Studies on Improvement of Seed Production Techniques in Salmonids and Osmerids

Shinya Mizuno

Salmon and Freshwater Fisheries Research Institute, Fisheries Research Department, Local Independent Administrative Agency Hokkaido Research Organization, 3-373, Kitakashiwagi, Eniwa, Hokkaido 061-1433, Japan

Abstract

Anadromous salmonids and osmerids are artificially propagated in Japan by release of their seeds. However, there are many unsolved problems in the techniques of their propagation. In the present monograph, studies on the improvement of seed production techniques in 4 fishes (masu salmon Oncorhynchus masou, chum salmon O. keta, shishamo smelt Spirinchus lanceolatus and Japanese smelt Hypomesus nipponensis) were outlined. Techniques to evaluate dorsal fin pigmentation during smoltification as an external seed quality, and to improve seed quality of hatchery-reared fish, and the discovery of metabolic problems in hatchery-reared fish were described in yearling masu salmon. In underyearling masu salmon, techniques to evaluate nutritional conditions using kidney melano-macrophage density was developed, and applied to the evaluation of the nutritional condition in hatchery-reared fish after release. In chum salmon fry, the development of techniques to monitor the physical condition and to find its appropriate culture conditions was reviewed. In egg cultures of shishamo and Japanese smelt, techniques to eliminate egg adhesiveness with treatments of kaolin or scallop shell powder suspension were established in order to improve hatching rates. In addition, the appropriate embryogenetic stage for the release of shishamo smelt embryos was demonstrated. Consequently, this monograph reveals that these techniques contribute directly to the development of artificial propagation in some salmonids and osmerids.

Keywords

culture condition, hatchery-reared fish, hatching rate, Hypomesus nipponensis, kaolin, Oncorhynchus keta, Oncorhynchus masou, physical condition, scallop shell powder, seed production, seed quality, Spirinchus lanceolatus


Received on January 10, 2012

Accepted on June 21, 2012

Online published on December 21, 2012

e-mail: mizuno-shinya@hro.or.jp


1. General introduction

Anadromous salmonids and osmerids including masu salmon Oncorhynchus masou, chum salmon O. keta, shishamo smelt Spirinchus lanceolatus and Japanese smelt Hypomesus nipponensis are important species for coastal and freshwater commercial fisheries in Hokkaido, the northern-most prefecture in Japan. These fish are not always abundant under natural reproduction systems (Nagata and Kaeriyama 2004; Torao 2005a, b; Miyakoshi 2006). Therefore, these fish are artificially propagated by releasing their seeds in order to increase their stock in various places in Hokkaido (Kusuda and Teranishi 1996; Kaeriyama 1999; Torisawa 1999).

Masu salmon, which inhabit both Pacific Ocean and the Japan Sea coasts of Hokkaido, spawn in upstream regions in autumn and hatch the winter of the same year (Kato 1991). The fish are separated into two types: anadromous and stream-resident types. The anadromous type migrates to the ocean in spring after spending 1 or 2 years in the streams. Before the seaward migration, they undergo smoltification, a series of behavioral, morphological, physiological and biochemical changes controlled by multiple hormonal pathways (Hoar 1988) that transform the freshwater parr into a seaward-migratory smolt. After 1 year of life in the ocean, the fish return to their natal stream in spring and spawn in autumn (Kubo 1980; Machidori and Kato 1984; Kato 1991). For the artificial propagation of masu salmon, hatchery-reared (hatchery) juveniles, originating from artificial insemination and fed a commercial food in hatcheries, are released into streams (Mayama 1992). Effects of masu salmon propagation, which are expressed as the recovery rate of released hatchery seed, are of 0.41 to 2.12% (Miyakoshi 2006), while the recovery rate of chum salmon artificial propagation was >10% (Taya 1989). In other words, masu salmon propagation costs more money compared to chum salmon propagation. It is essential for the sustainable artificial propagation of masu salmon to reduce the cost of seed production by increasing the survival rate of seed.

Hatchery masu salmon are released in two ways. One is called the smolt release method where yearling (1+) smoltified juveniles are released into downstream regions in spring, and the other is called the spring juvenile release method, where underyearling (0+) juveniles are released into upstream regions in spring (Mayama 1992). One of the keys to increasing survival rate of seed is stocking hatchery juveniles with high seed qualities determined from ethological, physiological and biochemical characteristics. However, seed quality of hatchery juveniles has not been perfectly evaluated in masu salmon. In addition, there has been no attempt to improve the seed quality of hatchery juveniles.

Chum salmon, which inhabit the whole region of Hokkaido, hatch in rivers in winter, and migrate to the ocean the following spring. All of the fish are anadromous. After 2 to 6 years of marine life, adults return to spawn in their natal rivers in autumn (Salo 1991). In the program, 0+ fry, which were produced in hatcheries in the same way as the masu salmon, are released into the river in spring (Kobayashi 1980). However, the increased stock due to the propagation-program has caused downsizing and aging (Kaeriyama and Edpaline 2004). This phenomenon is believed to be due to a population density-dependent effect: the growth of chum salmon fry is restrained by carrying capacity, such as the amount of food or habitat size, in the North Pacific (Kaeriyama et al. 2007). Hatchery fish have to coexist with wild fish in a narrow carrying capacity. Therefore, biological interactions between hatchery populations and wild populations are being studied (Kaeriyama and Edpaline 2004). Hilborn (1992) has warned that the excessive release of hatchery fish decreases the genetic diversity of Pacific salmon, including chum salmon. Accordingly, artificial propagation and the preservation of wild populations must be jointly considered in order to preserve the natural balance. Kaeriyama (2002) was concerned about the infection of wild fish by hatchery fish diseases. Considering this background, the reliable production of hatchery fry with good physical conditions is more important than an increased number of released hatchery fry for the sustainable artificial propagation of chum salmon.

Chum salmon fry are intensively cultured in high density conditions in hatchery ponds. In general, intensively cultured fish are commonly under high physiological stress resulting from deteriorated rearing conditions (Patinõ et al. 1986; Papoutsoglou et al. 1987). On the other hand, it has been impossible to monitor the physical conditions of hatchery fry cultured in high density conditions, and practical environmental conditions for the culture of fry in good physical conditions have not yet been found.

Shishamo smelt migrate to their natal river on the Pacific Ocean side of Hokkaido in early winter to spawn (Hikita 1930, 1958). Adults spawn 1.4 mm sized eggs in freshwater environments about 3 to 9 km upstream from the estuary (Ito 1959, 1963, 1964; Okada and Sasaki 1960; Okada et al. 1975, 1976; Omi 1978b). The larvae migrate to the estuary immediately after hatching carried by the snow-melt water during the following spring, and mature after 2 or 3 years of life in the ocean (Omi 1978a). All of the fish are anadromous (Miyaji et al. 1976). On the other hand, Japanese smelt, which are distributed around the whole of Hokkaido, consist of anadromous and lake-resident types (Hamada 1961; Katayama et al. 1999). Anadromous-type fish migrate to their natal stream and spawn 0.8 mm sized eggs in shallow streams from April to June (Shiraishi 1961; Torisawa 1999). The general lifespan of the fish is 1 to 2 years (Utoh and Sakazaki 1983, 1984, 1987; Torisawa 1999). For the artificial propagation of shishamo and Japanese smelt, larvae, obtained by artificial insemination, are released into the rivers or lakes through the drainage of hatchery waters (Iwai and Osama 1986; Kusuda and Teranishi 1996; Izuka 2003; Kitsukawa et al. 2003). However, the efficiency of the propagation has not yet been accurately evaluated. It is necessary to first produce a lot of seed to perform a pilot release, in order to establish effective seed-release methods and to elucidate the effects of releasing hatchery seed.

Some shishamo and Japanese smelt hatcheries intensively culture eggs using upwelling flow-type acrylic and cylindrical jar incubators (Kusuda and Teranishi 1996; Kitsukawa et al. 2006). However, it was found that eggs cultured in the jar incubators showed low hatching rates (Takeda et al. 2002). In a few shishamo smelt hatcheries, eyed-stage embryos are released into the river (Mizuno et al. 2004b, 2005) before they hatch. However, the most appropriate time for releasing eyed-stage embryos into the river is unknown.

In this monograph, I review research performed on the improvement of seed production techniques in salmonids and osmerids, with the aim to solve some of the problems mentioned above. In Section 2, three approaches to evaluating and improving the seed quality of hatchery masu salmon smolt are described. Section 3 reviews 2 studies on the development and application of techniques evaluating seed quality in hatchery masu salmon parr for spring juvenile release. Section 4 deals with 2 studies dealing with the establishment of techniques to monitor physical conditions and the elucidation of appropriate culture conditions in chum salmon fry. In Section 5, three attempts to develop techniques to improve survival rate in artificial propagation of shishamo and Japanese smelt were described. The final chapter describes the implications and perspective studies of the improvement of seed production techniques in salmonids and osmerids.

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2. Evaluation and improvement of seed qualities in 1+ hatchery masu salmon smolts

2-1. Introduction

External smoltification is mainly characterized by the black pigmentation of dorsal and caudal fin margins due to the diffusion of melanin granules in melanophores and by body silvering due to the deposition of guanine on the skin (Ura et al. 1994). A quantitative analysis of the body silvering have been developed in masu salmon (Chida and Kijima 1994; Ando et al. 2005), amago salmon O. rhodurus (Kuwada et al. 2000) and Atlantic salmon Salmo salar (Duston 1995; Haner et al. 1995). In contrast, quantification of the dorsal fin pigmentation has not yet been established in salmonids.

The most well-known internal changes occurring during smoltification is the development of seawater adaptability, which results from physiological and biochemical changes in the osmoregulatory organs such as the gill, kidney, intestine and esophagus (Clarke and Hirano 1995). Seawater adaptability, which possibly reflects the survival of juveniles after their entry into seawater, is often raised as a reliable indicator to judge the internal quality of hatchery smolt seed. A seawater challenge test, which examines the change in serum Na+ levels during a transfer from freshwater to seawater (Clarke and Blackburn 1977), and gill Na+,K+-ATPase activity (Zaugg and McLain 1971; Folmer and Dickhoff 1980; Boeuf and Harache 1982; Boeuf and Prunet 1985; McCormick 1993) are used to determine seawater adaptability. Until now, however, only the survival rate after the transfer from freshwater to seawater has been examined in the smolt seeds of masu salmon from hatcheries (Misaka et al. 1998b). Some previous studies have pointed out the problems of hatchery smolt: seawater adaptability is low (Shrimpton et al. 1994a), development of seawater adaptability is not synchronized with external smoltification (Wedemeyer et al. 1980), and hormonal regulation related to the development of seawater adaptability is incomplete (Shrimpton et al. 1994b; McCormick and Björnsson 1994).

Changes in metabolism are also generally accepted as an effect of internal smoltification. Metabolic changes during smoltification have been reported through metabolic enzyme activity, amounts of metabolites, and metabolic rates in Atlantic and coho salmon (O. kisutch). In Atlantic salmon, standard and active metabolic rate is higher in smolts compared to parr (Maxime et al. 1989). Atlantic salmon smolts have lower hepatic and muscle glycogen and increased blood glucose levels, which reflects an activation of glycolysis, absent in parr (Wendt and Saunders 1973). Sheridan et al. (1985) demonstrated that the low hepatic glycogen content was caused by a combination of reduced glycogen synthesis and increased glycogenolysis in coho salmon smolts. Muscle phosphofructokinase activity, a glycolytic enzyme, was high during smoltification in Atlantic salmon (Leonard and McCormick 2001). Citrate synthase activity, a citric acid cycle enzyme of liver, gill and kidney was enhanced during smoltification in Atlantic salmon (McCormick et al. 1989). In the gill and liver, the activity of respiratory enzymes increased during smoltification in Atlantic salmon (Langdon and Thorpe 1985; McCormick and Saunders 1987). These previous reports demonstrate that the activation of glycolysis, and of the citric acid cycle, and the increase in respiration are notable metabolic changes which occur during smoltification. However, there was little information on changes in metabolism and metabolic enzymes during smoltification in masu salmon. Consequently, seed quality related to seawater adaptability and metabolism has not yet been sufficiently evaluated in masu salmon smolt.

Anadromous salmonid juveniles migrating from a freshwater to a seawater environment are prone to high mortality (Parker 1968; Healey 1982; Bax 1983). One of the principal causes for this is predation by seabirds and large fish (Beamish and Neville 1995; Nagasawa 1998; Kawamura and Kudo 2000). Individual survival, then, is dependent on swimming ability, in order to escape from predators (Jayne and Lauder 1993). This swimming ability is more closely related to burst swimming, which expresses the sprint swimming in fish (Jayne and Lauder 1993), rather than to critical swimming, which reflects sustained swimming. Burst swimming is powered by white glycolytic fibres that constitute the bulk of the myotomal mass and that depend on an anaerobic metabolism (Domenici and Blake 1997; Franklin and Johnston 1997), whereas critical swimming principally relies upon aerobic red oxidative fibres (Bone et al. 1978; Gallaugher et al. 1995). Burst swimming results in the production of lactate and H+ by the muscle, which cause a marked reduction in plasma pH, as well as respiratory and metabolic acidosis (Turner et al. 1983; Milligan and Wood 1986). In the rainbow trout O. mykiss, increases in blood hemoglobin (Hb) concentrations following burst swimming have been observed (Milligan and Wood 1986, 1987; Pearson and Stevens 1991). The increased Hb concentrations have, in turn, been related to recovery from acidosis-induced fatigue (Milligan and Wood 1987; Wang et al. 1994). It is plausible, therefore, that there is a relationship between concentrations of Hb and the burst swimming capacity in salmonids, but this hypothesis has not yet been tested.

Woodward and Smith (1985) noted that hatchery salmon displayed a placid behavior and a poor swimming ability due to the rearing stress of high densities and low water flow. The swimming ability can be improved by chronic swimming exercises, resulting in increased Hb concentrations (Hochachka 1961; Burrows 1969; Zbanyszek and Smith 1984). Furthermore, increased water flow results in improved swimming performance in hatchery masu salmon in a circular tank (Azuma et al. 2002). However, it is often difficult or unpractical to extract enough water to generate a fast water flow or to make hatchery fish exercise in circular tanks, given the limited amount of utilizable water, and the fact that rectangular ponds are used by many Hokkaido Prefecture hatcheries. Accordingly, other methods must be developed to improve the swimming ability of hatchery fish. Increasing dietary iron is known to increase Hb concentrations in fish (Kawatsu 1972; Ikeda et al. 1973; Sakamoto and Yone 1978; Carriquiriborde et al. 2004), but its effect on burst swimming capacity has not yet been studied.

Subsection 2-2 focuses on the establishment of a quantifying system for the black pigmentation of the dorsal fin margin during smoltification using image analysis and on the elucidation of the relationships between the dorsal fin pigmentation and gill Na+,K+-ATPase activity. Subsection 2-3 deals with changes in the activities and transcription levels of several metabolic enzymes during smoltification, in both wild and hatchery fish, in order to show physiological qualities particular to hatchery smolt. In Subsection 2-4, differences in burst swimming capacity and physiological parameters other than those shown in Subsections 2-2 and 2-3 of both between wild and hatchery smolts, and the effects of diets supplemented with iron on these parameters in the hatchery smolt were investigated.

2-2. Development of techniques to evaluate dorsal fin pigmentation during smoltification

Smolting hatchery fish of the Donan Research Center of Salmon and Freshwater Fisheries Research Institute (SFRI) and smolting wild fish captured in the two rivers in Hokkaido were studied. One of the two rivers, the Ken-ichi River, was utilized for rearing the SFRI-hatchery fish. After lethal anesthesia of the fish, the depressed portion of the dorsal fin membrane between the fourth and fifth soft rays (Fig. 1) was observed with a light microscope. The grayscale digital picture of the portion was divided into black and white parts include all melanophores in the black part using an image analysis software (Fig. 2). The dorsal fin pigmentation level was expressed as the percentage of the black part over the whole picture size. The level of dorsal fin pigmentation increased from January to May during smoltification in both stocks of wild fish and the stock of hatchery fish (P < 0.05, One-way ANOVA) (Fig. 3), reflecting the progress of exterior smoltification. The levels converged at around 83–84% in May, which is the peak time of downstream migration to the sea in wild masu salmon. Moreover, there was no significant difference in the peak level between wild and hatchery fish (P > 0.05, One-way ANOVA). These findings reveal the establishment of a quantitative system for dorsal fin pigmentation with a level of 83–84% being an appropriate indicator for external seed quality in hatchery smolt. SFRI-hatchery smolt is regarded as excellent seed based on dorsal fin pigmentation.


Fig. 1. Observed portion of dorsal fin pigmentation. Panel A indicates the entire dorsal fin of wild masu salmon smolt in the Ken-ichi River. Panel B is magnified from the small rectangular area indicated in panel A. In panel B, the white square (100 μm × 100 μm) shows the portion of the dorsal fin observation. IV: fourth soft ray, V: fifth soft ray, VI: sixth soft ray. Scale bars show 1.00 mm. Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

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Fig. 2. Dorsal fin pigmentation during smoltification of wild masu salmon in the Utabetsu River. Upper pictures in grayscale, lower pictures after black and white transformation by an imaging software. The lines from 'a' to 'e' indicates dorsal fins in January, February, March, April and May, respectively. Scale bar shows 100 μm. Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

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Fig. 3. Quantitative changes in dorsal fin pigmentation during smoltification in wild and hatchery-reared masu salmon. The letter 'a' adjacent to a symbol indicates a significant difference with the initial value of each respective group (P < 0.05; One way ANOVA). Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

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Gill Na+,K+-ATPase activity increased from January to May during smoltification in all wild and hatchery fish (P < 0.05, One-way ANOVA) (Fig. 4). There were no significant differences in the activity between wild and hatchery fish at any given period (P > 0.05, One-way ANOVA). These results suggest that the SFRI-hatchery smolts have as high a seawater adaptability as wild fish. Figure 5 shows the relationship between dorsal fin pigmentation and gill Na+,K+-ATPase activity in all fish. A positive nonlinear correlation was found between the level of pigmentation of the dorsal fin and gill ATPase activity in both the wild and hatchery fish (P < 0.0001, Spearman's rank correlation coefficient) (Figs. 3, 4). The period of increased dorsal fin pigmentation preceded that of enhanced gill Na+,K+-ATPase activity in all fish. However, it has been generally accepted that external smoltification coincided with typical internal smoltification, such as the development of seawater adaptability, in other salmonids (Hoar 1988). The gap between the increase of dorsal fin pigmentation and gill Na+,K+-ATPase activity is possibly particular to masu salmon. However, the peak of dorsal fin pigmentation coincided with that of gill Na+,K+-ATPase activity. Therefore, we can use the level of dorsal fin pigmentation as an indicator in order to find the peak of smoltification in SFRI-hatchery masu salmon.


