in spring is generlly ccepted s chnge relted to smoltifiction in slmonids (McCormick nd Sunders 1987). In my study, the CF decresed during smoltifiction in ll htchery fish in 2003 nd 2004, nd wild fish in 2003 (P < 0.05, Onewy ANOVA), wheres it did not decrese in wild fish in 2004 (P > 0.05; Student s t-test) (Fig. 11). An sence of decrese of the CF during smoltifiction sometimes occurs in wild msu slmon (Mym 1992; Mizuno et l. 2004). This phenomenon cn e relted to the presence of insects, crried y snowmelt wter in spring, in the stomch (Mym 1992). This study showed tht htchery smolt controls hd low CF vlues with regrd to the totl iron content of the food remnnts in the stomch (P < 0.05, One-wy ANOVA) (Fig. 12), urst swimming cpcity (P < 0.05, Onewy ANOVA) (Fig. 13), H concentrtions (P < 0.05, One-wy ANOVA) (Fig. 14) nd in the ATP content in white muscle nd liver, compred to wild fish, in My (P < 0.05, One-wy ANOVA) (Fig. 15). These findings revel some new prolems of seed qulities in the SFRI-htchery smolt: shortge of iron, low swimming ility, low oxygen-crrying cpcity nd low energy production. Woodwrd nd Smith (1985) concluded tht low swimming ility in htchery rinow trout ws cused y plcid ehvior nd rering environment drsticlly different from the nturl hitt. This explntion is lso plusile for the SFRIhtchery smolt in high density conditions. Low H concentrtions re generlly oserved during nemi due to dietry iron insufficiency in htchery rook trout (Slvelinus fontinlis) (Kwtsu 1972) nd yellowtil (Seriol quinquerdit) (Iked et l. 1973). This considertion nd the present results (Fig. 12) point towrds possile nemi due to n iron-insufficient diet. Sullivn et l. (1985) proposed tht H chnges electrophoreticlly (i.e., different gene products or different processing of H proteins) from immture forms to mture forms during smoltifiction in coho slmon. Therefore, it is possile tht the low H concentrtions

10 112 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Totl iron content (mg iron/100g food remnnts in stomch) cd Wild Control Iron-1 Iron-2 Group Fig. 12. Totl iron content of the food remnnts in the stomch of wild nd htchery-rered msu slmon in My The letters,, c nd d indicte significnt differences etween the wild, htchery control, iron-1 nd iron-2 groups t given smpling time, respectively (P < 0.05; Onewy ANOVA). Modified from Aquculture, 273, Mizuno et l., Effects of diets supplemented with iron citrte on some physiologicl prmeters nd on urst swimming velocity in smoltifying htchery-rered msu slmon (Oncorhynchus msou), , 2007, with permission from Elsevier. d c BSC (fork length/s) Wild Control Iron-1 Iron-2 d c My 2003 c Apr Smpling time c My 2004 Fig. 13. Burst swimming cpcity (BSC) of wild nd htchery-rered msu slmon in My 2003, April 2004 nd My The letters,, c nd d indicte significnt differences etween the wild, htchery control, iron-1 nd iron-2 groups t given smpling time, respectively (P < 0.05; One-wy ANOVA). Modified from Aquculture, 273, Mizuno et l., Effects of diets supplemented with iron citrte on some physiologicl prmeters nd on urst swimming velocity in smoltifying htchery-rered msu slmon (Oncorhynchus msou), , 2007, with permission from Elsevier. H (g/dl) Wild Control Iron-1 Iron-2 c d c Wild Control Iron-1 C c c Jn. Fe. Apr. My Smpling time 5 Jn. Fe. Apr. My Smpling time Fig. 14. Hemogloin concentrtion (H) in wild nd htchery-rered msu slmon t ech smpling time of the 2003 nd 2004 experiments. The letters,, c nd d indicte significnt differences etween the wild, htchery control, iron-1 nd iron-2 groups t given smpling time, respectively (P < 0.05; One-wy ANOVA). Modified from Aquculture, 273, Mizuno et l., Effects of diets supplemented with iron citrte on some physiologicl prmeters nd on urst swimming velocity in smoltifying htchery-rered msu slmon (Oncorhynchus msou), , 2007, with permission from Elsevier. on SFRI-htchery smolt re ttriuted to insufficient chnges in the H proteins during smoltifiction, ecuse of low dietry iron content. Norml dietry levels of mg iron/kg dry weight of food hve een suggested for slmonids (Ogino et l. 1979; Desjrdins et l. 1987; Andersen et l. 1996). Iron levels in the diet of Atlntic slmon rnged from 51 to 515 mg iron/kg dry weight of food. It is evident tht the iron level of the control diet ( mg iron/kg diet) ws high compred to the norml slmonid dietry iron content. In the present study, there were no differences in the survivl rte mong control, iron-1 nd iron-2 groups in the 2003 experiment or etween the control nd iron-1 groups in the 2004 experiment (dt not shown), which suggests no effect of the supplemented iron on survivl. This is in ccord with previous results in Atlntic slmon (Bjørnevik nd Mge 1993; Andersen et l. 1996, 1998) nd rinow trout (Crriquiriorde et l. 2004). However, my study did show tht iron-1 supplement ( mg iron/kg diet), ut not iron-2 supplement ( mg iron/kg diet), improved urst

11 S. Mizuno / Aqu-BioSci. Monogr. 5: , ATP ( mol/g tissue) White muscle Wild Control Iron-1 c c Liver Wild Control Iron-1 c c 0.00 Fe. Mr. Apr. My Smpling time 0.00 Jn. Fe. Apr. My Smpling time Fig. 15. ATP content in the white muscle nd liver in wild nd htchery-rered msu slmon t ech smpling time of the 2004 experiment. The letters, nd c show significnt differences etween the wild, htchery control nd iron-1 groups t given smpling time, respectively (P < 0.05; One-wy ANOVA). Modified from Aquculture, 273, Mizuno et l., Effects of diets supplemented with iron citrte on some physiologicl prmeters nd on urst swimming velocity in smoltifying htchery-rered msu slmon (Oncorhynchus msou), , 2007, with permission from Elsevier. swimming cpcity (Fig. 13) nd incresed H concentrtions (Fig. 14) (P < 0.05, One-wy ANOVA). Incresed H concentrtions in response to suitle iron supplement hs een demonstrted in mny fish (Kwtsu 1972; Iked et l. 1973; Skmoto nd Yone 1978; Andersen et l. 1997; Crriquiriorde et l. 2004), wheres n excess of supplemented iron hs induced lower H concentrtions in rinow trout (Stndl et l. 1997) nd Atlntic slmon (Andersen et l. 1997). Excessive iron hs een shown to led to the oxidtion of dietry lipids nd/or polyunsturted fts, dversely ffecting the qulity of the diet (Desjrdins et l. 1987). Bker et l. (1997) oserved iron-induced heptotoxicity following the ccumultion of lipid peroxidtion product when diets were supplemented with iron sulfte (6300 mg iron/kg diet). Diets supplemented with iron sulfte (25 to 100, 175 to 1975 mg iron/kg diet) re effective in rising H concentrtions to high levels in Atlntic slmon (Andersen et l. 1997) nd rinow trout (Crriquiriorde et l. 2004). Shiu nd Su (2003) hve reveled tht iron citrte ws only hlf s effective s iron sulfte in meeting the iron requirements of juvenile tilpi (Oreochromis niloticus). In contrst, Vielm et l. (1999) reported tht citric cid stimulted the sorption of dietry iron. The effects of ironsupplements thus seem to depend on the iron source used, emphsizing the need to nlyze the effects of numer of iron sources in order to develop diet with suitle iron content for fish. ATP contents in the liver nd the white muscle incresed (P < 0.05, One-wy ANOVA) (Fig. 15), when n iron-1 diet ws dministered. ATP is primrily produced y cellulr respirtion. Iron is n importnt element for the electron trnsfer chin, since it is essentil for the tertiry folding of cytochrome, protein plying leding role in cellulr respirtion. Furthermore, the ctivtion of cellulr respirtion requires considerle concentrtions of H + nd electrons, which re provided in the form of NADH2 + or FADH2 from the citric cid cycle, which, in turn, depends on the concentrtions of citric cid. Mmmlin reserch hs reveled tht iron supplementtion results in n incresed ATP production in the form of cellulr respirtion nd NADH2 + production y the citric cid cycle in vitro (Horst et l. 1999). If fish re ssumed to hve the sme cellulr energy metolic system s mmmls, supplementl iron citrte my trigger ctivtion of oth cellulr respirtion nd the citric cid cycle, nd increse the ATP content of the liver nd white muscle. This study reveled n improvement of urst swimming cpcity fter 3 months of iron-1 supplement (P < 0.05, One-wy ANOVA) (Fig. 13), nd positive correltion etween the urst swimming cpcity nd oth H concentrtions nd ATP content in the white muscle efore urst swimming (P < 0.05, Person s correltion coefficient) (Fig. 16). It is generlly considered tht urst swimming is exhiited y contrction of the white muscle. The contrctile ctivity primrily depends on ATP genertion from phosphocretine nd neroic glycolysis (Bone et l. 1978; Doson et l. 1987; Altringhm nd Ellery 1999). After urst swimming, the ATP concentrtion in the white muscle decreses (Wng et l. 1994). On the sis of the present results, it is suggested tht the urst swimming cpcity is dependent on the ATP content in the white muscle efore urst swimming in msu slmon. Incresed H concentrtion hs een oserved in vriety of species following urst swimming, nd is relted to the recovery from metolic nd respirtory cidosis during neroic glycolysis (Millign nd Wood 1987; Wng et l. 1994). Acidosis depends on