Fig. 4. Changes in gill Na+,K+-ATPase activity during smoltification in wild and hatchery-reared masu salmon. The letter 'a' adjacent to a symbol indicates a significant difference with the initial value of each respective group (P < 0.05; One way ANOVA). Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

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Fig. 5. Correlation of gill Na+,K+-ATPase activity with dorsal fin pigmentation level, in wild and hatchery-reared smolting masu salmon. Spearman's rank correlation coefficient by rank test was used as statistical analysis. The normal, bold and dotted line show the nonlinear correlations [Pigment] = 61.0 + 16.0 ln [ATPase] (r2 = 0.752, P < 0.0001) in the Ken-ichi River, [Pigment] = 66.8 + 13.7 ln [ATPase] (r2 = 0.879, P < 0.0001) in the Utabetsu River and [Pigment] = 68.9 + 13.0 ln [ATPase] (r2 = 0.806, P < 0.0001) in the hatchery-reared groups, respectively. Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

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2-3. Differences in metabolism between 1+ wild and hatchery smolts

Wild and SFRI-hatchery smolting fish were sampled at the same time. The river where the wild fish were caught, was also utilized for rearing the hatchery fish. Serum, gill and liver of the fish were sampled. Serum glucose concentration and hepatic glycogen content decreased during smoltification in wild fish (P < 0.05, One-way ANOVA), while the decrease was not found in hatchery fish (P > 0.05, One-way ANOVA) (Fig. 6). Hepatic glycogen content of hatchery fish was significantly lower than in wild fish in March (P < 0.05, Student's t-test) (Fig. 6). These findings reflect low glycogenolysis during smoltification and low use of glycogen as an energy source in hatchery fish. Moreover, a decrease in hepatic pyruvate kinase (PRK) activity was observed from April to May in hatchery fish (P < 0.05, One-way ANOVA), while there was no decrease in the activity at the same stage in wild fish (P > 0.05, One-way ANOVA) (Fig. 7). An increase in gill PRK activity during smoltification was revealed in wild fish (P < 0.05, One-way ANOVA), while it did not increase in hatchery fish (P > 0.05, One-way ANOVA) (Fig. 7). These results suggest low glycolytic changes during smoltification and low use of glycolysis at the smolt stage in hatchery fish. Furthermore, this study discovered a temporal difference in increased hepatic citrate synthase (CS) activity between hatchery and wild fish, and the absence of an increased gill CS activity during smoltification in hatchery fish (P > 0.05, One-way ANOVA) (Fig. 8), which reveals that there is little change of the citric acid cycle at the smolt stage in hatchery fish. Hatchery smolt had lower liver cytochrome c oxidase (COX) activity compared to wild smolt in May (P < 0.05, Student's t-test), which possibly means that hatchery smolt cannot prepare for the activation of the hepatic respiratory chain before their seaward migration (Fig. 9). Liver and gill transcription levels of ATP synthase subunit 8 (AST) showed opposite changes between hatchery fish (decrease) and wild fish (increase) from April to May (P < 0.05, One-way ANOVA) (Fig. 10). Another notable point is that there are temporal differences in the increase in liver and gill AST transcription levels between hatchery and wild fish. Hatchery smolt indicated lower AST transcription levels in the liver and gill in May compared to the wild smolt (P < 0.05; Student's t-test) (Fig. 10). These findings suggest that hatchery fish cannot produce many respiratory chain enzymes before their seaward migration.


Fig. 6. Changes in serum glucose concentration (GL) and liver glycogen content (GC) during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks reveal significant differences compared to the value of the same parameter in March within each group of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

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Fig. 7. Changes in liver and gill pyruvate kinase (PRK) activity during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks reveal significant differences compared to the value of the same parameter in March within each group of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

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Fig. 8. Changes in liver and gill citrate synthase (CS) activities during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks reveal significant differences compared to the value of the same parameter in March within each group of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

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Fig. 9. Changes in liver cytochrome c oxidase (COX) activity during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences in the value between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks reveal significant differences compared to the value of the same parameter in March within each group of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

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Fig. 10. Changes in transcription levels of liver and gill ATP synthase subunit 8 (AST) during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences in the value between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks revealed significant differences compared to the value of the same parameter in March within each groups of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

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The present study found that SFRI-hatchery masu salmon had some problems in the carbohydrate metabolism, citric acid cycle, and respiratory chain, which suggests that the hatchery fish may not be able to adjust to marine environments after migrating from the river to the sea leading to high mortality rates. Walton (1986) reported that low carbohydrate and high protein food intake induced low activities of glycolytic enzymes and the citric acid cycle in rainbow trout. Brauge et al. (1994) demonstrated that elevated digestible carbohydrate intake resulted in glycaemia in rainbow trout. These findings imply that the problem of SFRI-hatchery fish is caused by a low carbohydrate diet. Meanwhile, Leonard and McCormick (2001) have found that there were significant fluctuations and temporal differences between hatchery fish and wild Atlantic salmon of the liver β-hydroxyacyl-coenzyme A dehydrogenase, heart phosphofructokinase and white muscle lactate dehydrogenase during smoltification, which indicated other metabolic problems in hatchery fish. McCormick and Björnsson (1994) have found higher levels of plasma cortisol, a stress-related hormone involved in stimulating or maintaining increased plasma glucose and gluconeogenesis, in hatchery smolt than in migrating hatchery smolt released into a stream, which may reflect that hatchery fish experience greater stress than wild fish. This consideration may demonstrate that metabolic problems of hatchery fish result from the stress of the artificial rearing environment such as feeding, artificial handling and high rearing densities. In addition, Misaka et al. (2004) elucidated that fasting induced a rapid decrease in hepatic glycogen in masu salmon, which suggests that the amount of food supplied impacts the hepatic glycogen content. Therefore, the small hepatic glycogen content of hatchery fish is possibly caused by stress related to low food supply in the present study. Acute handling stress, which often occurs during fish transportation in hatchery fish, induces a temporary increase in plasma cortisol and glucose levels (Carey and McCormick 1998). These earlier results suggest the considerable increase in plasma cortisol levels related to handling stress disturbs the carbohydrate metabolism in the SFRI-hatchery fish. Furthermore, high rearing densities resulted in reduced plasma levels of thyroid hormones and cortisol related to smoltification in smolting coho salmon (Patinõ et al. 1986) and in increased plasma glucose levels and low growth rate in rainbow trout (Procarione et al. 1999). Carbohydrate metabolism is directly or indirectly regulated by not only cortisol but also insulin, glucagon and epinephrine in fish (Plisetskaya et al. 1988; Plisetskaya 1990; Vijayan et al. 1993). Therefore, this consideration may reveal that predictable social stress related to high rearing densities disturbs the endocrine control of the carbohydrate metabolism in SFRI-hatchery fish. The best way to solve the metabolic problems of the hatchery masu salmon smolt is possibly to achieve an improvement of dietary quality, repletion, reduce handling and/or develop low density-rearing conditions.

2-4. Development of techniques to improve the seed quality of 1+ hatchery smolts

In the "2003 experiment", 1+ SFRI-hatchery fish were divided into 3 groups of 40 fish in January. Each group was housed in a separate tank and fed to satiation either the same diet as that supplied before January (control), a feebly iron-enriched diet (iron-1) or highly iron-enriched diet (iron-2), respectively. The iron-1 and iron-2 diets contained 2.50 and 7.50 g iron citrate per kg dry weight of control food, resulting in mean total iron levels in the control, iron-1 and iron-2 diets of 7.96 × 102, 1.51 × 103 and 3.17 × 103 mg/kg dry weight, respectively. Each of the 3 groups and smolting wild fish, which were captured in the river of SFRI, were sampled from January to May on the same days. In the "2004 experiment", forty thousand 1+ SFRI-hatchery fish were divided into 2 equal groups in February. The control diet was assigned to one group of fish, while the other group was fed the iron-1 diet. Each of the two groups and smolting wild fish were sampled as in the "2003 experiment". The sampled fish were used to calculate the condition factor (CF) (Bagenal and Tesch 1978), measure burst swimming capacity and for the sampling of stomach remnants, blood, liver and white muscle.

A decrease of the CF in spring is generally accepted as a change related to smoltification in salmonids (McCormick and Saunders 1987). In my study, the CF decreased during smoltification in all hatchery fish in 2003 and 2004, and wild fish in 2003 (P < 0.05, One-way ANOVA), whereas it did not decrease in wild fish in 2004 (P > 0.05; Student's t-test) (Fig. 11). An absence of decrease of the CF during smoltification sometimes occurs in wild masu salmon (Mayama 1992; Mizuno et al. 2004a). This phenomenon can be related to the presence of insects, carried by snowmelt water in spring, in the stomach (Mayama 1992). This study showed that hatchery smolt controls had low CF values with regard to the total iron content of the food remnants in the stomach (P < 0.05, One-way ANOVA) (Fig. 12), burst swimming capacity (P < 0.05, One-way ANOVA) (Fig. 13), Hb concentrations (P < 0.05, One-way ANOVA) (Fig. 14) and in the ATP content in white muscle and liver, compared to wild fish, in May (P < 0.05, One-way ANOVA) (Fig. 15). These findings reveal some new problems of seed qualities in the SFRI-hatchery smolt: a shortage of iron, low swimming ability, low oxygen-carrying capacity and low energy production. Woodward and Smith (1985) concluded that low swimming ability in hatchery rainbow trout was caused by a placid behavior and a rearing environment drastically different from the natural habitat. This explanation is also plausible for the SFRI-hatchery smolt in high density conditions. Low Hb concentrations are generally observed during anemia due to dietary iron insufficiency in hatchery brook trout (Salvelinus fontinalis) (Kawatsu 1972) and yellowtail (Seriola quinqueradiata) (Ikeda et al. 1973). This consideration and the present results (Fig. 12) point towards possible anemia due to an iron-insufficient diet. Sullivan et al. (1985) proposed that Hb changes electrophoretically (i.e., different gene products or different processing of Hb proteins) from immature forms to mature forms during smoltification in coho salmon. Therefore, it is possible that the low Hb concentrations on SFRI-hatchery smolt are attributed to insufficient changes in the Hb proteins during smoltification, because of low dietary iron content.


Fig. 11. Condition factor (CF) in wild and hatchery-reared masu salmon at each sampling time of the 2003 and 2004 experiments. The letters 'a', 'b', 'c' and 'd' indicate significant differences between the wild, hatchery control, iron-1 and iron-2 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Asterisks show significant differences compared to the value of the previous sampling time within the same group (P < 0.05; Student's t-test). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

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Fig. 12. Total iron content of the food remnants in the stomach of wild and hatchery-reared masu salmon in May 2003. The letters 'a', 'b', 'c' and 'd' indicate significant differences between the wild, hatchery control, iron-1 and iron-2 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

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Fig. 13. Burst swimming capacity (BSC) of wild and hatchery-reared masu salmon in May 2003, April 2004 and May 2004. The letters 'a', 'b', 'c' and 'd' indicate significant differences between the wild, hatchery control, iron-1 and iron-2 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

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Fig. 14. Hemoglobin concentration (Hb) in wild and hatchery-reared masu salmon at each sampling time of the 2003 and 2004 experiments. The letters 'a', 'b', 'c' and 'd' indicate significant differences between the wild, hatchery control, iron-1 and iron-2 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

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Fig. 15. ATP content in the white muscle and liver in wild and hatchery-reared masu salmon at each sampling time of the 2004 experiment. The letters 'a', 'b' and 'c' show significant differences between the wild, hatchery control and iron-1 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

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Normal dietary levels of 100–250 mg iron/kg dry weight of food have been suggested for salmonids (Ogino et al. 1979; Desjardins et al. 1987; Andersen et al. 1996). Iron levels in the diet of Atlantic salmon ranged from 51 to 515 mg iron/kg dry weight of food. It is evident that the iron level of the control diet (7.96 × 102 mg iron/kg diet) was high compared to the "normal" salmonid dietary iron content. In the present study, there were no differences in the survival rate among control, iron-1 and iron-2 groups in the "2003 experiment" or between the control and iron-1 groups in the "2004 experiment" (data not shown), which suggests no effect of the supplemented iron on survival. This is in accord with previous results in Atlantic salmon (Bjørnevik and Maage 1993; Andersen et al. 1996, 1998) and rainbow trout (Carriquiriborde et al. 2004). However, my study did show that iron-1 supplement (1.51 × 103 mg iron/kg diet), but not iron-2 supplement (3.17 × 103 mg iron/kg diet), improved burst swimming capacity (Fig. 13) and increased Hb concentrations (Fig. 14) (P < 0.05, One-way ANOVA). Increased Hb concentrations in response to a suitable iron supplement has been demonstrated in many fish (Kawatsu 1972; Ikeda et al. 1973; Sakamoto and Yone 1978; Andersen et al. 1997; Carriquiriborde et al. 2004), whereas an excess of supplemented iron has induced lower Hb concentrations in rainbow trout (Standal et al. 1997) and Atlantic salmon (Andersen et al. 1997). Excessive iron has been shown to lead to the oxidation of dietary lipids and/or polyunsaturated fats, adversely affecting the quality of the diet (Desjardins et al. 1987). Baker et al. (1997) observed iron-induced hepatotoxicity following the accumulation of a lipid peroxidation product when diets were supplemented with iron sulfate (6300 mg iron/kg diet). Diets supplemented with iron sulfate (25 to 100, 175 to 1975 mg iron/kg diet) are effective in raising Hb concentrations to high levels in Atlantic salmon (Andersen et al. 1997) and rainbow trout (Carriquiriborde et al. 2004). Shiau and Su (2003) have revealed that iron citrate was only half as effective as iron sulfate in meeting the iron requirements of juvenile tilapia (Oreochromis niloticus). In contrast, Vielma et al. (1999) reported that citric acid stimulated the absorption of dietary iron. The effects of iron-supplements thus seem to depend on the iron source used, emphasizing the need to analyze the effects of a number of iron sources in order to develop a diet with suitable iron content for fish. ATP contents in the liver and the white muscle increased (P < 0.05, One-way ANOVA) (Fig. 15), when an iron-1 diet was administered. ATP is primarily produced by cellular respiration. Iron is an important element for the electron transfer chain, since it is essential for the tertiary folding of cytochrome, a protein playing a leading role in cellular respiration. Furthermore, the activation of cellular respiration requires considerable concentrations of H+ and electrons, which are provided in the form of NADH2+ or FADH2 from the citric acid cycle, which, in turn, depends on the concentrations of citric acid. Mammalian research has revealed that iron supplementation results in an increased ATP production in the form of cellular respiration and NADH2+ production by the citric acid cycle in vitro (Horst et al. 1999). If fish are assumed to have the same cellular energy metabolic system as mammals, supplemental iron citrate may trigger activation of both cellular respiration and the citric acid cycle, and increase the ATP content of the liver and white muscle.

This study revealed an improvement of burst swimming capacity after 3 months of iron-1 supplement (P < 0.05, One-way ANOVA) (Fig. 13), and a positive correlation between the burst swimming capacity and both Hb concentrations and ATP content in the white muscle before burst swimming (P < 0.05, Pearson's correlation coefficient) (Fig. 16). It is generally considered that burst swimming is exhibited by contraction of the white muscle. The contractile activity primarily depends on ATP generation from phosphocreatine and anaerobic glycolysis (Bone et al. 1978; Dobson et al. 1987; Altringham and Ellerby 1999). After burst swimming, the ATP concentration in the white muscle decreases (Wang et al. 1994). On the basis of the present results, it is suggested that the burst swimming capacity is dependent on the ATP content in the white muscle before burst swimming in masu salmon. Increased Hb concentration has been observed in a variety of species following burst swimming, and is related to the recovery from metabolic and respiratory acidosis during anaerobic glycolysis (Milligan and Wood 1987; Wang et al. 1994). Acidosis depends on swimming speed, at least in yellowtail (Tsukamoto and Chiba 1981). Cytochrome c oxidase activity in the white muscle shows the strongest correlation with burst swimming capacity, suggesting that aerobic preparation of white muscle facilitates a rapid contraction after burst swimming (Martínez et al. 2004). Thus, aerobic metabolism seems to be related to the recovery from burst swimming fatigue, and we suggest that this, in turn, depends on pre-swimming Hb concentrations. The improved burst swimming capacity seen after 3 months of iron-1 supplement therefore seems to be due to increased Hb concentrations and ATP content in the white muscle before burst swimming.


Fig. 16. Relationship between the means of burst swimming capacity (BSC) and hemoglobin concentration (Hb) and between the means of BSC and ATP content in the white muscle. For simple regression analysis between the means of BSC and Hb concentrations, data in May 2003, April 2004 and May 2004 were plotted. The mean BSC was found to be significantly correlated with the mean Hb concentration (r2 = 0.538, P = 0.015, Pearson's correlation coefficient) and the mean ATP content (r2 = 0.784, P = 0.019, Pearson's correlation coefficient). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

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In the large-scale 2004 experiment, there was no significant difference in burst swimming capacity (P > 0.05, One-way ANOVA) (Fig. 13) or in Hb concentrations (P > 0.05, One-way ANOVA) (Fig. 14) between the iron-1 and wild groups in April and May. On the other hand, an absence of difference in the white muscle ATP content was found only in May (P > 0.05, One-way ANOVA) (Fig. 15). These findings demonstrate that it takes at least 3 months during smoltification to completely improve swimming ability of SFRI-hatchery fish using the iron-1 diet. In consequence, this section concludes that a supplement of 2.5 g iron citrate per kg of food for 3 months prior to release is a convenient method for improving the seed quality of smoltifying SFRI-hatchery masu salmon.

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3. Development and application of techniques to evaluate seed quality in 0+ hatchery masu salmon parr for spring juvenile release

3-1. Introduction

The success of the spring juvenile release program depends on the existence of sufficient food to sustain the growth of the juveniles in the stream. Growth and survival in stream juveniles are apparently density-dependent (Hume and Parkinson 1987). Increased density of stocked fry and fingerlings can increase the mortality of wild juveniles by competition for food in streams (Lichatowich and McIntyre 1988). Therefore, it is important to monitor the nutritional condition of the stocked juveniles in order to evaluate the advantages of the spring juvenile release program. In 0+ masu salmon, liver triglyceride (TG) contents, the storage lipid used as energy source in fish, are an appropriate index for the evaluation of the nutritional condition in wild and hatchery fish, since decreased TG levels were found by artificial starvation (Misaka et al. 2004). However, TG levels in the liver are not enough to assess nutritional condition of the stocked juveniles.

In the kidney and spleen of fish, there are cells called melano-macrophages that resemble macrophages in their ultrastructure, contain high amounts of melanin-type pigments, and are thought to act in the metabolism of toxic compounds (Roberts 1975; Agius 1979). In Osteichthyes, greater numbers of melano-macrophages are present than in taxonomically lower fish, including Agnatha and Chondrichthyes (Roberts 1975; Agius 1979; Wolke 1992). In salmonids, melano-macrophages are randomly distributed and irregularly aggregated throughout the kidneys (Agius 1980). It has been suggested that increased numbers of melano-macrophages were related to humoral and inflammatory responses, storage, destruction and detoxification of exogenous and endogenous substances, and iron recycling (Wolke 1992). Furthermore, it has been reported that starvation induces increased melano-macrophage density (MMD) in the kidney of some teleosts, including rainbow trout (Agius and Roberts 1981). However, the mechanisms which induce the changes are not well understood, and the relationship between MMD and mortality is unknown in salmonids.

In Subsection 3-2, histological effects of artificial starvation on kidney MMD were observed to determine whether the MMD would be a useful indicator of the nutritional condition of wild and hatchery juveniles. Subsection 3-3 examined the relationships between liver TG content and kidney MMD levels, and between each of these 2 parameters and fish density from spring to summer in hatchery juveniles stocked into a stream in spring, in order to clarify whether MMD levels are a practical way to monitor the nutritional condition of the stocked juveniles.

3-2. Development of techniques to evaluate nutritional condition using kidney MMD levels in 0+ juveniles

0+ hatchery juveniles were released into a river where wild salmonids are not found in May. After 1 month, a part of the released hatchery juveniles were recaptured from the river. At the same time, 0+ wild juveniles were caught from a different river. The hatchery and wild juveniles were placed in separate tanks, and maintained without food under the same conditions. Hatchery and wild feeding-control groups were maintained in the same conditions over the two months of the experiment. Mortality rates were examined in each group, and fish were randomly sampled from each group during the experiment. The hatchery juveniles released in the river before the experiment were sampled as the experimental control. Kidneys were dissected from all samples. Paraffin sections of the kidney were observed with a light microscope. The MMD levels were expressed as the mean percentage of melanin granule area to whole kidney area.

All masu salmon had only melano-macrophages with dark brown pigments (Fig. 17). Oguri (1976, 1985) and Agius (1980) have reported both yellow lipofuscin pigments and dark brown melanin pigments in rainbow trout, and indicated that pigment composition in the kidney is sometimes variable between individuals of a single fish species. My results reveal that melano-macrophage of 0+ hatchery and wild fish have high levels of melanin. In addition, this study indicated that starvation increased MMD in both wild and hatchery fish (P < 0.05, One-way ANOVA) (Fig. 18). Roberts (1978) and Agius and Roberts (1981) suggested that melano-macrophage centre enlargement during starvation was associated with damage to tissues, including kidney and spleen, in some fish. Therefore, it is suggested that the increased MMD levels in masu salmon were also caused by the acceleration of kidney catabolism during starvation. In mammals, increased deposition of pigments has been observed in various organs during cachexia (Dubin 1955), and appears to involve the peroxidation of polyunsaturated lipids of subcellular membranes (Chio et al. 1969). However, it is unclear whether pigment formation during starvation in masu salmon is regulated by this same mechanism. It is well-known that various environmental factors affect MMD levels (Blazer et al. 1987). Wolke (1992) has revealed that using MMD levels to monitor hyponutrition remains questionable because the actual reasons for increased MMD levels are uncertain. On the other hand, Mizuno et al. (2011) found significant correlations between kidney MMD and liver TG content. Therefore, it is considered that MMD reflects the nutritional condition of 0+ masu salmon. There were no significant differences in the MMD levels between the fed hatchery group and the group sampled from the river with respect to MMD levels on days 15 and 45 of the experiment (P > 0.05, One-way ANOVA) (Fig. 18). No significant differences in MMD levels (P > 0.05, One-way ANOVA) were found between hatchery and wild fish for either the fed or starved groups in the present study (Fig. 18). In consequence, these results demonstrate that there are no effects of environmental factors or of the fish origin on MMD levels from spring to summer.


Fig. 17. Histological observations of melano-macrophage in the kidney of hatchery-reared and wild masu salmon. Panels 'a' and 'b' designate kidneys from hatchery-reared and wild fish respectively, at the start of the experiment (day 0). Panels 'c' and 'd' show kidneys from the starved groups of hatchery-reared and wild fish respectively, 45 days after the start of the experiment. Panels 'e' and 'f' reveal kidneys from the fed groups of hatchery-reared and wild fish, respectively, and panel 'g' shows a kidney from the hatchery-reared fish sampled from the river. Panels e, f and g were shot 45 days after the start of the experiment. The arrowheads indicate melano-macrophages. Scale bars show 50.0 μm. Reprinted from Aquaculture, 209, Mizuno et al., Effects of starvation on melano-macrophages in the kidney of masu salmon (Oncorhynchus masou), 247–255, © 2002, with permission from Elsevier.

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Fig. 18. Changes in the level of melano-macrophage deposition (MMD) during the experiment in starved (○) and fed groups (□) of hatchery-reared masu salmon, in starved (●) and fed groups (■) of wild masu salmon, and in the hatchery-reared fish sampled from the river (△). Asterisks show significant differences in the MMD level between the starved group and the fed group at the same sampling time in each fish group (P < 0.05, One-way ANOVA). Cross marks designate significant differences in the MMD level from the hatchery-reared group sampled from the river (P < 0.05, One-way ANOVA). Modified from Aquaculture, 209, Mizuno et al., Effects of starvation on melano-macrophages in the kidney of masu salmon (Oncorhynchus masou), 247–255, © 2002, with permission from Elsevier.

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It is important to note that mortality rate increases progressively in the starved groups of both hatchery and wild fish, while fish in both fed groups and the fish sampled from the river maintain low levels of mortality throughout the experiment (Fig. 19). Increased mortality was observed between 30 and 45 days, when the mean MMD level reached 0.5% in the starved groups of both hatchery and wild fish. Therefore, these results demonstrate that a level of MMD of 0.5% could be potentially used as an index of ultimate hyponutrition in juvenile masu salmon.


Fig. 19. Changes in mortality during the experiment in starved (○) and fed groups (□) of hatchery-reared masu salmon and in starved (●) and fed groups (■) of wild masu salmon. Modified from Aquaculture, 209, Mizuno et al., Effects of starvation on melano-macrophages in the kidney of masu salmon (Oncorhynchus masou), 247–255, © 2002, with permission from Elsevier.

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3-3. Evaluation of nutritional conditions using kidney MMD levels in 0+ hatchery juveniles after release

There is a 550 m section between two dams on the Kami-Utabetsu River in southern Hokkaido (Fig. 20), which wild masu salmon do not inhabit. Six sampling stations were established in this section to examine fish density and the nutritional condition of stocked juveniles (Fig. 20). The surface area and maximum depth of stations during the experiment ranged from 3.49 to 100 m2 and from 0.45 to 1.27 m, respectively. Previous to this study, no 0+ juveniles could be found in this section. On May 1, ten thousand 0+ hatchery juveniles were released at station 1 (Fig. 20). On May 30 and June 30, fish were caught at each station. Fish numbers were estimated by the double-pass removal method (Seber and LeCren 1967). Sampled fish were used for the analysis of kidney MMD levels and liver TG content.


Fig. 20. Map showing the Utabetsu River in southern Hokkaido and sampling stations on the Kami-Utabetsu River. Small rectangular area shows sampling area of the Kami-Utabetsu river. Modified from Sci. Rep. Hokkaido Salmon Freshwater Fish. Res. Inst., 1, Mizuno et al., Assessment of nutritional conditions using kidney melano-macrophage density in hatchery-reared juvenile masu salmon Oncorhynchus masou released into a stream, 49–53, © 2011, Hokkaido Salmon and Freshwater Fisheries Research Institute.

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0+ juveniles were recaptured at all stations in all sampling times except for station 5 on July 30. Estimated fish density ranged from 0.31 to 2.18 fish/m2 in May and from 0.0404 to 1.96 fish/m2 in July (Fig. 21). Growth increments in wild and released 0+ juveniles are reported to decrease as population density increases (Nagata 1989). Additionally, Hume and Parkinson (1987) reported that densities >0.7 fish/m2 in a stream of British Columbia, Canada, resulted in increased mortality in rainbow trout, which may indicate that there are some juveniles in hypo-nutritional conditions in the present study. Mean kidney MMD was between 0.192 and 0.524% in May and between 0.299 and 0.503% in July (Fig. 21). Mean liver TG levels were between 0.494 and 1.02% in May and between 0.408 and 0.787% in July (Fig. 21). A significantly negative correlation was found between mean liver TG levels and mean kidney MMD levels (r = 0.601, P < 0.05, Spearman's rank correlation) (Fig. 22). These results demonstrate that mean kidney MMD is a negative indicator of the nutritional conditions, from spring to summer of hatchery juveniles released into a stream. In juvenile masu salmon, the point at which dead fish are observed in the juvenile population in an artificial rearing environment, is at a mean kidney MMD > 0.5% (Mizuno et al. 2002). In the present study, some juvenile populations are on the verge of death in May and July according to that MMD limit. Therefore, there is a possible death of juveniles in the population in a natural habitat. The correlation between fish density and MMD was significant in May and July (May: r = 0.504, P < 0.05; July: r = 0.592, P < 0.05; Spearman's rank correlation) (Fig. 21). The correlation between fish density and TG levels were significant both in May and July (May: r = 0.567, P < 0.05; July: r = 0.339, P < 0.05; Spearman's rank correlation) (Fig. 21). These results strongly suggest that the nutritional condition of released juvenile masu salmon depends on fish density from spring to summer. Measurements of MMD possibly contribute not only to evaluating the nutritional condition of juveniles but also to finding the appropriate amount of food supply to juveniles in hatcheries.


Fig. 21. Relationships between fish density and liver triglyceride (TG) levels, and between fish density and kidney melano-macrophage density (MMD), in juvenile masu salmon. There were no fish caught at station 5 in July. Modified from Sci. Rep. Hokkaido Salmon Freshwater Fish. Res. Inst., 1, Mizuno et al., Assessment of nutritional conditions using kidney melano-macrophage density in hatchery-reared juvenile masu salmon Oncorhynchus masou released into a stream, 49–53, © 2011, Hokkaido Salmon and Freshwater Fisheries Research Institute.

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Fig. 22. Relationship between mean triglyceride (TG) levels in the liver and mean melano-macrophage density (MMD) in the kidney in juvenile masu salmon. The plots in this figure express the means of TG and MMD in juvenile population at each station and time. Spearman's rank correlation coefficient was used as statistical analysis. The dotted line shows the linear correlation [MMD] = 0.622–0.359 [TG] (r = 0.601, P < 0.05). There were no juveniles caught at station 5 in July. Modified from Sci. Rep. Hokkaido Salmon Freshwater Fish. Res. Inst., 1, Mizuno et al., Assessment of nutritional conditions using kidney melano-macrophage density in hatchery-reared juvenile masu salmon Oncorhynchus masou released into a stream, 49–53, © 2011, Hokkaido Salmon and Freshwater Fisheries Research Institute.

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4. Establishment of techniques to monitor the physical condition and elucidation of appropriate culture conditions in chum salmon fry

4-1. Introduction

In chum salmon fry, rearing at high densities causes poor feeding and growth (Nogawa and Yagisawa 1994) and decreases seawater adaptability (Ban 2000). A heavy mortality of fry, which is caused by a bacterial gill disease, has been annually reported in some hatcheries in Hokkaido (Nomura 1994). The bacterial gill disease is due to an infection by Flavobacterium branchiophilum, which is observed in all water of fry cultures, after environmental deterioration due to high density culture (Borg 1960; Bullock 1972; Larmoyeux and Piper 1973). It is important to monitor the aggravation of the physical condition in the fry caused by high density culture before the infection of F. branchiophilum, in order to prevent the incidence of the bacterial gill disease. However, a method to monitor physical conditions has not yet been established for chum salmon seed. It is commonly accepted that the Japanese standards of rearing conditions for chum salmon fry are <20 kg/m3 for rearing density and >6 mg/l dissolved oxygen (DO) concentration (Nogawa and Yagisawa 1994), which was determined according to a conversion formula of optimum rearing density for salmonid advocated by Westers and Pratt (1977). However, this standard is not based on clear physiological effects. Therefore, the relationship between practical rearing conditions and the physical condition of the fry has not been shown.

In Subsection 4-2, the impacts of high density culture on a variety of physiological parameters were studied, in order to establish a method to monitor the physical condition of hatchery chum salmon fry. In addition, physical condition was monitored in the fry produced by some hatcheries. Subsection 4-3 examined the relationships between rearing density, DO and physical condition of chum salmon fry in two hatcheries, in order to determine appropriate rearing conditions for chum salmon fry.

4-2. Development of techniques to monitor the physical condition of hatchery fry

Chum salmon fry were introduced into 3 tanks at densities of 10, 20 and 40 kg/m3. The three groups were reared for 42 days with food. Fry were sampled from the original tank at the initial time and from each of the 3 tanks on every seventh day. In the samples, CF was calculated after fork length and body weight (BW) were measured. The plasma and fish body were used for physiological analyses. At each sampling time, fish density, DO and un-ionized ammonia concentration (UIA) were measured in each tank. After the final sampling, tolerance to starvation was examined in each of the 3 groups.

A reduced BW was observed in only the 40 kg/m3 group during the experiment (P < 0.05, One-way ANOVA) (Fig. 23A). This phenomenon is supported by the majority of papers, which have demonstrated that there is an adverse effect of increasing density on growth in salmonids (Ellis et al. 2002), possibly resulting from a reduction in food intake (Leatherland 1993; Alanärä and Brännäs 1996) and food conversion efficiency (Logan and Johnston 1992) owing to the deterioration of rearing conditions. It has been generally accepted that rearing environments leading to a reduced growth in salmonids had rearing densities >50 kg/m3 (Mäkinen and Ruohonen 1990), DO levels <5 mg/l (Brett 1979) and UIA concentrations >40 μg/l (Maede 1985). Moreover, the DO of the 40 kg/m3 group was kept at >5 mg/l during the experiment (Fig. 24B). The peak of UIA in the 40 kg/m3 group was of only 0.552 μg/l, although the UIA increased in the 40 kg/m3 group during the experiment. In consequence, it is suggested that the primary environmental cause of the reduced growth in this study is rearing density, since only rearing density applied to the aforementioned conditions in the 40 kg/m3 group (Fig. 24A).


Fig. 23. Changes in body weight (A) and condition factor (B) of the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letters at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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Fig. 24. Changes in rearing density (A) and dissolved oxygen (DO) concentration (B) of the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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The results of the CF, a crude measure reflecting levels of energy reserves (Goede and Barton 1990), demonstrated that 40 kg/m3 culture conditions adversely affected the CF in chum salmon (P < 0.05, One-way ANOVA) (Fig. 23B). This may reflect the results of the fasting-tolerance test, where the 40 kg/m3 group showed significantly low tolerance to starvation compared to the 10 and 20 kg/m3 groups (P < 0.05, Kaplan-Meier method followed by a Log-rank test) (Fig. 25). Many previous studies have reported an adverse effect of density on the CF in salmon species, with the exception of chum salmon (Pickering and Pottinger 1987; Mäkinen and Ruohonen 1990). However, our study made it clear that high density cultures caused poor energetic reserves in chum salmon, much as in other salmon. It has been well documented that plasma cholesterol and glucose concentrations mirror the nutritional condition of fish (Kiron and Maita 2003). In the present study, the 40 and 20 kg/m3 group showed significantly lower plasma cholesterol (P < 0.05, One-way ANOVA) (Fig. 26) and glucose concentrations (P < 0.05, One-way ANOVA) (Fig. 27) after 21 days compared to the 10 kg/m3 group. Leatherland and Cho (1985) reported that high density inhibited the increase in plasma glucose levels after feeding in rainbow trout, which resulted from a reduced ability to find food. Maita et al. (1998) found that there was a significant negative correlation between plasma cholesterol concentrations and mortality due to disease, in rainbow trout. These results imply that high density causes poor nutritional and physical conditions in chum salmon fry.


Fig. 25. Changes in the survival rate in the three density groups during the fasting-tolerance test. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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Fig. 26. Changes in plasma cholesterol concentrations in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with different alphabetical letters at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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Fig. 27. Changes in plasma glucose concentrations in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with different alphabetical letter at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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In the 40 kg/m3 group, increased plasma cortisol levels, a part of the neuro-endocrine stress response, were observed (P < 0.05, One-way ANOVA) (Fig. 28), which demonstrated that high density causes the initial stress in chum salmon. Pickering and Pottinger (1987) reported that plasma cortisol levels were elevated in the first 6 and 10 days after exposure to high rearing density in brown trout (S. trutta) and rainbow trout, respectively. This information may reflect the fact the effects of density on the initial increase in plasma cortisol levels are species dependent. Corticosteroids, including cortisol, are potent immunosuppressants (Barton et al. 1987; Wedemeyer 1996). North et al. (2006) found negative correlations between plasma cortisol levels and lysozyme activity, a non-specific immune trait with bacteriolytic effects, in rainbow trout. However, the 40 kg/m3 group did not show high cortisol levels at 42 days (P > 0.05, One-way ANOVA) (Fig. 28), when plasma lysozyme activity significantly decreased (P < 0.05, One-way ANOVA) (Fig. 29). These results demonstrate that prolonged stress due to high densities causes a decline in the immune system response, with no increased cortisol levels in chum salmon. Røed et al. (1993) and Balfry et al. (1997) revealed that there were species-specific variations in the stress-related lysozyme activity in salmonid fish. Therefore, the differences in the results between North et al. (2006) and this study may result from the different salmonid species considered.