12 114 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 BSC (fork length/s) V = H H (mg/dl) BSC (fork length/s) V = ATP ATP ( mol/g white muscle) Fig. 16. Reltionship etween the mens of urst swimming cpcity (BSC) nd hemogloin concentrtion (H) nd etween the mens of BSC nd ATP content in the white muscle. For simple regression nlysis etween the mens of BSC nd H concentrtions, dt in My 2003, April 2004 nd My 2004 were plotted. The men BSC ws found to e significntly correlted with the men H concentrtion (r 2 = 0.538, P = 0.015, Person s correltion coefficient) nd the men ATP content (r 2 = 0.784, P = 0.019, Person s correltion coefficient). Modified from Aquculture, 273, Mizuno et l., Effects of diets supplemented with iron citrte on some physiologicl prmeters nd on urst swimming velocity in smoltifying htcheryrered msu slmon (Oncorhynchus msou), , 2007, with permission from Elsevier. swimming speed, t lest in yellowtil (Tsukmoto nd Chi 1981). Cytochrome c oxidse ctivity in the white muscle shows the strongest correltion with urst swimming cpcity, suggesting tht eroic preprtion of white muscle fcilittes rpid contrction fter urst swimming (Mrtínez et l. 2004). Thus, eroic metolism seems to e relted to the recovery from urst swimming ftigue, nd we suggest tht this, in turn, depends on pre-swimming H concentrtions. The improved urst swimming cpcity seen fter 3 months of iron-1 supplement therefore seems to e due to incresed H concentrtions nd ATP content in the white muscle efore urst swimming. In the lrge-scle 2004 experiment, there ws no significnt difference in urst swimming cpcity (P > 0.05, One-wy ANOVA) (Fig. 13) or in H concentrtions (P > 0.05, One-wy ANOVA) (Fig. 14) etween the iron-1 nd wild groups in April nd My. On the other hnd, n sence of difference in the white muscle ATP content ws found only in My (P > 0.05, Onewy ANOVA) (Fig. 15). These findings demonstrte tht it tkes t lest 3 months during smoltifiction to completely improve swimming ility of SFRIhtchery fish using the iron-1 diet. In consequence, this section concludes tht supplement of 2.5 g iron citrte per kg of food for 3 months prior to relese is convenient method for improving the seed qulity of smoltifying SFRI-htchery msu slmon. 3. Development nd ppliction of techniques to evlute seed qulity in 0+ htchery msu slmon prr for spring juvenile relese 3-1. Introduction The success of the spring juvenile relese progrm depends on the existence of sufficient food to sustin the growth of the juveniles in the strem. Growth nd survivl in strem juveniles re pprently densitydependent (Hume nd Prkinson 1987). Incresed density of stocked fry nd fingerlings cn increse the mortlity of wild juveniles y competition for food in strems (Lichtowich nd McIntyre 1988). Therefore, it is importnt to monitor the nutritionl condition of the stocked juveniles in order to evlute the dvntges of the spring juvenile relese progrm. In 0+ msu slmon, liver triglyceride (TG) contents, the storge lipid used s energy source in fish, re n pproprite index for the evlution of the nutritionl condition in wild nd htchery fish, since decresed TG levels were found y rtificil strvtion (Misk et l. 2004). However, TG levels in the liver re not enough to ssess nutritionl condition of the stocked juveniles. In the kidney nd spleen of fish, there re cells clled melno-mcrophges tht resemle mcrophges in their ultrstructure, contin high mounts of melnintype pigments, nd re thought to ct in the metolism of toxic compounds (Roerts 1975; Agius 1979). In Osteichthyes, greter numers of melnomcrophges re present thn in txonomiclly lower fish, including Agnth nd Chondrichthyes (Roerts 1975; Agius 1979; Wolke 1992). In slmonids, melnomcrophges re rndomly distriuted nd irregulrly ggregted throughout the kidneys (Agius 1980). It hs een suggested tht incresed numers of melnomcrophges were relted to humorl nd inflmmtory responses, storge, destruction nd detoxifiction of exogenous nd endogenous sustnces, nd iron recycling (Wolke 1992). Furthermore, it hs een reported tht strvtion induces incresed melnomcrophge density (MMD) in the kidney of some teleosts, including rinow trout (Agius nd Roerts

13 S. Mizuno / Aqu-BioSci. Monogr. 5: , Fig. 17. Histologicl oservtions of melno-mcrophge in the kidney of htchery-rered nd wild msu slmon. Pnels nd designte kidneys from htchery-rered nd wild fish respectively, t the strt of the experiment (dy 0). Pnels c nd d show kidneys from the strved groups of htchery-rered nd wild fish respectively, 45 dys fter the strt of the experiment. Pnels e nd f revel kidneys from the fed groups of htchery-rered nd wild fish, respectively, nd pnel g shows kidney from the htchery-rered fish smpled from the river. Pnels e, f nd g were shot 45 dys fter the strt of the experiment. The rrowheds indicte melno-mcrophges. Scle rs show 50.0 µm. Reprinted from Aquculture, 209, Mizuno et l., Effects of strvtion on melno-mcrophges in the kidney of msu slmon (Oncorhynchus msou), , 2002, with permission from Elsevier.

14 116 S. Mizuno / Aqu-BioSci. Monogr. 5: , MMD (%) Mortlity (%) Dys fter experiment strt Fig. 18. Chnges in the level of melno-mcrophge deposition (MMD) during the experiment in strved ( ) nd fed groups ( ) of htchery-rered msu slmon, in strved ( ) nd fed groups ( ) of wild msu slmon, nd in the htchery-rered fish smpled from the river ( ). Asterisks show significnt differences in the MMD level etween the strved group nd the fed group t the sme smpling time in ech fish group (P < 0.05, One-wy ANOVA). Cross mrks designte significnt differences in the MMD level from the htchery-rered group smpled from the river (P < 0.05, Onewy ANOVA). Modified from Aquculture, 209, Mizuno et l., Effects of strvtion on melno-mcrophges in the kidney of msu slmon (Oncorhynchus msou), , 2002, with permission from Elsevier. 1981). However, the mechnisms which induce the chnges re not well understood, nd the reltionship etween MMD nd mortlity is unknown in slmonids. In Susection 3-2, histologicl effects of rtificil strvtion on kidney MMD were oserved to determine whether the MMD would e useful indictor of the nutritionl condition of wild nd htchery juveniles. Susection 3-3 exmined the reltionships etween liver TG content nd kidney MMD levels, nd etween ech of these 2 prmeters nd fish density from spring to summer in htchery juveniles stocked into strem in spring, in order to clrify whether MMD levels re prcticl wy to monitor the nutritionl condition of the stocked juveniles Development of techniques to evlute nutritionl condition using kidney MMD levels in 0+ juveniles 0+ htchery juveniles were relesed into river where wild slmonids re not found in My. After 1 month, prt of the relesed htchery juveniles were recptured Dys fter experiment strt Fig. 19. Chnges in mortlity during the experiment in strved ( ) nd fed groups ( ) of htchery-rered msu slmon nd in strved ( ) nd fed groups ( ) of wild msu slmon. Modified from Aquculture, 209, Mizuno et l., Effects of strvtion on melno-mcrophges in the kidney of msu slmon (Oncorhynchus msou), , 2002, with permission from Elsevier. from the river. At the sme time, 0+ wild juveniles were cught from different river. The htchery nd wild juveniles were plced in seprte tnks, nd mintined without food under the sme conditions. Htchery nd wild feeding-control groups were mintined in the sme conditions over the two months of the experiment. Mortlity rtes were exmined in ech group, nd fish were rndomly smpled from ech group during the experiment. The htchery juveniles relesed in the river efore the experiment were smpled s the experimentl control. Kidneys were dissected from ll smples. Prffin sections of the kidney were oserved with light microscope. The MMD levels were expressed s the men percentge of melnin grnule re to whole kidney re. All msu slmon hd only melno-mcrophges with drk rown pigments (Fig. 17). Oguri (1976, 1985) nd Agius (1980) hve reported oth yellow lipofuscin pigments nd drk rown melnin pigments in rinow trout, nd indicted tht pigment composition in the kidney is sometimes vrile etween individuls of single fish species. My results revel tht melnomcrophge of 0+ htchery nd wild fish hve high levels of melnin. In ddition, this study indicted tht strvtion incresed MMD in oth wild nd htchery fish (P < 0.05, One-wy ANOVA) (Fig. 18). Roerts (1978) nd Agius nd Roerts (1981) suggested tht melno-mcrophge centre enlrgement during strvtion ws ssocited with dmge to tissues, including