Fig. 28. Changes in plasma cortisol concentrations in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letter at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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Fig. 29. Changes in plasma lysozyme activity in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letter at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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In the present study, the 40 kg/m3 group showed that both the fastest increase and the slowest decrease in the somatic ATP content during the experiment, while increased later in the 20 kg/m3 group (P < 0.05, One-way ANOVA) (Fig. 30). Increased ATP content has been observed in masu salmon transforming from parr into smolt (Mizuno et al. 2007) and nutritionally supplemented chum salmon fry (Mizuno et al. 2008), which suggests that ATP content increases to satisfy the need for increased energy demand during the activation of metabolisms. However, my study found no evidence that increased ATP contents cooccurred with changes in plasma total cholesterol and glucose concentrations in the 40 and 20 kg/m3 groups (Figs. 27, 28, 30). On the other hand, an ATP-dependent system to excrete exogen substances has been found in teleost (Yanagi et al. 2004; Hirose and Nakada 2010). Accordingly, the increased ATP content found in the present study may be linked with increased energy demands resulting from an increased excretion of exogen substances as a result of a poor physical condition. Decreased ATP content has been shown to stop mobility in the sperm of bluegill Lepomis macrochirus (Burness et al. 2005). Therefore, the reduced ATP content found in the 40 kg/m3 group may demonstrate decreased vitality before death. Our study demonstrated that the 40 and 20 kg/m3 density groups had decreased AST transcription levels (P < 0.05, One-way ANOVA) (Fig. 31), one of the respiratory chain enzymes used to produce ATP aerobically. I suspect the respiratory-chain enzyme was negatively regulated by the ATP content, which is widely accepted in teleost (Burness et al. 2005). However, less ATP content and low AST transcription levels were found at the same time in the 40 kg/m3 group from 35 to 42 days (P < 0.05, One-way ANOVA) (Figs. 30, 31). This discrepancy may reflect that high densities disturb the balanced regulation between ATP content and transcription levels of respiratory chain enzymes.


Fig. 30. Changes in somatic adenosine triphosphate (ATP) content in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letters at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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Fig. 31. Changes in somatic ATP synthase transcription levels in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letters at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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Figure 32 shows a schematic representation of physiological changes during an acute aggravation of physical condition, which resulted from excessive rearing densities (>40 kg/m3). Values of physical condition indicators should be stable in physically good conditioned fish. The four parameters designated in this figure were not influenced by the duration of the experiment in the 10 kg/m3 group, which appeared to maintain the best physical condition during the experiment. It may be possible to estimate the physical condition of chum salmon fry using the stable parameter values of the 10 kg/m3 group as a baseline, because there was no effect of these parameters on fry growth during my experiment in the 10 kg/m3 group. The parameter baselines, which show the range of the mean value of the parameter during our experiment in the 10 kg/m3 group, are 11.4 to 19.4 ng/ml in plasma cortisol concentration, 8.16 to 21.0 pmol/g BW in somatic ATP content, 4.34 to 5.04 pmol competitor/μg total RNA in somatic AST transcription levels and 2.09 to 3.00 μg lysozyme/l in plasma lysozyme activity. The first sign of a bad physical condition was an increase in plasma cortisol concentration, followed by a dramatically increased ATP content and decreased AST transcription levels. Thereafter, the ATP content decreased, and finally lysozyme activity decreased. The 20 kg/m3 group, which was predicted to show a slower aggravation of physical conditions due to high density, showed an increased ATP content after 35 days and a decreased AST transcription level after 28 days. In contrast, increased cortisol concentrations and decreased lysozyme activity were not observed in the 20 kg/m3 group, which demonstrated that cortisol concentrations and lysozyme activity were not reliable parameters for estimating the physical condition of chum salmon fry. Consequently, this suggests that somatic ATP content and AST transcription levels are key parameters for monitoring the initial aggravation of the physical condition of chum salmon fry resulting from rearing at high densities, before the appearance of decreased growth and immune functions. The standard values of the ATP content and the AST transcription levels are regarded as 8.16 to 21.0 pmol/g BW and >4.34 pmol competitor/μg total RNA, respectively.


Fig. 32. Schematic representation of acute changes in physical condition during rearing at excessive density (>40 kg/m3). The four parameters designated in this figure were not influenced by the duration of the experiment in the 10 kg/m3 group, which appeared to maintain the best physical condition during the experiment. The first sign of an aggravated physical condition was an increase in plasma cortisol concentration and an increase in somatic ATP content and decrease in ATP synthase (AST) transcription levels. Thereafter, the ATP content and lysozyme activity decreased. Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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In order to routinely monitor the physical condition of hatchery fry for artificial propagation, hatchery fry were collected from 9 hatcheries (hatcheries I to IX) in different regions of Hokkaido, and wild fry were captured in southeastern Hokkaido in spring. Figure 33 shows the ATP content and AST transcription levels in the hatchery and wild fry. Mean ATP content and AST transcription levels ranged from 3.61 to 20.3 pmol/g BW and from 3.05 to 12.6 pmol competitor/μg total RNA, respectively. According to the standard values of both ATP content and AST transcription levels, fry from hatcheries I and II were regarded as being in bad physical condition, whereas fry from hatcheries III to IX and wild fry were considered in good physical condition, which shows that ATP content and AST transcription levels lead to the same results. Therefore, these results strongly demonstrate that ATP content and AST transcription levels are reliable parameters for estimating the physical condition of chum salmon fry.


Fig. 33. The somatic adenosine triphosphate (ATP) contents (left side) and ATP synthase transcription (AST) levels (right side) of hatchery-reared and wild chum salmon fry. The numbers I to IX represent the different hatcheries the chum salmon fry were collected at. Yellow areas indicate the range considered standard healthy values for each parameter.

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4-3. Appropriate culture conditions for hatchery chum salmon fry

Some ponds, containing introduced chum salmon fry were chosen in α and β hatcheries in Hokkaido, in April. Water temperature, pH, DO, UIA and fry density were examined in each pond. Fry were randomly sampled from each pond for the measurement of BW and the analyses of carcass ATP content and AST transcription levels.

In this study, the water temperature and pH of the ponds ranged between 5.2 and 9.0°C and between 6.50 and 6.83, respectively (Table 1). The UIA concentration was <0.1 μg/l in all ponds and the mean BW of the fry ranged from 0.81 to 2.36 g. Prior to this study, I had suggested that there was no effect of differences in water temperature between 5 and 10°C and in fry BW between 0.7 and 3.0 g on the two physical condition parameters analyzed in this study (Mizuno et al. 2008, 2010b). In chum salmon fry, negative physiological effects (unbalanced ion regulation) due to acid water have first been found at pH 5.0 (Watanabe et al. 1995). The lowest UIA concentration to show negative physiological impacts was 4 μg/l in salmonids (Maede 1985). Therefore, this information suggests that there was no effect of the different water temperatures, pH, UIA and fry body size on the two parameters of physical condition in the present study. The means of carcass ATP content and AST transcription levels ranged between 8.00 and 1.25 × 103 pmol/g BW and between 3.52 and 14.2 pmol competitor/μg total RNA, respectively (Table 1). According to the standard values of ATP content (8.16 to 21.0 pmol/g BW) and AST transcription levels (>4.34 pmol competitor/μg total RNA), α hatchery fry were regarded as being in bad physical condition in Ponds A, B and C on April 7, in Ponds C, D, E and F on April 21, and as being in good physical condition in Pond B on April 14 and 21. The fry in Pond C on April 14 were considered in impaired physical condition, since the AST transcription level was out of the range of physically good condition, while the ATP content level was within this range. This finding suggests that declined AST transcription levels precede increased ATP content during the aggravation of physical conditions in chum salmon fry. β hatchery-fry were considered in good physical condition in all ponds.


Table 1 Pond conditions and water quality, and condition of the chum salmon fry. Modified from Aquaculture Science, 58, Mizuno et al., Relationship between rearing conditions and health in chum salmon (Oncorhynchus keta) fry, 529–531, © 2010, Japanese Society for Aquaculture Research. Body weight (n = 15), somatic ATP content (n = 5) and somatic ATP synthase transcription level (n = 5) shown as means ± standard errors.

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Figure 34 shows the relationships between each of the two parameters of physical condition, rearing density and DO. The area regarded as a physically good condition for carcass ATP content (Fig. 34A) and AST transcription levels (Fig. 34B), was mainly found at rearing densities <30 kg/m3 and at DO levels >8 mg/l. Accordingly, these results demonstrate that appropriate rearing conditions for the culture of hatchery chum salmon fry in good physical condition were with rearing densities <30 kg/m3 and with DO levels >8 mg/l.


Fig. 34. Relationships between rearing density, dissolved oxygen concentration (DO) and somatic ATP content (A), and between rearing density, DO and somatic ATP synthase transcription levels (B). X- and Y-axes show rearing density and DO respectively. Contours represent somatic ATP content in the A graph and carcass ATP synthase transcription levels in the B graph. Modified from Aquaculture Science, 58, Mizuno et al., Relationship between rearing conditions and health in chum salmon (Oncorhynchus keta) fry, 529–531, © 2010, Japanese Society for Aquaculture Research.

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5. Development of techniques to improve the survival rate during the artificial propagation of osmerids

5-1. Introduction

Naturally spawned eggs of both shishamo and Japanese smelt stick to the surface of fine gravel and/or aquatic plants in the bottom of the river with adhesive proteins on the surface of the "inverted adhesive membrane", a part of the egg membrane (Fig. 35A). In intensive egg culture using jar incubators (Fig. 35B), the egg adhesiveness is eliminated with a tannic acid solution treatment just after artificial fertilization (Waltemyer 1976) to prevent clumping of adhesive eggs, which causes high mortality accompanied by suffocation and fungal growth (Doroshof et al. 1983). Adhesive proteins on the inverted adhesive membrane lose adhesiveness by treatment with tannic acid, which has functions in coagulating proteins. However, tannic acid-treated eggs show lower hatching rates. The low hatching rate is more conspicuous in the tannic acid-treated eggs cultured under iron-enriched environments (Takeda et al. 2002). Therefore, it is necessary to establish new methods to eliminate egg adhesiveness in order to improve the hatching rate in osmerids. Previous studies have reported methods to eliminate egg adhesiveness with mud in Japanese dace (Triborodon hakonensis) (Nakamura 1962) and with river silt in white sturgeon (Acipenser transmontanus) (Doroshof et al. 1983). Egg adhesiveness is lost when the adhesive portion of the egg is completely covered with mud or silt just after artificial fertilization. On the other hand, mud and river silt are not edible and the public sentiment on food safety is rising year after year in Japan. It is therefore necessary to consider the adoption of food or food additives for the development of new methods to eliminate egg adhesiveness.


Fig. 35. Osmerid egg (A) and jar incubators for culturing the eggs (B). Panel A shows a Japanese smelt egg cultured at 10°C for one day. Arrowhead shows the inverted adhesive membrane, a part of egg membrane. Scale bar shows 1.00 mm. Panel B shows 6l-jar incubators in Mukawa hatchery, southern Hokkaido.

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Kaolin, which is composed of Al3Si2O5(OH)4 , is a natural mineral clay and a certified food additive for cosmetics and medicines. This powder is a potential material for the elimination of egg adhesiveness, as it has a very low solubility. In the meantime, the success of artificial propagation of the Yezo giant scallop, Patinopecten yessoensis, has led to the present-day dramatic increase of its catch in Hokkaido. Increasing numbers of scallop shells, which are discharged during scallop processing, are serious problems of industrial waste (Kono et al. 2000). Some of the shells are crushed into a powder, which is used as a food additive for calcium supplements (Yamagishi et al. 2007; Liu et al. 2008). The scallop shell powder (SSP) is as insoluble in water as kaolin is. Therefore, it is also possible to utilize SSP as a material to eliminate egg adhesiveness.

Some shishamo hatcheries have to transport and release eyed-stage embryos into rivers, because the water outlets of hatcheries do not connect with rivers allowing the release of seeds. Naturally spawned shishamo smelt eggs, attached to small gravel are forced to flow from the river to the sea by snow-melt water (Omi 1978b). It is probable that eyed-stage embryos are exposed to seawater as soon as they are released. Accordingly, it is essential that only embryos with high seawater adaptability are released for the success of artificial propagation of shishamo smelt. It is well-known that there are many mitochondrion-rich cells, which play a main role in seawater adaptation after hatching, on the yolk sac membrane (Hayano et al. 1999). However, it is not clear which embryo stage has the best seawater tolerance. In consequence, appropriate time for releasing eyed-stage embryos is unknown for the Hokkaido shishamo hatcheries.

Subsection 5-2 examined whether kaolin suspension treatment eliminates the egg adhesiveness and results in a high hatching rate under an iron-enriched environment in shishamo smelt. In Subsection 5-3, the effects of treatment using SSP suspension on eliminating egg adhesiveness and hatching rate in Japanese smelt eggs were studied in order to demonstrate whether SSP is a useful material for eliminating egg adhesiveness. Finally, Subsection 5-4 investigated the development of seawater adaptability during embryogenesis to elucidate appropriate timing for the release of eyed-stage embryos in shishamo smelt.

5-2. Technique for eliminating egg adhesiveness using a kaolin suspension to achieve high hatching rates of shishamo smelt in an environment with a high iron concentration

The first experiment was performed to determine the appropriate kaolin concentration and treatment period for eliminating egg adhesiveness. All 25 individual kaolin concentrations (0.50, 1.0, 2.0, 5.0 and 10.0 g/l) and treatment periods (10 seconds, 1, 5, 15 and 30 minutes) were performed by adding a kaolin suspension to small-numbers fertilized eggs placed in 25 small acrylic plates. Treatments with river water (non-treatment) and a tannic acid solution were performed as experimental controls. After the acrylic plate containing the clump of eggs was laid on a petri dish, river water flowed gently over the dish. The rate of egg adhesiveness elimination is expressed as a percentage of the number of the eggs separated from the surface of the plate or dish over the total number of eggs. In the second experiment, the effects of the kaolin treatment on the survival rate of the eggs, hatching rate, mortality during hatching and seawater tolerance of larvae were investigated. A large-number of fertilized eggs were equally divided into 3 groups treated by river water as non-treatment, 5.0 g/l kaolin for 5 minutes and tannic acid. In the non-treatment group, the eggs were stuck to the acrylic plates. Each of the 3 groups was placed in a small acrylic mesh bag and reared in a 100l jar incubator at the Mukawa hatchery until just before hatching. Eggs were periodically sampled from each of the 3 groups, observed under a light microscope and used to investigate survival rate, hatching rate, mortality during hatching and egg pressure. Hatched larvae of each group were used for the experiment on seawater tolerance. Additionally, water from the Mukawa hatchery and from SFRI was collected to determine total iron concentrations.

Observation of the eggs revealed that the inverted adhesive membrane does not invert in the kaolin-treated eggs, as in the tannic acid-treated eggs (Fig. 36). These findings suggest that suspended kaolin particles completely cover the adhesive portion of the egg before the membrane inverts. The borderline between inverted adhesive membrane and the egg membrane just after fertilization was evident in the kaolin-treated eggs but not in the tannic acid-treated eggs (Fig. 36). Kaolin suspension treatment is a physical method of covering adhesive materials, whereas tannic acid treatment is a chemical method for solidifying the adhesive materials, as described by Kusuda and Teranishi (1996). Accordingly, it is suggested that the difference in the borderline appearance is caused by different mechanisms eliminating egg adhesiveness in kaolin and tannic acid treatments. In the first experiment, the kaolin suspension treatment showed a higher elimination of egg adhesiveness at a 5 g/l concentration for 5 to 30 minutes, and at a 10 g/l concentration for 10 seconds to 30 minutes, compared with the tannic acid treatment (P < 0.05, Chi-square test for independence) (Fig. 37). In consequence, the most effective treatment is to use a kaolin concentration of 5 g/l and a period of 5 minutes to reduce the cost and labor required.


Fig. 36. Observation of the eggs after non-treatment (A and D), tannic acid treatment (B and E) and kaolin treatment (C and F). The upper and lower halves show eggs on November 21, 2003 and March 23, 2004, respectively. The arrowheads indicate borderline between the egg membrane and the inverted adhesive membrane. Scale bar indicates 1.00 mm. Reprinted from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

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Fig. 37. Effects of the kaolin suspension treatment on elimination of egg adhesiveness in the first experiment. The letters 'a' and 'b' indicate significant differences in the value compared to the non-treatment and the tannic acid treatment, respectively (P < 0.05; Chi-square test for independence). Reprinted from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

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The total iron concentration in the river water of Mukawa hatchery was between 0.542 and 2.04 mg/l during this study (Fig. 38). In the rivers of Hokkaido Prefecture, the normal concentration is of 0.1 mg/l or less (Atoda and Imada 1972). According to Japanese standards on water quality for freshwater aquaculture, the suitable total iron concentration is also of 0.1 mg/l or less (Japan Fisheries Resource Conservation Association 2000). Therefore, the high iron concentration in Mukawa hatchery is very peculiar. The total amount of iron on the surface of eggs increased during this study regardless of the treatment types applied to eliminate egg adhesiveness (P < 0.05, One-way ANOVA) (Fig. 39). The amount of iron on the tannic acid-treated eggs was significantly larger than that on the kaolin-treated and the non-treated eggs, from February 25 to March 23, 2004 just before hatching (P < 0.05, One-way ANOVA). Therefore, these results demonstrate that the kaolin-suspension treatment reduces the amount of iron that binds to the egg surface compared with the tannic acid treatment. This variation between kaolin and tannic acid is probably due to difference in their chemical property: tannic acid can chemically bond iron, whereas kaolin cannot. Kaolin treated eggs showed significantly high hatching rates and low mortality during hatching compared to the tannic acid treated eggs (P < 0.05, One-way ANOVA) (Figs. 40A, B). However, there was no difference in the survival rate of eggs among kaolin treated, tannic acid treated and non-treated eggs on February 25 and March 1 and 23 (P > 0.05, One-way ANOVA). Accordingly, these results suggest that the low survival rate in the tannic acid-treated eggs is caused by an increased mortality during hatching. In the aquaculture of salmon, increased mortality during hatching is sometimes observed in the eggs with hardened membranes, produced after exposure of eggs with soft egg disease to a green tea treatment (Sasaki and Yoshimitsu 2008). In the present study, the egg pressure in tannic acid-treated egg was significantly higher than that of non-treated eggs just before hatching (P < 0.05, One-way ANOVA) (Fig. 41). This result reveals that the hardening of the egg membrane by the tannic acid treatment accounts for the increased mortality during hatching in this group of eggs. Takeda et al. (2002) discovered that an increased iron content on eggs is correlated with low hatching rates. Therefore, an increased amount of iron on the surface of the eggs is also possibly involved in the hardening of the tannic acid-treated egg membrane. Furthermore, there was no significant difference in the egg pressure between kaolin treated eggs and non-treated eggs (P > 0.05, One-way ANOVA) (Fig. 41), which reveals that kaolin suspension treatment does not raise egg pressure. Additionally, the present study showed no difference in the seawater tolerance of larvae among the 3 groups (P > 0.05, One-way ANOVA) (Fig. 40C), which shows that the kaolin treatment does not impact the seawater adaptability of larvae. In consequence, we found that a kaolin suspension treatment at 5 g/l concentration for 5 minutes is effective for eliminating egg adhesiveness and improving hatching rates in shishamo smelt.