15 S. Mizuno / Aqu-BioSci. Monogr. 5: , Kmi-Utetsu River The Se of Jpn Hokkido The Se of Okhotsk Upstrem dm Sttion 1 (Relese point) Sttion km Pcific Ocen Sttion 3 Sttion 4 Utetsu River Sttion 5 Pcific Ocen 0 5 km Sttion 6 Downstrem dm m Fig. 20. Mp showing the Utetsu River in southern Hokkido nd smpling sttions on the Kmi-Utetsu River. Smll rectngulr re shows smpling re of the Kmi-Utetsu river. Modified from Sci. Rep. Hokkido Slmon Freshwter Fish. Res. Inst., 1, Mizuno et l., Assessment of nutritionl conditions using kidney melno-mcrophge density in htcheryrered juvenile msu slmon Oncorhynchus msou relesed into strem, 49 53, 2011, Hokkido Slmon nd Freshwter Fisheries Reserch Institute. kidney nd spleen, in some fish. Therefore, it is suggested tht the incresed MMD levels in msu slmon were lso cused y the ccelertion of kidney ctolism during strvtion. In mmmls, incresed deposition of pigments hs een oserved in vrious orgns during cchexi (Duin 1955), nd ppers to involve the peroxidtion of polyunsturted lipids of sucellulr memrnes (Chio et l. 1969). However, it is uncler whether pigment formtion during strvtion in msu slmon is regulted y this sme mechnism. It is well-known tht vrious environmentl fctors ffect MMD levels (Blzer et l. 1987). Wolke (1992) hs reveled tht using MMD levels to monitor hyponutrition remins questionle ecuse the ctul resons for incresed MMD levels re uncertin. On the other hnd, Mizuno et l. (2011) found significnt correltions etween kidney MMD nd liver TG content. Therefore, it is considered tht MMD reflects the nutritionl condition of 0+ msu slmon. There were no significnt differences in the MMD levels etween the fed htchery group nd the group smpled from the river with respect to MMD levels on dys 15 nd 45 of the experiment (P > 0.05, One-wy ANOVA) (Fig. 18). No significnt differences in MMD levels (P > 0.05, One-wy ANOVA) were found etween htchery nd wild fish for either the fed or strved groups in the present study (Fig. 18). In consequence, these results demonstrte tht there re no effects of environmentl fctors or of the fish origin on MMD levels from spring to summer. It is importnt to note tht mortlity rte increses progressively in the strved groups of oth htchery nd wild fish, while fish in oth fed groups nd the fish smpled from the river mintin low levels of mortlity throughout the experiment (Fig. 19). Incresed mortlity ws oserved etween 30 nd 45 dys, when the men MMD level reched 0.5% in the strved groups of oth htchery nd wild fish. Therefore, these results demonstrte tht level of MMD of 0.5% could e potentilly used s n index of ultimte hyponutrition in juvenile msu slmon Evlution of nutritionl conditions using kidney MMD levels in 0+ htchery juveniles fter relese There is 550 m section etween two dms on the Kmi-Utetsu River in southern Hokkido (Fig. 20), which wild msu slmon do not inhit. Six smpling sttions were estlished in this section to exmine fish density nd the nutritionl condition of stocked juveniles (Fig. 20). The surfce re nd mximum depth of sttions during the experiment rnged from 3.49 to 100 m 2 nd from 0.45 to 1.27 m, respectively. Previous to this study, no 0+ juveniles could e found in this section. On My 1, ten thousnd 0+ htchery ju-

16 118 S. Mizuno / Aqu-BioSci. Monogr. 5: , My July My July TG (%) MMD (%) Fish density (fish/m 2 ) Fish density (fish/m 2 ) Fig. 21. Reltionships etween fish density nd liver triglyceride (TG) levels, nd etween fish density nd kidney melnomcrophge density (MMD), in juvenile msu slmon. There were no fish cught t sttion 5 in July. Modified from Sci. Rep. Hokkido Slmon Freshwter Fish. Res. Inst., 1, Mizuno et l., Assessment of nutritionl conditions using kidney melnomcrophge density in htchery-rered juvenile msu slmon Oncorhynchus msou relesed into strem, 49 53, 2011, Hokkido Slmon nd Freshwter Fisheries Reserch Institute. veniles were relesed t sttion 1 (Fig. 20). On My 30 nd June 30, fish were cught t ech sttion. Fish numers were estimted y the doule-pss removl method (Seer nd LeCren 1967). Smpled fish were used for the nlysis of kidney MMD levels nd liver TG content. 0+ juveniles were recptured t ll sttions in ll smpling times except for sttion 5 on July 30. Estimted fish density rnged from 0.31 to 2.18 fish/m 2 in My nd from to 1.96 fish/m 2 in July (Fig. 21). Growth increments in wild nd relesed 0+ juveniles re reported to decrese s popultion density increses (Ngt 1989). Additionlly, Hume nd Prkinson (1987) reported tht densities >0.7 fish/m 2 in strem of British Columi, Cnd, resulted in incresed mortlity in rinow trout, which my indicte tht there re some juveniles in hypo-nutritionl conditions in the present study. Men kidney MMD ws etween nd 0.524% in My nd etween nd 0.503% in July (Fig. 21). Men liver TG levels were etween nd 1.02% in My nd etween nd 0.787% in July (Fig. 21). A significntly negtive correltion ws found etween men liver TG levels nd men kidney MMD levels (r = 0.601, P < 0.05, Spermn s rnk correltion) (Fig. 22). These results demonstrte tht men kidney MMD is negtive indictor of the nutritionl conditions, from spring to summer of htchery juveniles relesed into strem. In juvenile msu slmon, the point t which ded fish re oserved in the juvenile popultion in n rtificil rering environment, is t men kidney MMD > 0.5% (Mizuno et l. 2002). In the present study, some juvenile popultions re on the verge of deth in My nd July ccording to tht MMD limit. Therefore, there is possile deth of juveniles in the popultion in nturl hitt. The correltion etween fish density nd MMD (%) TG (%) Fig. 22. Reltionship etween men triglyceride (TG) levels in the liver nd men melno-mcrophge density (MMD) in the kidney in juvenile msu slmon. The plots in this figure express the mens of TG nd MMD in juvenile popultion t ech sttion nd time. Spermn s rnk correltion coefficient ws used s sttisticl nlysis. The dotted line shows the liner correltion [MMD] = [TG] (r = 0.601, P < 0.05). There were no juveniles cught t sttion 5 in July. Modified from Sci. Rep. Hokkido Slmon Freshwter Fish. Res. Inst., 1, Mizuno et l., Assessment of nutritionl conditions using kidney melno-mcrophge density in htchery-rered juvenile msu slmon Oncorhynchus msou relesed into strem, 49 53, 2011, Hokkido Slmon nd Freshwter Fisheries Reserch Institute. MMD ws significnt in My nd July (My: r = 0.504, P < 0.05; July: r = 0.592, P < 0.05; Spermn s rnk correltion) (Fig. 21). The correltion etween fish density nd TG levels were significnt oth in My nd July (My: r = 0.567, P < 0.05; July: r = 0.339, P < 0.05; Spermn s rnk correltion) (Fig. 21). These results strongly suggest tht the nutritionl condition

17 S. Mizuno / Aqu-BioSci. Monogr. 5: , Fig. 23. Chnges in ody weight (A) nd condition fctor (B) of the three density groups during the experiment. Closed squre ( ), closed tringle ( ) nd open circle ( ) showed 40, 20 nd 10 kg/m 3 groups, respectively. The mrks with the different lpheticl letters t the sme smpling time were sttisticlly different (P < 0.05; One-wy ANOVA). Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. of relesed juvenile msu slmon depends on fish density from spring to summer. Mesurements of MMD possily contriute not only to evluting the nutritionl condition of juveniles ut lso to finding the pproprite mount of food supply to juveniles in htcheries. 4. Estlishment of techniques to monitor the physicl condition nd elucidtion of pproprite culture conditions in chum slmon fry 4-1. Introduction In chum slmon fry, rering t high densities cuses poor feeding nd growth (Nogw nd Ygisw 1994) nd decreses sewter dptility (Bn 2000). A hevy mortlity of fry, which is cused y cteril gill disese, hs een nnully reported in some htcheries in Hokkido (Nomur 1994). The cteril gill disese is due to n infection y Flvocterium rnchiophilum, which is oserved in ll wter of fry Fig. 24. Chnges in rering density (A) nd dissolved oxygen (DO) concentrtion (B) of the three density groups during the experiment. Closed squre ( ), closed tringle ( ) nd open circle ( ) showed 40, 20 nd 10 kg/m 3 groups, respectively. Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. cultures, fter environmentl deteriortion due to high density culture (Borg 1960; Bullock 1972; Lrmoyeux nd Piper 1973). It is importnt to monitor the ggrvtion of the physicl condition in the fry cused y high density culture efore the infection of F. rnchiophilum, in order to prevent the incidence of the cteril gill disese. However, method to monitor physicl conditions hs not yet een estlished for chum slmon seed. It is commonly ccepted tht the Jpnese stndrds of rering conditions for chum slmon fry re <20 kg/m 3 for rering density nd >6 mg/l dissolved oxygen (DO) concentrtion (Nogw nd Ygisw 1994), which ws determined ccording to conversion formul of optimum rering density for slmonid dvocted y Westers nd Prtt (1977). However, this stndrd is not sed on cler physiologicl effects. Therefore, the reltionship etween prcticl rering conditions nd the physicl condition of the fry hs not een shown. In Susection 4-2, the impcts of high density culture on vriety of physiologicl prmeters were studied, in order to estlish method to monitor the physicl condition of htchery chum slmon fry. In ddi-

18 120 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Fig. 25. Chnges in the survivl rte in the three density groups during the fsting-tolernce test. Closed squre ( ), closed tringle ( ) nd open circle ( ) showed 40, 20 nd 10 kg/m 3 groups, respectively. Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. tion, physicl condition ws monitored in the fry produced y some htcheries. Susection 4-3 exmined the reltionships etween rering density, DO nd physicl condition of chum slmon fry in two htcheries, in order to determine pproprite rering conditions for chum slmon fry Development of techniques to monitor the physicl condition of htchery fry Chum slmon fry were introduced into 3 tnks t densities of 10, 20 nd 40 kg/m 3. The three groups were rered for 42 dys with food. Fry were smpled from the originl tnk t the initil time nd from ech of the 3 tnks on every seventh dy. In the smples, CF ws clculted fter fork length nd ody weight (BW) were mesured. The plsm nd fish ody were used for physiologicl nlyses. At ech smpling time, fish density, DO nd un-ionized mmoni concentrtion (UIA) were mesured in ech tnk. After the finl smpling, tolernce to strvtion ws exmined in ech of the 3 groups. A reduced BW ws oserved in only the 40 kg/m 3 group during the experiment (P < 0.05, One-wy ANOVA) (Fig. 23A). This phenomenon is supported y the mjority of ppers, which hve demonstrted tht there is n dverse effect of incresing density on growth in slmonids (Ellis et l. 2002), possily resulting from reduction in food intke (Letherlnd 1993; Alnärä nd Brännäs 1996) nd food conversion efficiency (Logn nd Johnston 1992) owing to the deteriortion of rering conditions. It hs een generlly ccepted tht rering environments leding to reduced growth in slmonids hd rering densities >50 kg/m 3 (Mäkinen nd Ruohonen 1990), DO levels <5 mg/l (Brett 1979) nd UIA concentrtions >40 µg/l (Mede 1985). Moreover, the DO of the 40 kg/m 3 group ws kept t >5 mg/l during the experiment (Fig. 24B). The pek of UIA in the 40 kg/m 3 group ws of only µg/l, lthough the UIA incresed in the 40 kg/ m 3 group during the experiment. In consequence, it is suggested tht the primry environmentl cuse of the reduced growth in this study is rering density, since only rering density pplied to the forementioned conditions in the 40 kg/m 3 group (Fig. 24A). The results of the CF, crude mesure reflecting levels of energy reserves (Goede nd Brton 1990), demonstrted tht 40 kg/m 3 culture conditions dversely ffected the CF in chum slmon (P < 0.05, One-wy ANOVA) (Fig. 23B). This my reflect the results of the fsting-tolernce test, where the 40 kg/m 3 group showed significntly low tolernce to strvtion compred to the 10 nd 20 kg/m 3 groups (P < 0.05, Kpln- Meier method followed y Log-rnk test) (Fig. 25). Mny previous studies hve reported n dverse effect of density on the CF in slmon species, with the exception of chum slmon (Pickering nd Pottinger 1987; Mäkinen nd Ruohonen 1990). However, our study mde it cler tht high density cultures cused poor energetic reserves in chum slmon, much s in other slmon. It hs een well documented tht plsm cholesterol nd glucose concentrtions mirror the nutritionl condition of fish (Kiron nd Mit 2003). In the present study, the 40 nd 20 kg/m 3 group showed sig-

19 S. Mizuno / Aqu-BioSci. Monogr. 5: , Fig. 26. Chnges in plsm cholesterol concentrtions in the three density groups during the experiment. Closed squre ( ), closed tringle ( ) nd open circle ( ) showed 40, 20 nd 10 kg/m 3 groups, respectively. The mrks with different lpheticl letters t the sme smpling time were sttisticlly different (P < 0.05; One-wy ANOVA). Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. Fig. 27. Chnges in plsm glucose concentrtions in the three density groups during the experiment. Closed squre ( ), closed tringle ( ) nd open circle ( ) showed 40, 20 nd 10 kg/m 3 groups, respectively. The mrks with different lpheticl letter t the sme smpling time were sttisticlly different (P < 0.05; One-wy ANOVA). Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. nificntly lower plsm cholesterol (P < 0.05, One-wy ANOVA) (Fig. 26) nd glucose concentrtions (P < 0.05, One-wy ANOVA) (Fig. 27) fter 21 dys compred to the 10 kg/m 3 group. Letherlnd nd Cho (1985) reported tht high density inhiited the increse in plsm glucose levels fter feeding in rinow trout, which resulted from reduced ility to find food. Mit et l. (1998) found tht there ws significnt negtive correltion etween plsm cholesterol concentrtions nd mortlity due to disese, in rinow trout. These results imply tht high density cuses poor nutritionl nd physicl conditions in chum slmon fry. In the 40 kg/m 3 group, incresed plsm cortisol levels, prt of the neuro-endocrine stress response, were

20 122 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Fig. 28. Chnges in plsm cortisol concentrtions in the three density groups during the experiment. Closed squre ( ), closed tringle ( ) nd open circle ( ) showed 40, 20 nd 10 kg/m 3 groups, respectively. The mrks with the different lpheticl letter t the sme smpling time were sttisticlly different (P < 0.05; One-wy ANOVA). Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. Fig. 29. Chnges in plsm lysozyme ctivity in the three density groups during the experiment. Closed squre ( ), closed tringle ( ) nd open circle ( ) showed 40, 20 nd 10 kg/m 3 groups, respectively. The mrks with the different lpheticl letter t the sme smpling time were sttisticlly different (P < 0.05; One-wy ANOVA). Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. oserved (P < 0.05, One-wy ANOVA) (Fig. 28), which demonstrted tht high density cuses the initil stress in chum slmon. Pickering nd Pottinger (1987) reported tht plsm cortisol levels were elevted in the first 6 nd 10 dys fter exposure to high rering density in rown trout (S. trutt) nd rinow trout, respectively. This informtion my reflect the fct the effects of density on the initil increse in plsm cortisol levels re species dependent. Corticosteroids, including cortisol, re potent immunosuppressnts (Brton et l. 1987; Wedemeyer 1996). North et l. (2006) found negtive correltions etween plsm cortisol levels nd lysozyme ctivity, non-specific immune trit with cteriolytic effects, in rinow trout. However, the 40 kg/m 3 group did not show high cortisol levels t 42 dys (P > 0.05, One-wy ANOVA)