Fig. 38. Changes in total iron concentration in the river water of the Mukawa hatchery and the Salmon and Freshwater Fisheries Research Institute during this experiment. Modified from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

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Fig. 39. Changes in the total amount of iron on the egg surface in the non-treated, tannic acid-treated and kaolin-treated groups during the experiment. Asterisks express significant differences compared to the initial value of each group (P < 0.05; One way ANOVA). Cross marks show significant differences compared to the value of non-treatment group at the same sampling time (P < 0.05; One way ANOVA). The letter 'a' indicates significant differences with the value of the kaolin-treated group at the same sampling time (P < 0.05; One way ANOVA). Modified from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

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Fig. 40. Hatching rate (A), mortality during hatching (B) and survival rate of larvae after the seawater transfer (C) in non-treated, tannic acid-treated and kaolin-treated eggs. Columns with different alphabetical letters were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

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Fig. 41. Egg pressure in the non-treated, tannic acid-treated and kaolin-treated groups. Columns with different alphabetical letter were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

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5-3. Application of SSP as a material for eliminating egg adhesiveness in Japanese smelt

For the first experiment, a small-number of fertilized eggs were equally placed into seven acrylic dishes. Spring water (control), 5.0 g/l kaolin suspension and 1.0, 5.0, 10.0, 20.0 and 50.0 g/l SSP suspensions were poured into the seven dishes. All eggs were divided into two groups, adhesive and separated eggs, in the dish. The elimination rate of egg adhesiveness was calculated as the percentage of separated eggs over the total number of eggs in each dish. The eggs were cultured until completion of hatching and the hatching rate was examined in each group. For the second experiment, a large number of fertilized eggs were equally divided into four groups. Each of the four groups were treated with spring water (control), 5.0 g/l kaolin suspension, 5.0 or 20.0 g/l SSP suspension for 10 seconds and separately placed in small net bags. The four bags were kept in a 6l jar incubator. Eggs were sampled 20 days after fertilization for the analysis of survival and hatching rates and for observation with a light microscope.

The first experiments demonstrated that a treatment by more than 5 g/l SSP was effective in eliminating egg adhesiveness, compared to the conventional 5 g/l kaolin treatment (P < 0.05, Chi-square test for independence) (Fig. 42). It is generally accepted that the solubility of kaolin in water is 10 mg/l at 25°C, which is quite similar to the solubility of CaCO3 (15 mg/l) (Iguchi et al. 2001a). The specific gravity of kaolin is 2.6 g/cm3 (Iguchi et al. 2001b), while that of SSP is 2.7 g/cm3. This information suggests that the physical properties of SSP resemble that of kaolin. The hatching rates after all SSP treatments were not significantly different from that after the kaolin treatment (P > 0.05; Chi-square test for independence) (Fig. 43). This suggests that none of the SSP concentrations used had a bad influence upon the hatching rate.


Fig. 42. Egg adhesiveness elimination rate in Japanese smelt eggs treated with freshwater (control), kaolin suspension or SSP suspensions in the first experiment. Different alphabetical letters showed significant differences in the hatching rate between groups (P < 0.05; Chi-square test for independence). Modified from Aquaculture Science, 58, Mizuno et al., Effects of treatment using unbaked scallop shell powder suspension on eliminating egg adhesiveness, hatching rate and larval quality in Japanese smelt (Hypomesus nipponensis) eggs, 97–104, © 2010, Japanese Society for Aquaculture Research.

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Fig. 43. Hatching rate of Japanese smelt eggs treated with freshwater (control), kaolin suspension or SSP suspensions in the first experiment. Different alphabetical letters showed significant differences in the hatching rate between groups (P < 0.05; Chi-square test for independence). Modified from Aquaculture Science, 58, Mizuno et al., Effects of treatment using unbaked scallop shell powder suspension on eliminating egg adhesiveness, hatching rate and larval quality in Japanese smelt (Hypomesus nipponensis) eggs, 97–104, © 2010, Japanese Society for Aquaculture Research.

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Observation of the eggs showed that the inverted adhesive membrane was spread like a parachute in the control group, whereas it was rumpled in the kaolin- and SSP-treated groups (Fig. 44). These differences between the control and all suspension-treated groups probably reflect that SSP or kaolin stuck to the adhesive part and eliminated egg adhesiveness. Kaolin suspension treatment inverted the inverted adhesive membrane in Japanese smelt eggs in this study, while it did not invert the membrane in shishamo smelt (Mizuno et al. 2004b). This difference between Japanese and shishamo smelt possibly depends either on variations in the characteristics of the proteins of the inverted adhesive membrane, in the speed it takes to invert the inverted adhesive membrane after fertilization and/or in treatment time.


Fig. 44. Observation of freshwater-treated (A, control), 5 g/l kaolin-treated (B), 5 g/l (C) and 20 g/l (D) SSP-treated embryos 20 days after fertilization in the second experiment. Arrows indicate inverted adhesive membrane. Scale bars designate 1.0 mm. Control eggs (A) were unfastened from the palm tree skins before their observation. Modified from Aquaculture Science, 58, Mizuno et al., Effects of treatment using unbaked scallop shell powder suspension on eliminating egg adhesiveness, hatching rate and larval quality in Japanese smelt (Hypomesus nipponensis) eggs, 97–104, © 2010, Japanese Society for Aquaculture Research.

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In the second experiment, there were no significant differences in the survival rate and hatching rate among control, kaolin, 5 g/l and 20 g/l SSP (P > 0.05; One-way ANOVA). These results demonstrate that SSP treatments can be practically used for the elimination of egg adhesiveness in intensive egg cultures using jar incubators. On the other hand, it is necessary to keep in mind the internal particulate damage in the use of minute particles such as SSP, which implies that the use of as few particles as possible is safer. Therefore, we should use a 5 g/l SSP treatment, which is the minimum SSP concentration for effective treatment, for the elimination of egg adhesiveness.

5-4. Appropriate timing for the release of eyed-stage embryos in shishamo smelt

The present study used eggs spawned naturally on the bottom of the tank by homing-migrating adults in Mukawa hatchery, which releases eyed-stage embryos into the Mukawa River. According to previous studies of the embryogenetic stages of shishamo smelt (Hikita 1958), the embryogenetic stage was regarded as stage 15 when a lens appeared in the optic vesicles on February 10, stage 16 when the end of embryo's tail did not reach its head and the color of optic vesicles began darken on February 25, stage 17 when it's tail reached its head on March 10, stage 18 when the color of the optic vesicles darkened completely and the embryo tail overlapped its head on March 23 and stage 19 when the embryo moved in the egg and a silver color became distinct in the eyes on April 2 by observation with a light microscope (Fig. 45). Stages 15, 16, 17, 18 and 19 corresponded to 133, 140, 154, 188 and 243°C in cumulative temperature, respectively. Live eggs in each stage were divided into 3 groups. Each group was put into either freshwater, 17.0 psu artificial brackish water or 34.0 psu artificial seawater. Hatching rate was examined in each of the 3 dishes. Additionally, live eggs were used for the analysis of Na+,K+-ATPase activity at each stage.


Fig. 45. Observation of the eyed-stage embryos used in this study. Panels (a), (b), (c), (d) and (e) show embryos at 133 (Stage 15), 140 (Stage 16), 154 (Stage 17), 188 (Stage 18) and 243°C (Stage 19) cumulative temperature, respectively. The arrowheads designate the position of the end of the tail. Scale bar shows 1.00 mm. Reprinted with permission of John Wiley & Sons, Inc. from Aquaculture Research, 36, Mizuno et al., Changes in seawater tolerance during the development of eyed-stage embryos in shishamo smelt Spirinchus lanceolatus (Hikita), 615–619, Fig. 1, © 2005, Wiley-Liss, Inc., a Wiley Company.

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Figure 46 indicates that the hatching rate increased significantly during embryogenesis in both 17 psu brackish water and 34 psu seawater. In response to the increased hatching rate, egg Na+,K+-ATPase activity also increased significantly during embryogenesis (P < 0.05, One-way ANOVA) (Fig. 47). Correlation analyses between the hatching rate and Na+,K+-ATPase activity showed there are significant unlinear positive correlations between these 2 parameters under both 17 psu brackish water and 34 psu seawater environments (17 psu: r = 0.903, P < 0.05; 34 psu: r = 0.833, P < 0.05; Pearson's correlation coefficient) (Fig. 48). These results suggest that increased egg Na+,K+-ATPase activity during embryogenesis is linked to the increased hatching rate in seawater environments during embryogenesis. Hayano et al. (1999) have reported that the activation of mitochondrion-rich cells on the yolk-sac membrane of embryos is linked to the development of seawater adaptability of the embryo of shishamo smelt, since immunoreactions against anti Na+,K+-ATPase α-subunit were enhanced in the cells, and cell size became larger during seawater adaptation. The activation of mitochondrion-rich cells on the yolk-sac during seawater adaptation is also observed in chum salmon (Kaneko et al. 1993) and tilapia (Ayson et al. 1994, Shiraishi et al. 1997). Larvae of shishamo smelt hatch with a yolk sac and without complete gills (Hikita 1958), which are one of the main osmoregulatory organs in fish (Payan et al. 1984). Accordingly, increased egg Na+,K+-ATPase activity in shishamo smelt is probably caused by the activation of Na+,K+-ATPase on the yolk sac membrane.


Fig. 46. Impact of seawater environments on the hatching rate of embryos at various embryogenetic stages. The letter 'a' shows significant differences in the hatching rate compared to 133°C (Stage 15) in each environment (P < 0.05; Chi-square test for independence). The letter 'b' designates significant differences in the rate compared to the freshwater group at each embryogenetic stage (P < 0.05; Chi-square test for independence). The letter 'c' expresses significant differences in the rate between 17 psu brackish water and 34 psu seawater at each embryogenetic stage (P < 0.05; Chi-square test for independence). Modified with permission of John Wiley & Sons, Inc. from Aquaculture Research, 36, Mizuno et al., Changes in seawater tolerance during the development of eyed-stage embryos in shishamo smelt Spirinchus lanceolatus (Hikita), 615–619, Table 1, © 2005, Wiley-Liss, Inc., a Wiley Company.

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Fig. 47. Changes in egg Na+,K+-ATPase activity during the development of eyed-stage embryos. Asterisks indicate significant differences in the activity compared to 133.3°C (Stage 15) (P < 0.05; One-way ANOVA). Modified with permission of John Wiley & Sons, Inc. from Aquaculture Research, 36, Mizuno et al., Changes in seawater tolerance during the development of eyed-stage embryos in shishamo smelt Spirinchus lanceolatus (Hikita), 615–619, Fig. 2, © 2005, Wiley-Liss, Inc., a Wiley Company.

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Fig. 48. Relationships between mean egg Na+,K+-ATPase activity and hatching rate in freshwater (FW), 17 psu brackish water (BW) and 34 psu seawater (SW) environments of various eyed-stage embryos. Modified with permission of John Wiley & Sons, Inc. from Aquaculture Research, 36, Mizuno et al., Changes in seawater tolerance during the development of eyed-stage embryos in shishamo smelt Spirinchus lanceolatus (Hikita), 615–619, Fig. 3, © 2005, Wiley-Liss, Inc., a Wiley Company.

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The hatching rate in freshwater was very high at all embryogenetic stages, with the brackish water and seawater groups showing lower hatching rates at most embryogenetic stages (P < 0.05; Chi-square test for independence) (Fig. 46). However, the rates with 17 psu brackish water were equal to the rates of the freshwater group at stages 18 and 19 (P > 0.05; Chi-square test for independence) (Fig. 46). Consequently, these results demonstrate that eyed-stage embryos of shishamo smelt have no complete 34 psu seawater tolerance at any embryogenetic stage, while they have perfect tolerance to 17 psu brackish water after stage 18. If eyed-stage embryos are released into the river for artificial propagation of shishamo smelt, we should release embryos after at least stage 18.

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6. General discussion: Towards a sustainable artificial propagation of salmonids and osmerids

6-1. Implications of this monograph for artificial propagation

In Section 2, a quantitative system of dorsal fin pigmentation during smoltification was first established in smolting masu salmon, and a standard value was determined for the peak of smoltification (Subsection 2-2). The SFRI-hatchery smolts showed good seed qualities using dorsal fin pigmentation as external quality proxy and gill Na+,K+-ATPase activity as internal quality proxy (Subsection 2-2). If the level of dorsal fin pigmentation is routinely examined in SFRI-hatchery masu salmon, the hatchery workers can accurately determine the peak of smoltification without the slaughter of seed. On the other hand, Subsection 2-3 revealed that SFRI-hatchery smolt seed had some problems regarding the quality of the carbohydrate metabolism, citric acid cycle and respiratory chain (Mizuno et al. 2012). Subsection 2-4 indicated that SFRI-hatchery smolt had defects in seed quality such as anemia, low energy levels and slow swimming performances. These problems were solved by supplying diets supplemented with iron citrate for 3 months prior to release (Subsection 2-4). At present, this technique is adopted to improve seed quality of the SFRI-hatchery smolt. As a result of this, SFRI has succeeded in producing high-quality seed hatchery smolt. The improved hatchery seed are tentatively released from public hatcheries to confirm the effects of releasing the improved smolt on the recovery rate of adults. The upward tendency of the recovery rate is found in some hatcheries (Hokkaido Fish Hatchery 2008). If positive effects of releasing improved hatchery seed on the recovery rate are confirmed, techniques to improve the seed quality of hatchery smolt will prevail among all hatcheries in Hokkaido. In addition, the author has also elucidated that diets supplemented with iron are effective in enhancing the metabolism of chum salmon (Mizuno et al. 2008).

Section 3 demonstrated that the mean level of kidney MMD is a practical indicator to reflect the nutritional condition from spring to summer in hatchery juveniles released by the spring juvenile release method (Subsections 3-2, 3-3). Subsection 3-3 showed that the nutritional condition of released hatchery juveniles has a negative correlation with fish density in the river. At present, this technique is applied to estimate the appropriate number of hatchery seed for spring juvenile release in each river by monitoring the nutritional condition of the seed after release (Mizuno et al. 2011). In the near future, this research should contribute to determining, from a scientific point of view, the appropriate number of hatchery seed to be released in all rivers of Hokkaido.

In Section 4, ATP content and AST transcription level were used as parameters to monitor the physical condition of hatchery chum salmon fry (Subsection 4-2). Monitoring the physical condition using these indices, Subsection 4-3 revealed that appropriate rearing conditions for chum salmon fry culture were rearing densities <30 kg/m3 and at DO levels >8 mg/l. At present, these techniques are used to monitor the physical condition of chum salmon fry produced by some private hatcheries (Mizuno et al. 2010c). If the physical condition of hatchery fry is diagnosed as being bad, the SFRI can coach the private hatcheries, so as to culture fry according to known appropriate rearing conditions.

In Section 5, new methods to eliminate egg adhesiveness were established in order to improve the hatching rates, using kaolin in shishamo smelt and SSP in Japanese smelt (Subsections 5-2, 5-3). Furthermore, Subsection 5-4 showed that appropriate time for releasing eyed-stage embryo of shishamo smelt is from embryogenetic stage 18. At present, these techniques are commonly used by some private hatcheries of shishamo and Japanese smelt (Mizuno et al. 2010a) because the SFRI popularized the techniques through workshops. As a result of this, my techniques are connected with an improvement of the hatching rates and survival rates after release during the artificial propagation of osmerids.

Consequently, this section manifested that my studies can, in a practical way, contribute to the development of the artificial propagation of salmonids and osmerids.

6-2. Perspectives for studies on the improvement of seed production techniques

In masu salmon, it is important to find and resolve the physiological factors influencing seed quality, in order to provide more effective artificial propagation in the future. The author reported ultrastructural changes in gill mitochondrion-rich cells (Mizuno et al. 2000), increased transcription levels of gill cortisol receptors (Mizuno et al. 2001a), an increased number and increased hormonal regulation of kidney juxtaglomerular cells (Mizuno et al. 2001b, c) during smoltification in masu salmon. An impending study is to test the differences in these physiological parameters between hatchery and wild fish. Additionally, Maynard et al. (1996) reported that diets supplemented with aquatic insects can increase the post-release foraging effectiveness of hatchery chinook salmon (O. tshawytscha). Olla and Davis (1989) found that learning predation and not being handled enhanced predator avoidance in hatchery coho salmon. Munakata et al. (2000) suggested that post-release adaptation to the natural environment affected endocrine control by growth hormone and exocrine pancreatic activity. Thus, it is also necessary to study seed quality related to some physiological factors, social behavior, learning ability and the relations between endocrine factors and post-release behavior, for the success of artificial masu salmon propagation. Additionally, high mortality in eyed-stage embryos (Misaka et al. 1998a) and high sensitivity to viral and bacterial diseases (Kimura 1994; Yoshimizu 1994) are also notable problems in the seed of masu salmon. It is also important to elucidate whether these embryogenetic and pathological problems impact on the seed quality of masu salmon.