21 S. Mizuno / Aqu-BioSci. Monogr. 5: , Fig. 30. Chnges in somtic denosine triphosphte (ATP) content in the three density groups during the experiment. Closed squre ( ), closed tringle ( ) nd open circle ( ) showed 40, 20 nd 10 kg/m 3 groups, respectively. The mrks with the different lpheticl letters t the sme smpling time were sttisticlly different (P < 0.05; One-wy ANOVA). Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. (Fig. 28), when plsm lysozyme ctivity significntly decresed (P < 0.05, One-wy ANOVA) (Fig. 29). These results demonstrte tht prolonged stress due to high densities cuses decline in the immune system response, with no incresed cortisol levels in chum slmon. Røed et l. (1993) nd Blfry et l. (1997) reveled tht there were species-specific vritions in the stress-relted lysozyme ctivity in slmonid fish. Therefore, the differences in the results etween North et l. (2006) nd this study my result from the different slmonid species considered. In the present study, the 40 kg/m 3 group showed tht oth the fstest increse nd the slowest decrese in the somtic ATP content during the experiment, while incresed lter in the 20 kg/m 3 group (P < 0.05, Onewy ANOVA) (Fig. 30). Incresed ATP content hs een oserved in msu slmon trnsforming from prr into smolt (Mizuno et l. 2007) nd nutritionlly supplemented chum slmon fry (Mizuno et l. 2008), which suggests tht ATP content increses to stisfy the need for incresed energy demnd during the ctivtion of metolisms. However, my study found no evidence tht incresed ATP contents cooccurred with chnges in plsm totl cholesterol nd glucose concentrtions in the 40 nd 20 kg/m 3 groups (Figs. 27, 28, 30). On the other hnd, n ATP-dependent system to excrete exogen sustnces hs een found in teleost (Yngi et l. 2004; Hirose nd Nkd 2010). Accordingly, the incresed ATP content found in the present study my e linked with incresed energy demnds resulting from n incresed excretion of exogen sustnces s result of poor physicl condition. Decresed ATP content hs een shown to stop moility in the sperm of luegill Lepomis mcrochirus (Burness et l. 2005). Therefore, the reduced ATP content found in the 40 kg/m 3 group my demonstrte decresed vitlity efore deth. Our study demonstrted tht the 40 nd 20 kg/m 3 density groups hd decresed AST trnscription levels (P < 0.05, One-wy ANOVA) (Fig. 31), one of the respirtory chin enzymes used to produce ATP eroiclly. I suspect the respirtory-chin enzyme ws negtively regulted y the ATP content, which is widely ccepted in teleost (Burness et l. 2005). However, less ATP content nd low AST trnscription levels were found t the sme time in the 40 kg/m 3 group from 35 to 42 dys (P < 0.05, One-wy ANOVA) (Figs. 30, 31). This discrepncy my reflect tht high densities distur the lnced regultion etween ATP content nd trnscription levels of respirtory chin enzymes. Figure 32 shows schemtic representtion of physiologicl chnges during n cute ggrvtion of physicl condition, which resulted from excessive rering densities (>40 kg/m 3 ). Vlues of physicl condition indictors should e stle in physiclly good conditioned fish. The four prmeters designted in this figure were not influenced y the durtion of the experiment in the 10 kg/m 3 group, which ppered to mintin the est physicl condition during the experiment. It my e possile to estimte the physicl condition of chum slmon fry using the stle prmeter vlues of the 10 kg/m 3 group s seline, ecuse there ws no effect of these prmeters on fry growth during my experiment in the 10 kg/m 3 group. The prmeter se

22 124 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Fig. 31. Chnges in somtic ATP synthse trnscription levels in the three density groups during the experiment. Closed squre ( ), closed tringle ( ) nd open circle ( ) showed 40, 20 nd 10 kg/m 3 groups, respectively. The mrks with the different lpheticl letters t the sme smpling time were sttisticlly different (P < 0.05; One-wy ANOVA). Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. lines, which show the rnge of the men vlue of the prmeter during our experiment in the 10 kg/m 3 group, re 11.4 to 19.4 ng/ml in plsm cortisol concentrtion, 8.16 to 21.0 pmol/g BW in somtic ATP content, 4.34 to 5.04 pmol competitor/µg totl RNA in somtic AST trnscription levels nd 2.09 to 3.00 µg lysozyme/ l in plsm lysozyme ctivity. The first sign of d physicl condition ws n increse in plsm cortisol concentrtion, followed y drmticlly incresed ATP content nd decresed AST trnscription levels. Therefter, the ATP content decresed, nd finlly lysozyme ctivity decresed. The 20 kg/m 3 group, which ws predicted to show slower ggrvtion of physicl conditions due to high density, showed n incresed ATP content fter 35 dys nd decresed AST trnscription level fter 28 dys. In contrst, incresed cortisol concentrtions nd decresed lysozyme ctivity were not oserved in the 20 kg/m 3 group, which demonstrted tht cortisol concentrtions nd lysozyme ctivity were not relile prmeters for estimting the physicl condition of chum slmon fry. Consequently, this suggests tht somtic ATP content nd AST trnscription levels re key prmeters for monitoring the initil ggrvtion of the physicl condition of chum slmon fry resulting from rering t high densities, efore the ppernce of decresed growth nd immune functions. The stndrd vlues of the ATP content nd the AST trnscription levels re regrded s 8.16 to 21.0 pmol/g BW nd >4.34 pmol competitor/µg totl RNA, respectively. In order to routinely monitor the physicl condition of htchery fry for rtificil propgtion, htchery fry Prmeter level Good Phse of physicl condition Lysozyme ctivity Cortisol concentrtion ATP content AST trnscription level Fig. 32. Schemtic representtion of cute chnges in physicl condition during rering t excessive density (>40 kg/ m 3 ). The four prmeters designted in this figure were not influenced y the durtion of the experiment in the 10 kg/ m 3 group, which ppered to mintin the est physicl condition during the experiment. The first sign of n ggrvted physicl condition ws n increse in plsm cortisol concentrtion nd n increse in somtic ATP content nd decrese in ATP synthse (AST) trnscription levels. Therefter, the ATP content nd lysozyme ctivity decresed. Modified from Aquculture Science, 58, Mizuno et l., Physiologicl impcts of high rering density on chum slmon Oncorhynchus ket fry, , 2010, Jpnese Society for Aquculture Reserch. were collected from 9 htcheries (htcheries I to IX) in different regions of Hokkido, nd wild fry were cptured in southestern Hokkido in spring. Figure 33 shows the ATP content nd AST trnscription levels in the htchery nd wild fry. Men ATP content nd AST trnscription levels rnged from 3.61 to 20.3 Bd

23 S. Mizuno / Aqu-BioSci. Monogr. 5: , Fig. 33. The somtic denosine triphosphte (ATP) contents (left side) nd ATP synthse trnscription (AST) levels (right side) of htchery-rered nd wild chum slmon fry. The numers I to IX represent the different htcheries the chum slmon fry were collected t. Yellow res indicte the rnge considered stndrd helthy vlues for ech prmeter. Fig. 34. Reltionships etween rering density, dissolved oxygen concentrtion (DO) nd somtic ATP content (A), nd etween rering density, DO nd somtic ATP synthse trnscription levels (B). X- nd Y-xes show rering density nd DO respectively. Contours represent somtic ATP content in the A grph nd crcss ATP synthse trnscription levels in the B grph. Modified from Aquculture Science, 58, Mizuno et l., Reltionship etween rering conditions nd helth in chum slmon (Oncorhynchus ket) fry, , 2010, Jpnese Society for Aquculture Reserch. pmol/g BW nd from 3.05 to 12.6 pmol competitor/µg totl RNA, respectively. According to the stndrd vlues of oth ATP content nd AST trnscription levels, fry from htcheries I nd II were regrded s eing in d physicl condition, wheres fry from htcheries III to IX nd wild fry were considered in good physicl condition, which shows tht ATP content nd AST trnscription levels led to the sme results. Therefore, these results strongly demonstrte tht ATP content nd AST trnscription levels re relile prmeters for estimting the physicl condition of chum slmon fry Approprite culture conditions for htchery chum slmon fry Some ponds, contining introduced chum slmon fry were chosen in α nd β htcheries in Hokkido, in April. Wter temperture, ph, DO, UIA nd fry density were exmined in ech pond. Fry were rndomly smpled from ech pond for the mesurement of BW nd the nlyses of crcss ATP content nd AST trnscription levels. In this study, the wter temperture nd ph of the ponds rnged etween 5.2 nd 9.0 C nd etween 6.50

24 126 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Tle 1. Pond conditions nd wter qulity, nd condition of the chum slmon fry. Modified from Aquculture Science, 58, Mizuno et l., Reltionship etween rering conditions nd helth in chum slmon (Oncorhynchus ket) fry, , 2010, Jpnese Society for Aquculture Reserch. Smpling time Htchery Pond nme Pond condition Wter qulity Fry condition Volume (m 3 ) Fry numer Rering density (kg/m 3 ) Wter temperture ( C) ph Dissolved oxygen concentrtion (mg/l) Unionized mmoni concentrtion (µg/l) Body weight (g) Somtic ATP content (pmol/g wet ody weight) Somtic ATP synthse trnscription level (pmol/µg totl RNA) Apr. 7, 2008 α A < ± ± ± Apr. 7, 2008 α B < ± ± ± Apr. 7, 2008 α C < ± ± ± Apr. 14, 2008 α B < ± ± ± Apr. 14, 2008 α C < ± ± ± Apr. 21, 2008 α B < ± ± ± Apr. 21, 2008 α C < ± ± ± Apr. 21, 2008 α D < ± ± ± Apr. 21, 2008 α E < ± ± ± Apr. 21, 2008 α F < ± ± ± Apr. 26, 2010 β G < ± ± ± Apr. 26, 2010 β H < ± ± ± Apr. 26, 2010 β I < ± ± ± Apr. 26, 2010 β J < ± ± ± Apr. 26, 2010 β K < ± ± ± Apr. 26, 2010 β L < ± ± ± Apr. 26, 2010 β M < ± ± ± Body weight (n = 15), somtic ATP content (n = 5) nd somtic ATP synthse trnscription level (n = 5) shown s mens ± stndrd errors.