In chum salmon, production of the best physically conditioned seed will be desired in the future. The technique to monitor the physical condition of chum salmon fry is not always commonly used in all private hatcheries in Hokkaido. Accordingly, it is the coming challenge to simplify the techniques of that everyone can monitor the physical condition of fry. In addition, countermeasures against ectoparasitic protozoans (Urawa and Awakura 1994) and some bacterial diseases (Nomura 1994) are also notable assignments, because these diseases can not always be eradicated. It is also important to establish a treatment for these diseases and to clarify the impacts of the diseases on the physical condition of chum salmon fry.

In Japanese smelt, transplantation of eggs from Hokkaido to Honshu, the central part of Japan, has been performed until now (Ikeda 2008). However, it has recently been suggested that the artificial propagation of osmerids possibly causes a decrease in the genetic diversity of wild osmerid populations (Ikeda 2008). For the sustainable artificial propagation of osmerids, the propagation should be performed locally at first. Secondly, improvement of the seed quality should be more important than the increase in the number of released seed in future artificial propagation of osmerids. It is essential to establish techniques to perfectly evaluate seed quality in shishamo and Japanese smelt in the future. Additionally, egg saprolegniasis, which breaks out from dead eggs, has been a serious problem (Izuka 2005) and the provision for saprolegniasis is an urgent topic for the improvement of seed production techniques in osmerids. It is very important to study whether the new methods of eliminating egg adhesiveness prevent saprolegniasis.

Consequently, I conclude that it is essential for the sustainable artificial propagation in salmonids and osmerids to promote studies on the improvement of seed production techniques.

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Acknowledgments

I am grateful to Dr. Katsumi Aida, Professor Emeritus at the University of Tokyo, for his extensive support and encouragement for the preparation of this monograph. I also acknowledge the technical direction and valuable discussion of a staff member of the Salmon and Freshwater Fisheries Research Institute, and staff members of Hidaka Salmon Propagation Association, Mukawa Fishery Cooperative Association and Lake Toya Fishery Cooperative Association, for their assistance in sample collection.

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References

Agius C. The role of melano-macrophage centres in iron storage in normal and diseased fish. J. Fish Dis. 1979; 2: 337–343.

Agius C. Phylogenic development of melano-macrophage centres in fish. J. Zool. Lond. 1980; 191: 11–31.

Agius C, Roberts RJ. Effects of starvation on the melano-macrophage centres of fish. J. Fish Biol. 1981; 19: 161–169.

Alanärä A, Brännäs E. Dominance in demand feeding behavior in Arctic charr and rainbow trout: the effects of stocking density. J. Fish Biol. 1996; 48: 242–254.

Altringham JD, Ellerby DJ. Fish swimming: patterns in muscle function. J. Exp. Biol. 1999; 202: 3397–3403.

Andersen F, Maage A, Julshamn K. An estimation of dietary iron requirement of Atlantic salmon, Salmo salar L., parr. Aquacult. Nutr. 1996; 2: 41–47.

Andersen F, Lorentzen M, Waagbo R, Maage A. Bioavailability and interactions with other micronutrients of three dietary iron sources in Atlantic salmon, Salmo salar, smolts. Aquacult. Nutr. 1997; 3: 239–246.

Andersen F, Lygren B, Maage A, Waagbø R. Interactions between two dietary levels of iron and two forms of ascorbic acid and the effect on growth, antioxidant status and some non-specific immune parameters in Atlantic salmon (Salmo salar) smolts. Aquaculture 1998; 161: 437–451.

Ando D, Kitamura T, Mizuno S. Quantitative analysis of body silvering during smoltification in masu salmon using chromameter. North Am. J. Aquacult. 2005; 67: 160–166.

Atoda M, Imada K. Studies on the aquatic insect fauna and the environmental conditions of the Shakotan river, the Kenichi river and the Otoshibe river, Hokkaido. Sci. Rep. Hokkaido Fish Hatchery 1972; 27: 97–149 (in Japanese with English abstract).

page top


Ayson FG, Kaneko T, Hasegawa S, Hirano T. Development of mitochondrion-rich cells in the yolk-sac membrane of embryos and larvae of tilapia, Oreochromis mossambicus, in fresh water and seawater. J. Exp. Zool. 1994; 270: 129–135.

Azuma T, Noda S, Yada T, Ototake M, Nagoya H, Moriyama S, Yamada H, Nakanishi T, Iwata M. Profiles in growth, smoltification, immune function and swimming performance of 1-year-old masu salmon Oncorhynchus masou masou reared in water flow. Fish. Sci. 2002; 68: 1282–1294.

Bagenal TB, Tesch FW. Age and growth (IBP Handbook No. 3). In: Bagenal TB (ed.). Methods for Assessment of Fish Production in Fresh Water. Blackwell, Oxford. 1978; 101–136.

Baker RTM, Martin P, Davies SJ. Ingestion of sub-lethal levels of iron sulfate by African catfish affects growth, tissue lipid peroxidation. Aquat. Toxicol. 1997; 40: 51–61.

Balfry SK, Heath DD, Iwama GK. Genetic analysis of lysozyme activity and resistance to vibriosis in farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum). Aquaculture Res. 1997; 28: 893–899.

Ban M. Effects of density, dissolved oxygen concentration and the amount of flowing water on seawater adaptability in chum salmon. Tech. Rep. Natl. Salmon Res. Center. 2000; 167: 1–7.

Barton BA, Schreck CB, Barton LD. Effects of chronic cortisol administration and daily acute stress on growth, physiological conditions and stress responses in juvenile rainbow trout. Dis. Aquat. Org. 1987; 2: 173–185.

Bax NJ. Early marine mortality of marked juvenile chum salmon (Oncorhynchus keta) released into Hood Canal, Puget Sound, Washington, in 1980. Can. J. Fish. Aquat. Sci. 1983; 40: 426–435.

Beamish RJ, Neville CM. Pacific salmon and Pacific herring mortalities in the Fraser River plume caused by river lamprey (Lampetra ayresi). Can. J. Fish. Aquat. Sci. 1995; 52: 644–650.

Bjørnevik M, Maage A. Effects of dietary iron supplementation on tissue iron concentration and haematology in Atlantic salmon (Salmo salar). Fisk. Dir. Skr. Ser. Ernaering 1993; 6: 35–45.

page top


Blazer VS, Wolke RE, Brown J, Powell CA. Piscine macrophage aggregate parameters as health monitors: Effect of age, sex, relative weight, season and site quality in largemouth bass (Micropterus salmoides). Aquat. Toxicol. 1987; 10: 199–215.

Boeuf G, Harache Y. Criteria for adaptation of salmonids to high salinity seawater in France. Aquaculture 1982; 28: 163–176.

Boeuf G, Prunet P. Measurements of gill (Na+,K+)-ATPase activity and plasma thyroid hormones during smoltification in Atlantic salmon (Salmo salar L.). Aquaculture 1985; 45: 111–119.

Bone Q, Kiceniuk J, Jones DR. On the role of different fiber types in fish myotomes at intermediate swimming speeds. Fish. Bull. 1978; 76: 691–699.

Borg AF. Studies on myxobacteria associated with diseases of salmonid fishes. J. Wildlife Dis. 1960; 8: 1–85.

Brauge C, Medale F, Corraze G. Effect of dietary carbohydrate levels on growth. body composition and glycaemia in rainbow trout, Oncorhynchus mykiss, reared in seawater. Aquaculture 1994; 123: 109–120.

Brett JR. Environmental factors and growth. In: Hoar WS, Randall DJ, Brett JR (eds.). Fish Physiology. Academic Press, New York. 1979; 599–675.

Bullock GL. Studies on selected myxobacteria pathogenic for fishes and on bacterial gill disease in hatchery-reared salmonids. U.S. Bureau Sport Fish. Wildl. Tech. Papers 1972; 60 pp.

Burness G, Moyes CD, Montgomerie R. Mortility, ATP levels and metabolic enzyme activity of sperm from bluegill (Lepomis macrochirus). Comp. Biochem. Physiol. 2005; A140: 11–17.

Burrows RE. The influence of fingering quality on adult salmon survivals. Trans. Amer. Fish. Soc. 1969; 98: 777–784.

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Carey JB, McCormick SD. Atlantic salmon smolts are more responsive to an acute handling and confinement stress than parr. Aquaculture 1998; 168: 237–253.

Carriquiriborde P, Handy RD, Davies SJ. Physiological modulation of iron metabolism in rainbow trout (Oncorhynchus mykiss) fed low and high iron diets. J. Exp. Biol. 2004; 207: 75–86.

Chida K, Kijima A. Colorimetric measurement of the masu salmon for smoltification. Fish Genet. Breed. Sci. 1994; 20: 55–61 (in Japanese).

Chio KS, Reiss U, Fletcher B, Tappel AL. Peroxidation of subcellular organelles: Formation of lipofuscin-like fluorescent pigments. Science 1969; 166: 1535–1536.

Clarke WC, Blackburn J. A seawater challenge test to measure smolting of juvenile salmon. Can. Fish. Mar. Serv. Tech. Rep. 1977; 705: 1–11.

Clarke WC, Hirano T. Osmoregulation. In: Groot CL, Margolis L, Clarke WC (eds.). Physiological Ecology of Pacific Salmon. UBC Press, Vancouver. 1995; 317–377.

Desjardins LM, Hicks BD, Hilton JW. Iron catalyzed oxidation of trout diets and its effect on growth and physiological response of rainbow trout. Fish Physiol. Biochem. 1987; 3: 173–182.

Dobson GP, Parkhouse WS, Hochachka PW. Regulation of anaerobic ATP-generating pathways in trout fast-twitch skeletal muscle. Am. J. Physiol. 1987; 253: R186–R194.

Domenici P, Blake RW. The kinematics and performance of fish fast-start swimming. J. Exp. Biol. 1997; 200: 1165–1178.

Doroshof SI, Clark WH, Lutes PB, Swallow RL, Beer KE, McGuire AB, Cochran MD. Artificial propagation of the white sturgeon, Acipenser transmontanus Richardson. Aquaculture 1983; 32: 93–104.

Dubin IN. Idiopathic hemochromatosis and transfusion siderosis. Am. J. Clin. Path. 1955; 25: 514–542.

page top


Duston J. A light-reflectance meter to quantify silvering during smolting in Atlantic salmon. J. Fish Biol. 1995; 46: 912–914.

Ellis T, North B, Scott AP, Bromage NR, Porter M, Gadd D. The relationship between stocking density and welfare in farmed rainbow trout. J. Fish Biol. 2002; 61: 493–531.

Folmer LC, Dickhoff WW. The parr-smolt transformation (smoltification) and seawater adaptation in salmonids. A review of selected literature. Aquaculture 1980; 21: 1–37.

Franklin CE, Johnston IA. Muscle power output during escape responses in an Antarctic fish. J. Exp. Biol. 1997: 200: 703–712.

Gallaugher P, Thorarensen H, Farrell AP. Hematocrit in oxygen transport and swimming in rainbow trout (Oncorhynchus mykiss). Respir. Physiol. 1995; 102: 279–292.

Goede RW, Barton BA. Organismic indices and an autopsy-based assessment as indicators of health and condition of fish. Am. Fish. Soc. Symp. 1990; 8: 93–108.

Hamada K. Taxonomic and ecological studies of the genus Hypomesus of Japan. Mem. Fac. Fish. Hokkaido Univ. 1961; 9: 1–56.

Haner PV, Faler JC, Schrock RM, Rondorf DW, Maule AG. Skin reflectance as a nonlethal measure of smoltification for juvenile salmonids. N. Am. J. Fish. Manage. 1995; 15: 814–822.

Hayano H, Murakami Y, Kojima H, Kaneko T. Chloride cells in the yolk-sac membrane of eyed-stage embryos of long-finned smelt, Sprinchus lanceolatus. Sci. Rep. Hokkaido Fish Hatchery 1999; 53: 67–72 (in Japanese with English abstract).

Healey MC. Timing and relative intensity of size-selective mortality of juvenile chum salmon (Oncorhynchus keta) during early sea life. Can. J. Fish. Aquat. Sci. 1982; 39: 426–435.

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Hikita T. On the morphology, ecology and distribution of long-finned smelt, Sprinchus lanceolatus (Hikita) in Hokkaido and Sakhalin. Doubutsugaku Zasshi 1930; 42: 358–360.

Hikita T. On the development of long-finned smelt, Sprinchus lanceolatus (Hikita). Sci. Rep. Hokkaido Fish Hatchery 1958; 13: 39–49 (in Japanese).

Hilborn R. Hatcheries and future of salmon in the Northwest. Fisheries 1992; 17: 5–8.

Hirose S, Nakada T. From blood typing to a transport metabolism at a crossroad. Focus on ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae. Am. J. Physiol. Cell. Physiol. 2010; 298: 209–210.

Hoar WS. The physiology of smolting salmonids. In: Hoar WS, Randall DJ (eds.). Fish Physiology Vol. 11B. Academic Press, New York. 1988; 275–344.

Hochachka PW. The effect of physical training on oxygen debt and glycogen reserves in trout. Can. J. Zool. 1961; 39: 767–776.

Hokkaido Fish Hatchery. Effects of release of smolt improved seed qualities on recapture rate. Report on Achievement of Service for Hokkaido Fish Hatchery. 2008; 24–26.

Horst O, Erich G, Gnter W. Iron-dependent changes in cellular energy metabolism: influence on citric acid cycle and oxidative phosphorylation. Biochimica. Biophysica. Acta 1999; 1413: 99–107.

Hume JMB, Parkinson EA. Effect of stocking density on the survival, growth, and dispersal of steelhead trout fry (Salmo gairdneri). Can. J. Fish. Aquat. Sci. 1987; 44: 271–281.

Iguchi H, Iwamura S, Umezawa Y, Oshima Y, Saito T, Seki K, Nagakura S. Calcium carbonate. In: Nagakura S, Iguchi H, Ezawa H, Iwamura S, Sato F, Kubo R (eds.). Physics and Chemistry Dictionary. Iwanami Shoten, Tokyo. 2001a; TA274 (in Japanese).

Iguchi H, Iwamura S, Umezawa Y, Oshima Y, Saito T, Seki K, Nagakura S. Kaolin. In: Nagakura S, Iguchi H, Ezawa H, Iwamura S, Sato F, Kubo R (eds.). Physics and Chemistry Dictionary. Iwanami Shoten, Tokyo. 2001b; KA118 (in Japanese).

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Ikeda M. New problems on artificial propagation of freshwater fisheries resource from aspect of DNA analysis. In: Kitada S, Kaeriyama M, Hamasaki K, Taniguchi N (eds.). Artificial Propagation and Preservation of Fisheries Resources. Seizando, Tokyo. 2008; 105–127 (in Japanese).

Ikeda Y, Ozaki H, Uematsu K. Effect of enriched diet with iron in culture of yellow tail. Bull. J. Tokyo Univ. Fish. 1973; 59: 91–99 (in Japanese with English abstract).

Ito K. Ecological studies of the long finned smelt, Spirinchus lanceolatus (Hikita). I. On the measuring comparison of egg number from several localities, and relationship between body weight, age and mature ovarian egg. Sci. Rep. Hokkaido Fish Hatchery 1959; 14: 47–55 (in Japanese).

Ito K. Ecological studies of the long finned smelt, Spirinchus lanceolatus (Hikita). III. On the local forms. Sci. Rep. Hokkaido Fish Hatchery 1963; 18: 27–40 (in Japanese).

Ito K. Ecological studies of the long finned smelt, Spirinchus lanceolatus (Hikita). II. Observation on the natural spawning. Sci. Rep. Hokkaido Fish Hatchery 1964; 19: 17–26 (in Japanese).

Iwai T, Osama H. Effects of salinity on the developing eggs and early survival of the hatched pond smelt, Hypomesus olidus (Pallas). The Aquiculture 1986; 95: 95–102 (in Japanese).

Izuka T. Motility and fertilizing capacity of chilled testicular spermatozoa in wakasagi Hypomesus nipponensis. Bull. Kanagawa Pref. Fish. Res. Inst. 2003; 8: 13–16 (in Japanese).

Izuka T. Japanese smelt. In: Takashima F, Murai M (eds.). Fisheries Aquaculture System. Kouseisha Koseikaku, Tokyo. 2005; 103–113 (in Japanese).

Japan Fisheries Resource Conservation Association. Iron. In: Standard of Water Supply for Fishery Culture. Japan Fisheries Resource Conservation Association, Tokyo. 2000; 68.

Jayne BC, Lauder GV. Red and white muscle activity and kinetics of the escape response of the bluegill sunfish during swimming. J. Comp. Physiol. 1993; A175: 495–508.

page top


Kaeriyama M. Hatchery programs and stock management of salmonid populations in Japan. In: Howell BR, Moksness E, Sväsand T (eds.). Stock Enhancement and Sea Ranching. Blackwell, Oxford. 1999; 153–167.

Kaeriyama M. Salmonology. Seizando, Tokyo. 2002; 128 pp. (in Japanese).

Kaeriyama M, Edpaline RR. Evaluation of the biological interaction between wild and hatchery population for sustainable fisheries management of Pacific salmon. In: Leber KM, Kitada S, Blankenship HL, Sväsand T (eds.). Stock Enhancement and Sea Ranching II. Development, Pitfalls and Opportunities. Blackwell, Oxford. 2004; 247–259.

Kaeriyama M, Yatsu A, Noto M, Saitoh S. Spatial and temporal changes in the growth patterns and survival of Hokkaido chum salmon populations in 1970–2001. N. Pac. Anadr. Fish Comm. Bull. 2007; 4: 251–256.

Kaneko T, Hasegawa S, Takagi Y, Tagawa M, Hirano T. Hypoosmoregulatory ability of eyed-stage embryos of chum salmon. Mar. Biol. 1993; 122: 165–170.

Katayama S, Sugawara Y, Omori M, Okata A. Maturation and spawning processes of anadromous and resident pond smelt in Lake Ogawara. Icthyol. Res. 1999; 46: 7–18.

Kato F. Life histories of masu and amago salmon (Oncorhynchus masou and Oncorhynchus rhodurus). In: Groot C, Margolis L (eds.). Pacific Salmon Life Histories. UBC Press, Vancouver. 1991; 447–520.

Kawamura H, Kudo S. Seabird predation on juvenile chum salmon. N. Pac. Anadr. Fish Comm. Tech. Rep. 2000; 2: 9–10.