25 S. Mizuno / Aqu-BioSci. Monogr. 5: , (A) (B) Fig. 35. Osmerid egg (A) nd jr incutors for culturing the eggs (B). Pnel A shows Jpnese smelt egg cultured t 10 C for one dy. Arrowhed shows the inverted dhesive memrne, prt of egg memrne. Scle r shows 1.00 mm. Pnel B shows 6l-jr incutors in Mukw htchery, southern Hokkido. nd 6.83, respectively (Tle 1). The UIA concentrtion ws <0.1 µg/l in ll ponds nd the men BW of the fry rnged from 0.81 to 2.36 g. Prior to this study, I hd suggested tht there ws no effect of differences in wter temperture etween 5 nd 10 C nd in fry BW etween 0.7 nd 3.0 g on the two physicl condition prmeters nlyzed in this study (Mizuno et l. 2008, 2010). In chum slmon fry, negtive physiologicl effects (unlnced ion regultion) due to cid wter hve first een found t ph 5.0 (Wtne et l. 1995). The lowest UIA concentrtion to show negtive physiologicl impcts ws 4 µg/l in slmonids (Mede 1985). Therefore, this informtion suggests tht there ws no effect of the different wter tempertures, ph, UIA nd fry ody size on the two prmeters of physicl condition in the present study. The mens of crcss ATP content nd AST trnscription levels rnged etween 8.00 nd pmol/g BW nd etween 3.52 nd 14.2 pmol competitor/µg totl RNA, respectively (Tle 1). According to the stndrd vlues of ATP content (8.16 to 21.0 pmol/g BW) nd AST trnscription levels (>4.34 pmol competitor/µg totl RNA), α htchery fry were regrded s eing in d physicl condition in Ponds A, B nd C on April 7, in Ponds C, D, E nd F on April 21, nd s eing in good physicl condition in Pond B on April 14 nd 21. The fry in Pond C on April 14 were considered in impired physicl condition, since the AST trnscription level ws out of the rnge of physiclly good condition, while the ATP content level ws within this rnge. This finding suggests tht declined AST trnscription levels precede incresed ATP content during the ggrvtion of physicl conditions in chum slmon fry. β htchery-fry were considered in good physicl condition in ll ponds. Figure 34 shows the reltionships etween ech of the two prmeters of physicl condition, rering density nd DO. The re regrded s physiclly good condition for crcss ATP content (Fig. 34A) nd AST trnscription levels (Fig. 34B), ws minly found t rering densities <30 kg/m 3 nd t DO levels >8 mg/l. Accordingly, these results demonstrte tht pproprite rering conditions for the culture of htchery chum slmon fry in good physicl condition were with rering densities <30 kg/m 3 nd with DO levels >8 mg/l. 5. Development of techniques to improve the survivl rte during the rtificil propgtion of osmerids 5-1. Introduction Nturlly spwned eggs of oth shishmo nd Jpnese smelt stick to the surfce of fine grvel nd/or qutic plnts in the ottom of the river with dhesive proteins on the surfce of the inverted dhesive memrne, prt of the egg memrne (Fig. 35A). In intensive egg culture using jr incutors (Fig. 35B), the egg dhesiveness is eliminted with tnnic cid solution tretment just fter rtificil fertiliztion (Wltemyer 1976) to prevent clumping of dhesive eggs, which cuses high mortlity ccompnied y suffoction nd fungl growth (Doroshof et l. 1983). Adhesive proteins on the inverted dhesive memrne lose dhesiveness y tretment with tnnic cid, which hs functions in cogulting proteins. However, tnnic cid-treted eggs show lower htching rtes. The low htching rte is more conspicuous in the tnnic cid-treted eggs cultured under iron-enriched environments (Tked et l. 2002). Therefore, it is necessry to estlish new methods to eliminte egg dhe-

26 128 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Fig. 36. Oservtion of the eggs fter non-tretment (A nd D), tnnic cid tretment (B nd E) nd kolin tretment (C nd F). The upper nd lower hlves show eggs on Novemer 21, 2003 nd Mrch 23, 2004, respectively. The rrowheds indicte orderline etween the egg memrne nd the inverted dhesive memrne. Scle r indictes 1.00 mm. Reprinted from Aquculture, 242, Mizuno et l., Elimintion of dhesiveness in the eggs of shishmo smelt Spirinchus lnceoltus using kolin tretment to chieve high htching rte in n environment with high iron concentrtion, , 2004, with permission from Elsevier. siveness in order to improve the htching rte in osmerids. Previous studies hve reported methods to eliminte egg dhesiveness with mud in Jpnese dce (Triorodon hkonensis) (Nkmur 1962) nd with river silt in white sturgeon (Acipenser trnsmontnus) (Doroshof et l. 1983). Egg dhesiveness is lost when the dhesive portion of the egg is completely covered with mud or silt just fter rtificil fertiliztion. On the other hnd, mud nd river silt re not edile nd the pulic sentiment on food sfety is rising yer fter yer in Jpn. It is therefore necessry to consider the doption of food or food dditives for the development of new methods to eliminte egg dhesiveness. Kolin, which is composed of Al3Si2O5(OH)4, is nturl minerl cly nd certified food dditive for cosmetics nd medicines. This powder is potentil mteril for the elimintion of egg dhesiveness, s it hs very low soluility. In the mentime, the success of rtificil propgtion of the Yezo gint scllop, Ptinopecten yessoensis, hs led to the present-dy drmtic increse of its ctch in Hokkido. Incresing numers of scllop shells, which re dischrged during scllop processing, re serious prolems of industril wste (Kono et l. 2000). Some of the shells re crushed into powder, which is used s food dditive for clcium supplements (Ymgishi et l. 2007; Liu et l. 2008). The scllop shell powder (SSP) is s insolule in wter s kolin is. Therefore, it is lso possile to utilize SSP s mteril to eliminte egg dhesiveness. Some shishmo htcheries hve to trnsport nd relese eyed-stge emryos into rivers, ecuse the wter outlets of htcheries do not connect with rivers llowing the relese of seeds. Nturlly spwned shishmo smelt eggs, ttched to smll grvel re forced to flow from the river to the se y snow-melt wter (Omi 1978). It is prole tht eyed-stge emryos re exposed to sewter s soon s they re relesed. Accordingly, it is essentil tht only emryos with high sewter dptility re relesed for the success of rtificil propgtion of shishmo smelt. It is well-known tht there re mny mitochondrion-rich cells, which ply min role in sewter dpttion fter htching, on the yolk sc memrne (Hyno et l. 1999). However, it is not cler which emryo stge hs the est sewter tolernce. In consequence, pproprite time for relesing eyed-stge emryos is unknown for the Hokkido shishmo htcheries. Susection 5-2 exmined whether kolin suspension tretment elimintes the egg dhesiveness nd results in high htching rte under n iron-enriched environment in shishmo smelt. In Susection 5-3, the effects of tretment using SSP suspension on eliminting egg dhesiveness nd htching rte in Jpnese

27 S. Mizuno / Aqu-BioSci. Monogr. 5: , Egg dhesiveness elimintion rte (%) 0.5 g/l 1 g/l 2 g/l 5 g/l 10 g/l Kolin tretment Fig. 37. Effects of the kolin suspension tretment on elimintion of egg dhesiveness in the first experiment. The letters nd indicte significnt differences in the vlue compred to the non-tretment nd the tnnic cid tretment, respectively (P < 0.05; Chi-squre test for independence). Reprinted from Aquculture, 242, Mizuno et l., Elimintion of dhesiveness in the eggs of shishmo smelt Spirinchus lnceoltus using kolin tretment to chieve high htching rte in n environment with high iron concentrtion, , 2004, with permission from Elsevier. smelt eggs were studied in order to demonstrte whether SSP is useful mteril for eliminting egg dhesiveness. Finlly, Susection 5-4 investigted the development of sewter dptility during emryogenesis to elucidte pproprite timing for the relese of eyed-stge emryos in shishmo smelt Technique for eliminting egg dhesiveness using kolin suspension to chieve high htching rtes of shishmo smelt in n environment with high iron concentrtion The first experiment ws performed to determine the pproprite kolin concentrtion nd tretment period for eliminting egg dhesiveness. All 25 individul kolin concentrtions (0.50, 1.0, 2.0, 5.0 nd 10.0 g/l) nd tretment periods (10 seconds, 1, 5, 15 nd 30 minutes) were performed y dding kolin suspension to smll-numers fertilized eggs plced in 25 smll crylic pltes. Tretments with river wter (nontretment) nd tnnic cid solution were performed s experimentl controls. After the crylic plte contining the clump of eggs ws lid on petri dish, river wter flowed gently over the dish. The rte of egg dhesiveness elimintion is expressed s percentge of the numer of the eggs seprted from the surfce of the plte or dish over the totl numer of eggs. In the second experiment, the effects of the kolin tretment on the survivl rte of the eggs, htching rte, mortl- ity during htching nd sewter tolernce of lrve were investigted. A lrge-numer of fertilized eggs were eqully divided into 3 groups treted y river wter s non-tretment, 5.0 g/l kolin for 5 minutes nd tnnic cid. In the non-tretment group, the eggs were stuck to the crylic pltes. Ech of the 3 groups ws plced in smll crylic mesh g nd rered in 100l jr incutor t the Mukw htchery until just efore htching. Eggs were periodiclly smpled from ech of the 3 groups, oserved under light microscope nd used to investigte survivl rte, htching rte, mortlity during htching nd egg pressure. Htched lrve of ech group were used for the experiment on sewter tolernce. Additionlly, wter from the Mukw htchery nd from SFRI ws collected to determine totl iron concentrtions. Oservtion of the eggs reveled tht the inverted dhesive memrne does not invert in the kolintreted eggs, s in the tnnic cid-treted eggs (Fig. 36). These findings suggest tht suspended kolin prticles completely cover the dhesive portion of the egg efore the memrne inverts. The orderline etween inverted dhesive memrne nd the egg memrne just fter fertiliztion ws evident in the kolin-treted eggs ut not in the tnnic cid-treted eggs (Fig. 36). Kolin suspension tretment is physicl method of covering dhesive mterils, wheres tnnic cid tretment is chemicl method for solidifying the dhesive mterils, s descried y Kusud nd Ternishi