Kawatsu H. Studies on the anemia of fish—V. Dietary iron deficient anemia in brook trout, Salvelinus fontinalis. Bull. Freshwater Fish. Res. Lab. 1972; 22: 59–67.

Kimura T. History of fish pathology in Hokkaido. Sci. Rep. Hokkaido Fish Hatchery 1994; 48: 3–10.

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Kiron V, Maita M. Fish health. In: Nakagawa H, Sato M (eds.). Micronutrients and Health of Cultured Fish. Koseisha Koseikaku, Tokyo. 2003; 107–118 (in Japanese).

Kitsukawa M, Ohba M, Hirose H, Hirose K. Efficient egg collection by spontaneous spawning in a tank of the Japanese smelt, Hypomesus nipponensis in Lake Ashinoko, Kanagawa Prefecture. Suisanzoshoku 2003; 51: 401–405 (in Japanese with English abstract).

Kitsukawa M, Ohba M, Kudoh S. Use of a new jar hatchery to control the hatching of adhesive-eliminated eggs of Japanese smelt, Hypomesus nipponensis. Aquaculture Sci. 2006; 54: 231–236 (in Japanese with English abstract).

Kobayashi T. Salmon propagation in Japan. In: Thorpe JE (ed.). Salmon Ranching. Academic Press, London. 1980; 91–107.

Kono S, Iwashita A, Shimizu H, Minoshima H. Studies on the utilization of scallop waste. Food. Sci. Technol. Res. 2000; 4: 21–30 (in Japanese with English abstract).

Kubo T. Studies on the life history of the masu salmon (Oncorhynchus masou) in Hokkaido. Sci. Rep. Hokkaido Salmon Hatchery 1980; 34: 1–95.

Kusuda S, Teranishi T. Eliminating adhesiveness of shishamo smelt (Spirinchus lanceolatus) eggs during artificial propagation. Uo to Mizu 1996; 33: 37–42 (in Japanese).

Kuwada T, Matsuda H, Tuzuku N. Measurement of body color on smoltification in amago salmon Oncorhynchus masou ishikawae by colorimeter. Sci. Rep. Gifu Pref. Fish. Exp. Sta. 2000; 45: 23–31 (in Japanese).

Langdon JS, Thorpe JE. The ontogeny of smoltification: developmental patterns of gill Na+,K+-ATPase, SDH and chloride cells in juvenile Atlantic salmon, Salmo salar L. Aquaculture 1985; 45: 83–96.

Larmoyeux JD, Piper RG. Effects of water reuse on rainbow trout in hatcheries. Prog. Fish-Cult. 1973; 35: 2–8.

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Leatherland JF. Stocking density and cohort sampling effects on endocrine interactions in rainbow trout. Aquaculture Int. 1993; 1: 137–156.

Leatherland JF, Cho CY. Effects of rearing density on thyroid and interrenal gland activity and plasma and hepatic metabolite levels in rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 1985; 27: 583–592.

Leonard JBK, McCormick SD. Metabolic enzyme activity during smolting in stream- and hatchery-reared Atlantic salmon (Salmo salar). Can. J. Fish. Aquat. Sci. 2001; 58: 1585–1593.

Lichatowich JA, McIntyre JD. Use of hatcheries in the management of pacific anadromous salmonids. Am. Fish. Soc. Symp. 1988; 1: 131–136.

Liu YC, Torita A, Hasegawa Y. Scallop shell extract inhibits squalene monohydroperoxide-induced skin erythema and wrinkle formation in rat. Fish. Sci. 2008; 74: 217–219.

Logan SH, Johnston WE. Economics of commercial trout production. Aquaculture, 1992; 100: 25–46.

Machidori S, Kato F. Spawning populations and marine life of masu salmon (Oncorhynchus masou). Int. North Pac. Fish. Comm. Bull. 1984; 43: 1–138.

Maede JW. Allowable ammonia for fish culture. Prog. Fish-Cult. 1985; 47: 135–145.

Maita M, Aoki H, Yamagata Y, Satoh S, Okamoto N, Watanabe T. Plasma biochemistry and disease resistance in yellowtail fed a non fish meal diet. Fish Pathol. 1998; 104: 389–393.

Mäkinen T, Ruohonen K. The effects of rearing density on the growth of Finnish rainbow trout (Oncorhynchus mykiss Walbaum 1792). J. Appl. Icthyol. 1990; 6: 193–203.

page top


Martínez M, Bédard M, Dutil JD, Guderley H. Does condition of Atlantic cod (Gadus morhua) have a greater impact upon swimming performance at Ucrit or sprint speeds? J. Exp. Biol. 2004; 207: 2979–2990.

Maxime V, Boeuf G, Pennec JP, Peyraud C. Comparative study of the energetic metabolism of Atlantic salmon (Salmo salar) parr and smolts. Aquaculture 1989; 82: 163–171.

Mayama H. Studies on the freshwater life and propagation technology of masu salmon, Oncorhynchus masou (Brevoort). Sci. Rep. Hokkaido Salmon Hatchery 1992; 46: 1–156 (in Japanese with English abstract).

Maynard DJ, McDowell GC, Tezak EP, Flagg TA. Effects of diets supplemented with live food on the foraging behavior of cultured fall chinook salmon. Prog. Fish-Cult. 1996; 58: 187–191.

McCormick SD. Methods for non-lethal gill biopsy and measurement of Na+,K+-ATPase activity. Can. J. Fish. Aquat. Sci. 1993; 50: 656–658.

McCormick SD, Björnsson BT. Physiological and hormonal differences among Atlantic salmon parr and smolts reared in the wild and hatchery smolts. Aquaculture 1994; 121: 235–244.

McCormick SD, Saunders RL. Preparatory physiological adaptations for marine life of salmonids: osmoregulation, growth, and metabolism. Am. Fish. Soc. Symp. 1987; 1: 211–229.

McCormick SD, Saunders RL, MacIntyre AD. Mitochondrial enzyme and Na+, K+-ATPase activity, and ion regulation during parr-smolt transformation of Atlantic salmon (Salmo salar). Fish. Physiol. Biochem. 1989; 6: 231–241.

Milligan CL, Wood CM. Tissue intra acid-base status and the fate of lactate after exhaustive exercise in the rainbow trout. J. Exp. Biol. 1986; 123: 123–144.

Milligan CL, Wood CM. Regulation of blood oxygen transport and red cell pH after exhaustive activity in rainbow trout (Salmo gairdneri) and starry flounder (Platichthys stellatus). J. Exp. Biol. 1987; 133: 263–282.

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Misaka N, Kudo S, Aoyama T, Sakamoto H. Technical studies on artificial fertilization method of domesticated masu salmon in Kumaishi hatchery. Uo to Mizu 1998a; 35: 39–44 (in Japanese).

Misaka N, Naito K, Kawamura H. Seawater adaptability index to evaluate the quality of hatchery-reared masu salmon smolt. Uo to Mizu 1998b; 35: 233–239 (in Japanese).

Misaka N, Mizuno S, Miyakoshi Y, Takeuchi K, Takami T, Kasahara N. Changes of triglyceride and glycogen levels in the liver of underyearling masu salmon Oncorhynchus masou during starvation. Nippon Suisan Gakkaishi 2004; 70: 168–174 (in Japanese with English abstract).

Miyaji D, Kawanabe H, Mizuno N. Shishamo Spirinchus lanceolatus. In: Miyaji D, Kawanabe H, Mizuno N (eds.). Coloured Illustrations of the Freshwater Fishes of Japan. Hoikusha Publishing, Osaka. 1976; 57.

Miyakoshi Y. Evaluation of stock enhancement programs and stock assessment for masu salmon in Hokkaido, northern Japan. Sci. Rep. Hokkaido Fish Hatchery 2006; 60: 1–64.

Mizuno S, Ura K, Okubo T, Chida Y, Misaka N, Adachi S, Yamauchi K. Ultrastructural changes in gill chloride cells during smoltification in wild and hatchery-reared masu salmon Oncorhynchus masou. Fish. Sci. 2000; 66: 670–677.

Mizuno S, Ura K, Onodera Y, Fukada H, Misaka N, Hara A, Adachi S, Yamauchi K. Changes in transcript levels of gill cortisol receptor during smoltification in wild masu salmon, Oncorhynchus masou. Zool. Sci. 2001a; 18: 853–860.

Mizuno S, Misaka N, Kasahara N. Morphological changes in juxtaglomerular cells of the kidney during smoltification in masu salmon Oncorhynchus masou. Fish. Sci. 2001b; 67: 538–540.

Mizuno S, Misaka N, Kasahara N. Effects of cortisol and angiotensin II on the number and size of juxtaglomerular cells in masu salmon, Oncorhynchus masou. Fish Physiol. Biochem. 2001c; 25: 249–254.

Mizuno S, Misaka N, Miyakoshi Y, Takeuchi K, Kasahara, N. Effects of starvation on melano-macrophages in the kidney of masu salmon (Oncorhynchus masou). Aquaculture 2002; 209: 247–255.

page top


Mizuno S, Misaka N, Ando D, Kitamura T. Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou. Aquaculture 2004a; 229: 433–450.

Mizuno S, Sasaki Y, Omoto N, Imada K. Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration. Aquaculture 2004b; 242: 713–726.

Mizuno S, Sasaki Y, Imada K. Changes in seawater tolerance during the development of eyed-stage embryos in shishamo smelt Spirinchus lanceolatus (Hikita). Aqaculture Res. 2005; 36: 615–619.

Mizuno S, Misaka N, Ando D, Torao M, Urabe H, Kitamura T. Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou). Aquaculture 2007; 273: 284–297.

Mizuno S, Misaka N, Teranishi T, Ando D, Koyama T, Araya K, Miyamoto M, Nagata M. Physiological effects of an iron citrate dietary supplement on chum salmon (Oncorhynchus keta) fry. Aquaculture Sci. 2008; 56: 531–542.

Mizuno S, Teranishi T, Sasaki N, Koide N. Effects of treatment using unbaked scallop shell powder suspension on eliminating egg adhesiveness, hatching rate and larval quality in Japanese smelt (Hypomesus nipponensis) eggs. Aquaculture Sci. 2010a; 58: 97–104.

Mizuno S, Nakajima M, Naito K, Koyama T, Saneyoshi H, Kobayashi M, Koide N, Ueda H. Physiological impacts of high rearing density in chum salmon fry. Aquaculture Sci. 2010b; 58: 387–399.

Mizuno S, Hatakeyama M, Nakajima M, Naito K, Koyama T, Saneyoshi H, Kobayashi M, Koide N, Misaka N, Ueda H. Relationship between rearing conditions and health in chum salmon (Oncorhynchus keta) fry. Aquaculture Sci. 2010c; 58: 529–531.

Mizuno S, Misaka N, Miyakoshi Y. Assessment of nutritional conditions using kidney melano-macrophage density in hatchery-reared juvenile masu salmon Oncorhynchus masou released into a stream. Sci. Rep. Hokkaido Salmon Freshwater Fish. Res. Inst. 2011; 1: 49–53.

Mizuno S, Urabe H, Aoyama T, Omori H, Iijima A, Kasugai K, Torao M, Misaka N, Koide N, Ueda H. Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou). Aquaculture 2012; 362–363: 109–120.

page top


Munakata A, Björnsson BT, Jönsson E, Amano M, Ikuta K, Kitamura S, Kurokawa T, Aida K. Post-release adaptation process of hatchery-reared honmasu salmon parr. J. Fish Biol. 2000; 56: 163–172.

Nagasawa K. Fish and seabird predation on juvenile chum salmon (Oncorhynchus keta) in Japanese coastal waters, and an evaluation of the impact. N. Pac. Anadr. Fish Comm. Bull. 1998; 1: 480–495.

Nagata M. The occurrence of bimodality in the length frequency distribution, and its relation to growth and density in a juvenile masu salmon population in a Hokkaido stream. In: Kawanabe H, Yamazaki F, Noakes DLG (eds.). Biology of Charrs and Masu Salmon, Physiology and Ecology Japan Special Issue 29 Vol. I. Kyoto University Press, Kyoto. 1989; 141–150.

Nagata M, Kaeriyama M. Salmonid status and conservation in Japan. In: Gallaugher P, Wood L (eds.). Proceedings from the World Summit on Salmon. Simon Fraser University, Burnaby. 2004; 89–97.

Nakamura K. Method to eliminate egg adhesion in Japanese dace. The Aquaculture 1962; 14: 37–40 (in Japanese).

Nogawa H, Yagisawa I. Optimum environmental condition for rearing juvenile chum salmon (Oncorhynchus keta): A review. Sci. Rep. Hokkaido Salmon Hatchery 1994; 48: 31–39 (in Japanese with English abstract).

Nomura T. Bacterial diseases of freshwater fishes of Hokkaido. Sci. Rep. Hokkaido Fish Hatchery 1994; 48: 39–46.

North BP, Turnbull JF, Ellis T, Porter MJ, Migaud H, Bron J, Bromage NR. The impacts of stocking density on the welfare of rainbow trout (Oncorhynchus mykiss). Aquaculture 2006; 255: 466–479.

Ogino C, Takeuchi T, Takeda H, Watanebe T. Availability of dietary phosphorus in carp and rainbow trout. Bull. Japan. Soc. Sci. Fish. 1979; 45: 1527–1532.

Oguri M. Histochemical observations in the dark brown pigment granules found in the kidney tissue of rainbow trout. Bull. Japan. Soc. Sci. Fish. 1976; 42: 1223–1227.

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Oguri M. Pigment granules in the renal interstitial tissue of marine teleosts. Bull. Japan. Soc. Sci. Fish. 1985; 51: 1447–1449.

Okada H, Kudo S, Hayashi K. Studies on the spawning habitat of the long finned smelt, Spirinchus lanceolatus (Hikita)—I. On the spawning behavior in the aquarium. Sci. Rep. Hokkaido Fish Hatchery 1975; 30: 31–38 (in Japanese with English abstract).

Okada H, Kudo S, Hayashi K. On the action of males anal fin of long finned smelt, Spirinchus lanceplatus (Hikita), in spawning behavior. Sci. Rep. Hokkaido Fish Hatchery 1976; 31: 155–161 (in Japanese with English abstract).

Okada S, Sasaki T. On the propagation of Sprinchus lanceolatus at Kushiro-river. Sci. Rep. Hokkaido Fish. Exp. Sta. 1960; 17: 14–19 (in Japanese).

Olla B, Davis MW. The role of learning and stress in predator avoidance of hatchery-reared coho salmon (Oncorhynchus kisutch) juveniles. Aquaculture 1989; 76: 209–214.

Omi H. The normal embryonic development and the effect of temperature on its speed of the long finned smelt, Sprinchus lanceolatus (Hikita). Sci. Rep. Hokkaido Fish. Exp. Sta. 1978a; 35: 10–20 (in Japanese).

Omi H. On the distribution of spawned eggs and the seaward migration of the newly hatched larvae of the long-finned smelt, Sprinchus lanceolatus at some rivers in Kushiro Hokkaido. Sci. Rep. Hokkaido Fish. Exp. Sta. 1978b; 35: 21–28 (in Japanese).

Papoutsoglou SE, Papaparaskeva-Papoutsoglou E, Alexis MN. Effect of density on growth rate and production of rainbow trout (Salmo gairdneri Rich.) over a full rearing period. Aquaculture, 1987; 66: 9–17.

Parker RR. Mortality of juvenile salmon in central British Columbia coastal waters. Fish. Res. Bd. Can. Man. Rep. 1968; 956.

Patiño R, Schreck CB, Banks JL, Zaugg WS. Effects of rearing conditions on the developmental physiology of smolting coho salmon. Trans. Am. Fish. Soc. 1986; 115: 828–837.

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Payan P, Girard JP, Mayer-Gostan N. Branchial iron movements in teleosts: The roles of respiratory and chloride cells. In: Hoar WS, Randall DJ (eds.). Fish Physilogy XB. Academic Press, Florida. 1984; 39–60.

Pearson MP, Stevens ED. Size and hematological impact of the splenic erythrocyte reservoir in rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem. 1991; 9: 39–50.

Pickering AD, Pottinger TG. Crowding causes prolonged leucopenia in salmonid fish, despite interrenal acclimation. J. Fish Biol. 1987; 30: 701–712.

Plisetskaya EM. Recent studies of fish pancreatic hormones: selected topics. Zool. Sci. 1990; 7: 335–353.

Plisetskaya EM, Swanson P, Bernard MG, Dickhoff WW. Insulin in coho salmon (Oncorhynchus kisutch) during the parr to smolt transformation. Aquaculture 1988; 72: 151–164.

Procarione LS, Barry TP, Malison JA. Effects of high rearing densities and loading rates on the growth and stress responses of juvenile rainbow trout. North Am. J. Aquacult. 1999; 61: 91–96.

Roberts RJ. Melanin-containing cells of teleost fish and their relation to disease. In: Ribelin WE, Migaki G (eds.). The Pathology of Fishes. University of Wisconsin Press, Madison. 1975; 399–428.

Roberts RJ. The pathophysiology and systematic pathology of teleosts. In: Roberts RJ (ed.). Fish Pathology. Balliére Tindall, London. 1978; 55–91.

Røed KH, Fjalestad KT, Strømsheim A. Genetic variation in lysozyme activity and spontaneous haemolytic activity in Atlantic salmon (Salmo salar). Aquaculture 1993; 114: 19–31.

Sakamoto S, Yone Y. Iron deficiency symptoms of carp. Bull. Japan. Soc. Sci. Fish. 1978; 44: 1157–1160.

page top


Salo EO. Life histories of chum salmon (Oncorhynchus keta). In: Groot C, Margolis L (eds.). Pacific Salmon Life Histories. UBC Press, Vancouver. 1991; 231–309.

Sasaki K, Yoshimitsu S. Control of soft egg disease of chum salmon by green tea extract. J. Fish. Technol. 2008; 1: 43–47 (in Japanese with English abstract).

Seber GAF, LeCren ED. Estimating population parameters from catches large relative to the population. J. Anim. Ecol. 1967; 36: 631–643.

Sheridan MA, Woo NY, Bern HA. Changes in the rates of glycogenesis, glycogenolysis, lipogenesis, and lipolysis in selected tissues of the coho salmon (Oncorhynchus kisutch) associated with parr-smolt transformation. J. Exp. Zool. 1985; 236: 35–44.