28 130 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Totl iron concentrtion (mg/l) Slmon Fish. Res. Inst. Mukw Htchery Nov. Dec. Jn. Fe. Mr Smpling time Totl mount of iron on the surfce of eggs (mg/ 20 eggs) Non-tretment Tnnic cid Kolin Nov.21 Dec.10 Jn.7 Fe.10 Fe.25 Mr.10 Mr Smpling time Fig. 38. Chnges in totl iron concentrtion in the river wter of the Mukw htchery nd the Slmon nd Freshwter Fisheries Reserch Institute during this experiment. Modified from Aquculture, 242, Mizuno et l., Elimintion of dhesiveness in the eggs of shishmo smelt Spirinchus lnceoltus using kolin tretment to chieve high htching rte in n environment with high iron concentrtion, , 2004, with permission from Elsevier. (1996). Accordingly, it is suggested tht the difference in the orderline ppernce is cused y different mechnisms eliminting egg dhesiveness in kolin nd tnnic cid tretments. In the first experiment, the kolin suspension tretment showed higher elimintion of egg dhesiveness t 5 g/l concentrtion for 5 to 30 minutes, nd t 10 g/l concentrtion for 10 seconds to 30 minutes, compred with the tnnic cid tretment (P < 0.05, Chi-squre test for independence) (Fig. 37). In consequence, the most effective tretment is to use kolin concentrtion of 5 g/l nd period of 5 minutes to reduce the cost nd lor required. The totl iron concentrtion in the river wter of Mukw htchery ws etween nd 2.04 mg/l during this study (Fig. 38). In the rivers of Hokkido Prefecture, the norml concentrtion is of 0.1 mg/l or less (Atod nd Imd 1972). According to Jpnese stndrds on wter qulity for freshwter quculture, the suitle totl iron concentrtion is lso of 0.1 mg/ l or less (Jpn Fisheries Resource Conservtion Assocition 2000). Therefore, the high iron concentrtion in Mukw htchery is very peculir. The totl mount of iron on the surfce of eggs incresed during this study regrdless of the tretment types pplied to eliminte egg dhesiveness (P < 0.05, One-wy ANOVA) (Fig. 39). The mount of iron on the tnnic cid-treted eggs ws significntly lrger thn tht on the kolin-treted nd the non-treted eggs, from Ferury 25 to Mrch 23, 2004 just efore htching (P < 0.05, One-wy ANOVA). Therefore, these results demonstrte tht the kolin-suspension tretment reduces Fig. 39. Chnges in the totl mount of iron on the egg surfce in the non-treted, tnnic cid-treted nd kolin-treted groups during the experiment. Asterisks express significnt differences compred to the initil vlue of ech group (P < 0.05; One wy ANOVA). Cross mrks show significnt differences compred to the vlue of non-tretment group t the sme smpling time (P < 0.05; One wy ANOVA). The letter indictes significnt differences with the vlue of the kolin-treted group t the sme smpling time (P < 0.05; One wy ANOVA). Modified from Aquculture, 242, Mizuno et l., Elimintion of dhesiveness in the eggs of shishmo smelt Spirinchus lnceoltus using kolin tretment to chieve high htching rte in n environment with high iron concentrtion, , 2004, with permission from Elsevier. the mount of iron tht inds to the egg surfce compred with the tnnic cid tretment. This vrition etween kolin nd tnnic cid is proly due to difference in their chemicl property: tnnic cid cn chemiclly ond iron, wheres kolin cnnot. Kolin treted eggs showed significntly high htching rtes nd low mortlity during htching compred to the tnnic cid treted eggs (P < 0.05, One-wy ANOVA) (Figs. 40A, B). However, there ws no difference in the survivl rte of eggs mong kolin treted, tnnic cid treted nd non-treted eggs on Ferury 25 nd Mrch 1 nd 23 (P > 0.05, One-wy ANOVA). Accordingly, these results suggest tht the low survivl rte in the tnnic cid-treted eggs is cused y n incresed mortlity during htching. In the quculture of slmon, incresed mortlity during htching is sometimes oserved in the eggs with hrdened memrnes, produced fter exposure of eggs with soft egg disese to green te tretment (Sski nd Yoshimitsu 2008). In the present study, the egg pressure in tnnic cid-treted egg ws significntly higher thn tht of non-treted eggs just efore htching (P < 0.05, One-wy ANOVA) (Fig. 41). This result revels tht the hrdening of the egg memrne y the tnnic cid tretment ccounts for the incresed mortlity during htching in this group

29 S. Mizuno / Aqu-BioSci. Monogr. 5: , Htching rte (%) Mortlity during htching (%) Survivl rte of lrve fter sewter trnsfer (%) (A) (B) (C) of eggs. Tked et l. (2002) discovered tht n incresed iron content on eggs is correlted with low htching rtes. Therefore, n incresed mount of iron on the surfce of the eggs is lso possily involved in the hrdening of the tnnic cid-treted egg memrne. Furthermore, there ws no significnt difference in the egg pressure etween kolin treted eggs nd nontreted eggs (P > 0.05, One-wy ANOVA) (Fig. 41), which revels tht kolin suspension tretment does Tretment Fig. 40. Htching rte (A), mortlity during htching (B) nd survivl rte of lrve fter the sewter trnsfer (C) in non-treted, tnnic cid-treted nd kolin-treted eggs. Columns with different lpheticl letters were sttisticlly different (P < 0.05; One-wy ANOVA). Modified from Aquculture, 242, Mizuno et l., Elimintion of dhesiveness in the eggs of shishmo smelt Spirinchus lnceoltus using kolin tretment to chieve high htching rte in n environment with high iron concentrtion, , 2004, with permission from Elsevier. Egg pressure (kp) Non-tretment Tnnic cid Kolin Tretment Fig. 41. Egg pressure in the non-treted, tnnic cid-treted nd kolin-treted groups. Columns with different lpheticl letter were sttisticlly different (P < 0.05; One-wy ANOVA). Modified from Aquculture, 242, Mizuno et l., Elimintion of dhesiveness in the eggs of shishmo smelt Spirinchus lnceoltus using kolin tretment to chieve high htching rte in n environment with high iron concentrtion, , 2004, with permission from Elsevier. not rise egg pressure. Additionlly, the present study showed no difference in the sewter tolernce of lrve mong the 3 groups (P > 0.05, One-wy ANOVA) (Fig. 40C), which shows tht the kolin tretment does not impct the sewter dptility of lrve. In consequence, we found tht kolin suspension tretment t 5 g/l concentrtion for 5 minutes is effective for eliminting egg dhesiveness nd improving htching rtes in shishmo smelt Appliction of SSP s mteril for eliminting egg dhesiveness in Jpnese smelt For the first experiment, smll-numer of fertilized eggs were eqully plced into seven crylic dishes. Spring wter (control), 5.0 g/l kolin suspension nd 1.0, 5.0, 10.0, 20.0 nd 50.0 g/l SSP suspensions were poured into the seven dishes. All eggs were divided into two groups, dhesive nd seprted eggs, in the dish. The elimintion rte of egg dhesiveness ws clculted s the percentge of seprted eggs over the totl numer of eggs in ech dish. The eggs were cultured until completion of htching nd the htching rte ws exmined in ech group. For the second experiment, lrge numer of fertilized eggs were eqully divided into four groups. Ech of the four groups were treted with spring wter (control), 5.0 g/l kolin sus-

30 132 S. Mizuno / Aqu-BioSci. Monogr. 5: , 2012 Egg dhesiveness elimintion rte (%) c c c c c d de e April 27 April 28 Tretment Experiment time Fig. 42. Egg dhesiveness elimintion rte in Jpnese smelt eggs treted with freshwter (control), kolin suspension or SSP suspensions in the first experiment. Different lpheticl letters showed significnt differences in the htching rte etween groups (P < 0.05; Chi-squre test for independence). Modified from Aquculture Science, 58, Mizuno et l., Effects of tretment using unked scllop shell powder suspension on eliminting egg dhesiveness, htching rte nd lrvl qulity in Jpnese smelt (Hypomesus nipponensis) eggs, , 2010, Jpnese Society for Aquculture Reserch. pension, 5.0 or 20.0 g/l SSP suspension for 10 seconds nd seprtely plced in smll net gs. The four gs were kept in 6l jr incutor. Eggs were smpled 20 dys fter fertiliztion for the nlysis of survivl nd htching rtes nd for oservtion with light microscope. The first experiments demonstrted tht tretment y more thn 5 g/l SSP ws effective in eliminting egg dhesiveness, compred to the conventionl 5 g/l kolin tretment (P < 0.05, Chi-squre test for independence) (Fig. 42). It is generlly ccepted tht the soluility of kolin in wter is 10 mg/l t 25 C, which is quite similr to the soluility of CCO 3 (15 mg/l) (Iguchi et l. 2001). The specific grvity of kolin is 2.6 g/cm 3 (Iguchi et l. 2001), while tht of SSP is 2.7 g/cm 3. This informtion suggests tht the physicl properties of SSP resemle tht of kolin. The htching rtes fter ll SSP tretments were not significntly different from tht fter the kolin tretment (P > 0.05; Chi-squre test for independence) (Fig. 43). This suggests tht none of the SSP concentrtions used hd d influence upon the htching rte. Oservtion of the eggs showed tht the inverted dhesive memrne ws spred like prchute in the control group, wheres it ws rumpled in the kolinnd SSP-treted groups (Fig. 44). These differences etween the control nd ll suspension-treted groups proly reflect tht SSP or kolin stuck to the dhesive prt nd eliminted egg dhesiveness. Kolin suspension tretment inverted the inverted dhesive memrne in Jpnese smelt eggs in this study, while it did not invert the memrne in shishmo smelt (Mizuno et l. 2004). This difference etween Jpnese nd shishmo smelt possily depends either on vritions in the chrcteristics of the proteins of the inverted dhesive memrne, in the speed it tkes to invert the inverted dhesive memrne fter fertiliztion nd/or in tretment time. In the second experiment, there were no significnt differences in the survivl rte nd htching rte mong control, kolin, 5 g/l nd 20 g/l SSP (P > 0.05; Onewy ANOVA). These results demonstrte tht SSP tretments cn e prcticlly used for the elimintion of egg dhesiveness in intensive egg cultures using jr incutors. On the other hnd, it is necessry to keep in mind the internl prticulte dmge in the use of minute prticles such s SSP, which implies tht the use of s few prticles s possile is sfer. Therefore, we should use 5 g/l SSP tretment, which is the minimum SSP concentrtion for effective tretment, for the elimintion of egg dhesiveness.

31 S. Mizuno / Aqu-BioSci. Monogr. 5: , Htching rte (%) April 27 April 28 Tretment Experiment time Fig. 43. Htching rte of Jpnese smelt eggs treted with freshwter (control), kolin suspension or SSP suspensions in the first experiment. Different lpheticl letters showed significnt differences in the htching rte etween groups (P < 0.05; Chi-squre test for independence). Modified from Aquculture Science, 58, Mizuno et l., Effects of tretment using unked scllop shell powder suspension on eliminting egg dhesiveness, htching rte nd lrvl qulity in Jpnese smelt (Hypomesus nipponensis) eggs, , 2010, Jpnese Society for Aquculture Reserch Approprite timing for the relese of eyedstge emryos in shishmo smelt The present study used eggs spwned nturlly on the ottom of the tnk y homing-migrting dults in Mukw htchery, which releses eyed-stge emryos into the Mukw River. According to previous studies of the emryogenetic stges of shishmo smelt (Hikit 1958), the emryogenetic stge ws regrded s stge 15 when lens ppered in the optic vesicles on Ferury 10, stge 16 when the end of emryo s til did not rech its hed nd the color of optic vesicles egn drken on Ferury 25, stge 17 when it s til reched its hed on Mrch 10, stge 18 when the color of the optic vesicles drkened completely nd the emryo til overlpped its hed on Mrch 23 nd stge 19 when the emryo moved in the egg nd silver color ecme distinct in the eyes on April 2 y oservtion with light microscope (Fig. 45). Stges 15, 16, 17, 18 nd 19 corresponded to 133, 140, 154, 188 nd 243 C in cumultive temperture, respectively. Live eggs in ech stge were divided into 3 groups. Ech group ws put into either freshwter, 17.0 psu rtificil rckish wter or 34.0 psu rtificil sewter. Htching rte ws exmined in ech of the 3 dishes. Additionlly, live eggs were used for the nlysis of N +,K + -ATPse ctivity t ech stge. Figure 46 indictes tht the htching rte incresed significntly during emryogenesis in oth 17 psu rckish wter nd 34 psu sewter. In response to the incresed htching rte, egg N +,K + -ATPse ctivity lso incresed significntly during emryogenesis (P < 0.05, One-wy ANOVA) (Fig. 47). Correltion nlyses etween the htching rte nd N +,K + -ATPse ctivity showed there re significnt unliner positive correltions etween these 2 prmeters under oth 17 psu rckish wter nd 34 psu sewter environments (17 psu: r = 0.903, P < 0.05; 34 psu: r = 0.833, P < 0.05; Person s correltion coefficient) (Fig. 48). These results suggest tht incresed egg N +,K + -ATPse ctivity during emryogenesis is linked to the incresed htching rte in sewter environments during emryogenesis. Hyno et l. (1999) hve reported tht the ctivtion of mitochondrion-rich cells on the yolk-sc memrne of emryos is linked to the development of sewter dptility of the emryo of shishmo smelt, since immunorections ginst nti N +,K + -ATPse α- suunit were enhnced in the cells, nd cell size ecme lrger during sewter dpttion. The ctivtion of mitochondrion-rich cells on the yolk-sc during sewter dpttion is lso oserved in chum slmon (Kneko et l. 1993) nd tilpi (Ayson et l. 1994, Shirishi et l. 1997). Lrve of shishmo smelt htch with yolk sc nd without complete gills (Hikit

32 S. Mizuno / Aqu-BioSci. Monogr. 5: , A B C D 1958), which re one of the min osmoregultory orgns in fish (Pyn et l. 1984). Accordingly, incresed egg N+,K+-ATPse ctivity in shishmo smelt is proly cused y the ctivtion of N+,K+-ATPse on the yolk sc memrne. The htching rte in freshwter ws very high t ll emryogenetic stges, with the rckish wter nd sewter groups showing lower htching rtes t most emryogenetic stges (P < 0.05; Chi-squre test for independence) (Fig. 46). However, the rtes with 17 psu rckish wter were equl to the rtes of the freshwter group t stges 18 nd 19 (P > 0.05; Chi-squre test for independence) (Fig. 46). Consequently, these results demonstrte tht eyed-stge emryos of shishmo smelt hve no complete 34 psu sewter tolernce t ny emryogenetic stge, while they hve perfect tolernce to 17 psu rckish wter fter stge 18. If eyed-stge emryos re relesed into the river for rtificil propgtion of shishmo smelt, we should relese emryos fter t lest stge 18. Fig. 44. Oservtion of freshwter-treted (A, control), 5 g/ l kolin-treted (B), 5 g/l (C) nd 20 g/l (D) SSP-treted emryos 20 dys fter fertiliztion in the second experiment. Arrows indicte inverted dhesive memrne. Scle rs designte 1.0 mm. Control eggs (A) were unfstened from the plm tree skins efore their oservtion. Modified from Aquculture Science, 58, Mizuno et l., Effects of tretment using unked scllop shell powder suspension on eliminting egg dhesiveness, htching rte nd lrvl qulity in Jpnese smelt (Hypomesus nipponensis) eggs, , 2010, Jpnese Society for Aquculture Reserch. 6. Generl discussion: Towrds sustinle rtificil propgtion of slmonids nd osmerids 6-1. Implictions of this monogrph for rtificil propgtion In Section 2, quntittive system of dorsl fin pigmenttion during smoltifiction ws first estlished in smolting msu slmon, nd stndrd vlue ws Fig. 45. Oservtion of the eyed-stge emryos used in this study. Pnels (), (), (c), (d) nd (e) show emryos t 133 (Stge 15), 140 (Stge 16), 154 (Stge 17), 188 (Stge 18) nd 243 C (Stge 19) cumultive temperture, respectively. The rrowheds designte the position of the end of the til. Scle r shows 1.00 mm. Reprinted with permission of John Wiley & Sons, Inc. from Aquculture Reserch, 36, Mizuno et l., Chnges in sewter tolernce during the development of eyed-stge emryos in shishmo smelt Spirinchus lnceoltus (Hikit), , Fig. 1, 2005, Wiley-Liss, Inc., Wiley Compny.