Shiau SY, Su LW. Ferric citrate is half as effective as ferrous sulfate in meeting the iron requirement of juvenile tilapia, Oreochromis niloticus × O. aureus. J. Nutr. 2003; 133: 483–488.

Shiraishi K, Kaneko T, Hasegawa S, Hirano T. Development of multicellular complexes of chloride cells in the yolk-sac membrane of tilapia (Oreochromis mossambicus) embryos and larvae in seawater. Cell Tiss. Res. 1997; 288: 583–590.

Shiraishi Y. The fisheries biology and population dynamics of pond-smelt, Hypomesus olidus (Pallas). Bull. Freshwater Fish. Res. Lab. 1961; 10: 1–263 (in Japanese with English abstract).

Shrimpton JM, Bernier NJ, Iwama GK, Randall DJ. Differences in measurements of smolt development between wild and hatchery-reared juvenile coho salmon (Oncorhynchus kisutch) before and after saltwater exposure. Can. J. Fish. Aquat. Sci. 1994a; 51: 2170–2178.

Shrimpton JM, Bernier NJ, Randall DJ. Changes in cortisol dynamics in wild and hatchery-reared juvenile coho salmon (Oncorhynchus kisutch) during smoltification. Can. J. Fish. Aquat. Sci. 1994b; 51: 2179–2187.

Standal H, Rorvik KA, Lien H, Andersen O. Effects of acute iron overload on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Biol. Trace Elem. Res. 1997; 59: 13–22.

page top


Sullivan CV, Dickhoff WW, Mahnken CV, Hershberger WK. Changes in the hemoglobin system of the coho salmon Oncorhynchus kisutch during smoltification and triiodothyronine and propylthiouracil treatment. Comp. Biochem. Physiol. 1985; A81: 807–813.

Takeda N, Kusuda S, Teranishi T, Imada K. Effect of iron concentrations on hatching rates of adhesive-eliminated Osmerid fish (Sprinchus lanceolatus) eggs from the surface. Sci. Rep. Hokkaido Fish Hatchery. 2002; 56: 107–113 (in Japanese with English abstract).

Taya K. Economical approach to stock enhancement in fisheries. In: Agriculture, Forestry and Fisheries Research Council (ed.). The Plan for Marine Ranching. Koseisha Koseikaku, Tokyo. 1989; 568–600.

Torao M. Japanese smelt Hypomesus nipponensis (McAllister). Uo to Mizu 2005a; 41: 95–98.

Torao M. Shishamo smelt Spirinchus lanceolatus (Hikita). Uo to Mizu 2005b; 41: 99–101.

Torisawa M. Life history polymorphism and the population dynamics of wakasagi (Hypomesus nipponensis) in Lake Abashiri, Hokkaido, Japan. Sci. Rep. Hokkaido Fish. Exp. Stn. 1999; 56: 1–117 (in Japanese with English abstract).

Tsukamoto K, Chiba K. Oxygen consumption of yellowtail, Seriola quinqueradiata, in relation to swimming speed. Nippon Suisan Gakkaishi 1981; 47: 673.

Turner JD, Wood CM, Clark D. Lactate and proton dynamics in the rainbow trout (Salmo gairdneri). J. Exp. Biol. 1983; 104: 247–268.

Ura K, Hara A, Yamauchi K. Serum thyroid hormone, guanine and protein profiles during smoltification and after thyroxine treatment in the masu salmon, Oncorhynchus masou. Comp. Biochem. Physiol. 1994; 107A: 607–612.

Urawa S, Awakura T. Protozon diseases of freshwater fishes in Hokkaido. Sci. Rep. Hokkaido Fish Hatchery 1994; 48: 47–58.

page top


Utoh H, Sakazaki S. Life history of the pond smelt, Hypomesus transpacificus McAllister, in Lake Abashiri 1. Review of fishery and study on the life history of the pond smelt in Lake Abashiri. J. Hokkaido Fish. Exp. Sta. 1983; 40: 147–156 (in Japanese).

Utoh H, Sakazaki S. Life history of the pond smelt, Hypomesus transpacificus McAllister, in Lake Abashiri 2. On the pond smelt caught from the spawning streams and the lake during the spawning season. J. Hokkaido Fish. Exp. Sta. 1984; 40: 447–459 (in Japanese).

Utoh H, Sakazaki S. Life history of the pond smelt, Hypomesus transpacificus McAllister, in Lake Abashiri 3. Seaward and upstream migration. Sci. Rep. Hokkaido Fish. Exp. Sta. 1987; 29: 1–16 (in Japanese).

Vielma J, Ruohonen K, Lall SP. Supplemental citric acid and particle size of fish bone-meal influence the availability of minerals in rainbow trout Oncorhynchus mykiss (Walbaum). Aquacult. Nutr. 1999; 5, 65–71.

Vijayan MM, Foster GD, Moon TW. Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven, Hemitripterus americanus. Fish Physiol. Biochem. 1993; 12: 327–335.

Waltemyer DL. Tannin as an agent to eliminate adhesiveness of walleye eggs during artificial propagation. Trans. Am. Fish. Soc. 1976; 105: 731–736.

Walton MJ. Metabolic effects of feeding a high protein/low carbohydrate diet as compared to a low protein/high carbohydrate diet to rainbow trout Salmo gairdneri. Fish Physiol. Biochem. 1986; 1: 7–15.

Wang Y, Heigemhauser GJF, Wood CM. Integrated responses to exhaustive exercise and recovery in rainbow trout white muscle: acid-base, phosphogen, carbohydrate, lipid, ammonia, fluid volume and electrolyte metabolism. J. Exp. Biol. 1994; 195: 227–258.

Watanabe T, Ida H, Iwata M. Serum ion regulation in Pacific salmon exposed to short term acid water stress during seaward migratory and post migratory seasons. Fish. Sci. 1995; 62: 353–354.

Wedemeyer GA. Physiology of Fish in Intensive Culture Systems. Chapman & Hall, New York. 1996; 227 pp.

page top


Wedemeyer GA, Saunders RL, Clarke WC. Environmental factors affecting smoltification and early marine survival of anadromous salmonids. Mar. Fish. Rev. 1980; 42: 1–14.

Wendt CAG, Saunders RL. Changes in carbohydrate metabolism in young Atlantic salmon in response to various forms of stress. Int. Atl. Salmon Found. Spec. Publ. 1973; 4: 55–82.

Westers H, Pratt KM. Rational design of hatcheries for intensive salmonid culture, based on metabolic characteristics. Prog. Fish-Cult. 1977; 39: 157–165.

Wolke RE. Piscine macrophage aggregates: A review. Annual Rev. Fish Diseases 1992; 2: 91–108.

Woodword JJ, Smith LS. Exercise training and the stress response in rainbow trout, Salmo gairdneri Richardson. J. Fish. Biol. 1985; 26: 435–447.

Yamagishi T, Kani H, Yoshida M, Uchiyama T, Nagano N, Minoshima H, Wada K, Shoji Y. Improvement of skid resistance of traffic paint by scallop shell. Rep. Hokkaido Indust. Res. Inst. 2007; 306: 55–60 (in Japanese with English abstract).

Yanagi S, Kudo H, Doi Y, Yamauchi K, Ueda H. Immunohistochemical demonstration of salmon olfactory glutathione S-transferase class pi (N24) in the olfactory system of lacustrine sockeye salmon during ontogenesis and cell proliferation. Anat. Embryol. 2004; 208: 231–238.

Yoshimizu M. Viral diseases of freshwater fishes in Hokkaido. Sci. Rep. Hokkaido Fish Hatchery 1994; 48: 15–24.

Zaugg WS, McLain LR. Changes in gill ATPase activity associated with parr smolt transformation in steelhead trout (Salmo gairdneri), coho salmon (Oncorhynchus kisutch) and spring chinook salmon (O. tshawytscha). J. Fish. Res. Bd. Canada 1971; 19, 167–171.

Zbanyszek R, Smith L. Changes in carbonic anhydrase activity in coho salmon smolts resulting from physical training and transfer into seawater. Comp. Biochem. Physiol. 1984; 79A, 229–233.

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List of Figures

Fig. 1 Observed portion of dorsal fin pigmentation. Panel A indicates the entire dorsal fin of wild masu salmon smolt in the Ken-ichi River. Panel B is magnified from the small rectangular area indicated in panel A. In panel B, the white square (100 μm × 100 μm) shows the portion of the dorsal fin observation. IV: fourth soft ray, V: fifth soft ray, VI: sixth soft ray. Scale bars show 1.00 mm. Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

Fig. 2 Dorsal fin pigmentation during smoltification of wild masu salmon in the Utabetsu River. Upper pictures in grayscale, lower pictures after black and white transformation by an imaging software. The lines from 'a' to 'e' indicates dorsal fins in January, February, March, April and May, respectively. Scale bar shows 100 μm. Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

Fig. 3 Quantitative changes in dorsal fin pigmentation during smoltification in wild and hatchery-reared masu salmon. The letter 'a' adjacent to a symbol indicates a significant difference with the initial value of each respective group (P < 0.05; One way ANOVA). Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

Fig. 4 Changes in gill Na+,K+-ATPase activity during smoltification in wild and hatchery-reared masu salmon. The letter 'a' adjacent to a symbol indicates a significant difference with the initial value of each respective group (P < 0.05; One way ANOVA). Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

Fig. 5 Correlation of gill Na+,K+-ATPase activity with dorsal fin pigmentation level, in wild and hatchery-reared smolting masu salmon. Spearman's rank correlation coefficient by rank test was used as statistical analysis. The normal, bold and dotted line show the nonlinear correlations [Pigment] = 61.0 + 16.0 ln [ATPase] (r2 = 0.752, P < 0.0001) in the Ken-ichi River, [Pigment] = 66.8 + 13.7 ln [ATPase] (r2 = 0.879, P < 0.0001) in the Utabetsu River and [Pigment] = 68.9 + 13.0 ln [ATPase] (r2 = 0.806, P < 0.0001) in the hatchery-reared groups, respectively. Reprinted from Aquaculture, 229, Mizuno et al., Quantitative changes of black pigmentation in the dorsal fin margin during smoltification in masu salmon, Oncorhynchus masou, 433–450, © 2004, with permission from Elsevier.

Fig. 6 Changes in serum glucose concentration (GL) and liver glycogen content (GC) during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks reveal significant differences compared to the value of the same parameter in March within each group of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

Fig. 7 Changes in liver and gill pyruvate kinase (PRK) activity during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks reveal significant differences compared to the value of the same parameter in March within each group of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

Fig. 8 Changes in liver and gill citrate synthase (CS) activities during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks reveal significant differences compared to the value of the same parameter in March within each group of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

Fig. 9 Changes in liver cytochrome c oxidase (COX) activity during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences in the value between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks reveal significant differences compared to the value of the same parameter in March within each group of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

Fig. 10 Changes in transcription levels of liver and gill ATP synthase subunit 8 (AST) during smoltification in wild and hatchery-reared masu salmon. Different small alphabetical letters indicate significant differences in the value between wild and hatchery-reared fish at each sampling time (P < 0.05; Student's t-test). Asterisks revealed significant differences compared to the value of the same parameter in March within each groups of fish (P < 0.05; One-way ANOVA). Cross marks indicate a significant change from the value of the same parameter one month before for the same group of fish (P < 0.05; Student's t-test). Modified from Aquaculture, 362–363, Mizuno et al., Changes in activity and transcript level of liver and gill metabolic enzymes during smoltification in wild and hatchery-reared masu salmon (Oncorhynchus masou), 109–120, © 2010, with permission from Elsevier.

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Fig. 11 Condition factor (CF) in wild and hatchery-reared masu salmon at each sampling time of the 2003 and 2004 experiments. The letters 'a', 'b', 'c' and 'd' indicate significant differences between the wild, hatchery control, iron-1 and iron-2 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Asterisks show significant differences compared to the value of the previous sampling time within the same group (P < 0.05; Student's t-test). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

Fig. 12 Total iron content of the food remnants in the stomach of wild and hatchery-reared masu salmon in May 2003. The letters 'a', 'b', 'c' and 'd' indicate significant differences between the wild, hatchery control, iron-1 and iron-2 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

Fig. 13 Burst swimming capacity (BSC) of wild and hatchery-reared masu salmon in May 2003, April 2004 and May 2004. The letters 'a', 'b', 'c' and 'd' indicate significant differences between the wild, hatchery control, iron-1 and iron-2 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

Fig. 14 Hemoglobin concentration (Hb) in wild and hatchery-reared masu salmon at each sampling time of the 2003 and 2004 experiments. The letters 'a', 'b', 'c' and 'd' indicate significant differences between the wild, hatchery control, iron-1 and iron-2 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

Fig. 15 ATP content in the white muscle and liver in wild and hatchery-reared masu salmon at each sampling time of the 2004 experiment. The letters 'a', 'b' and 'c' show significant differences between the wild, hatchery control and iron-1 groups at a given sampling time, respectively (P < 0.05; One-way ANOVA). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

Fig. 16 Relationship between the means of burst swimming capacity (BSC) and hemoglobin concentration (Hb) and between the means of BSC and ATP content in the white muscle. For simple regression analysis between the means of BSC and Hb concentrations, data in May 2003, April 2004 and May 2004 were plotted. The mean BSC was found to be significantly correlated with the mean Hb concentration (r2 = 0.538, P = 0.015, Pearson's correlation coefficient) and the mean ATP content (r2 = 0.784, P = 0.019, Pearson's correlation coefficient). Modified from Aquaculture, 273, Mizuno et al., Effects of diets supplemented with iron citrate on some physiological parameters and on burst swimming velocity in smoltifying hatchery-reared masu salmon (Oncorhynchus masou), 284–297, © 2007, with permission from Elsevier.

Fig. 17 Histological observations of melano-macrophage in the kidney of hatchery-reared and wild masu salmon. Panels 'a' and 'b' designate kidneys from hatchery-reared and wild fish respectively, at the start of the experiment (day 0). Panels 'c' and 'd' show kidneys from the starved groups of hatchery-reared and wild fish respectively, 45 days after the start of the experiment. Panels 'e' and 'f' reveal kidneys from the fed groups of hatchery-reared and wild fish, respectively, and panel 'g' shows a kidney from the hatchery-reared fish sampled from the river. Panels e, f and g were shot 45 days after the start of the experiment. The arrowheads indicate melano-macrophages. Scale bars show 50.0 μm. Reprinted from Aquaculture, 209, Mizuno et al., Effects of starvation on melano-macrophages in the kidney of masu salmon (Oncorhynchus masou), 247–255, © 2002, with permission from Elsevier.

Fig. 18 Changes in the level of melano-macrophage deposition (MMD) during the experiment in starved (○) and fed groups (□) of hatchery-reared masu salmon, in starved (●) and fed groups (■) of wild masu salmon, and in the hatchery-reared fish sampled from the river (△). Asterisks show significant differences in the MMD level between the starved group and the fed group at the same sampling time in each fish group (P < 0.05, One-way ANOVA). Cross marks designate significant differences in the MMD level from the hatchery-reared group sampled from the river (P < 0.05, One-way ANOVA). Modified from Aquaculture, 209, Mizuno et al., Effects of starvation on melano-macrophages in the kidney of masu salmon (Oncorhynchus masou), 247–255, © 2002, with permission from Elsevier.

Fig. 19 Changes in mortality during the experiment in starved (○) and fed groups (□) of hatchery-reared masu salmon and in starved (●) and fed groups (■) of wild masu salmon. Modified from Aquaculture, 209, Mizuno et al., Effects of starvation on melano-macrophages in the kidney of masu salmon (Oncorhynchus masou), 247–255, © 2002, with permission from Elsevier.

Fig. 20 Map showing the Utabetsu River in southern Hokkaido and sampling stations on the Kami-Utabetsu River. Small rectangular area shows sampling area of the Kami-Utabetsu river. Modified from Sci. Rep. Hokkaido Salmon Freshwater Fish. Res. Inst., 1, Mizuno et al., Assessment of nutritional conditions using kidney melano-macrophage density in hatchery-reared juvenile masu salmon Oncorhynchus masou released into a stream, 49–53, © 2011, Hokkaido Salmon and Freshwater Fisheries Research Institute.

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Fig. 21 Relationships between fish density and liver triglyceride (TG) levels, and between fish density and kidney melano-macrophage density (MMD), in juvenile masu salmon. There were no fish caught at station 5 in July. Modified from Sci. Rep. Hokkaido Salmon Freshwater Fish. Res. Inst., 1, Mizuno et al., Assessment of nutritional conditions using kidney melano-macrophage density in hatchery-reared juvenile masu salmon Oncorhynchus masou released into a stream, 49–53, © 2011, Hokkaido Salmon and Freshwater Fisheries Research Institute.

Fig. 22 Relationship between mean triglyceride (TG) levels in the liver and mean melano-macrophage density (MMD) in the kidney in juvenile masu salmon. The plots in this figure express the means of TG and MMD in juvenile population at each station and time. Spearman's rank correlation coefficient was used as statistical analysis. The dotted line shows the linear correlation [MMD] = 0.622–0.359 [TG] (r = 0.601, P < 0.05). There were no juveniles caught at station 5 in July. Modified from Sci. Rep. Hokkaido Salmon Freshwater Fish. Res. Inst., 1, Mizuno et al., Assessment of nutritional conditions using kidney melano-macrophage density in hatchery-reared juvenile masu salmon Oncorhynchus masou released into a stream, 49–53, © 2011, Hokkaido Salmon and Freshwater Fisheries Research Institute.

Fig. 23 Changes in body weight (A) and condition factor (B) of the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letters at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

Fig. 24 Changes in rearing density (A) and dissolved oxygen (DO) concentration (B) of the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

Fig. 25 Changes in the survival rate in the three density groups during the fasting-tolerance test. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

Fig. 26 Changes in plasma cholesterol concentrations in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with different alphabetical letters at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

Fig. 27 Changes in plasma glucose concentrations in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with different alphabetical letter at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

Fig. 28 Changes in plasma cortisol concentrations in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letter at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

Fig. 29 Changes in plasma lysozyme activity in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letter at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

Fig. 30 Changes in somatic adenosine triphosphate (ATP) content in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letters at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

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Fig. 31 Changes in somatic ATP synthase transcription levels in the three density groups during the experiment. Closed square (■), closed triangle (▲) and open circle (○) showed 40, 20 and 10 kg/m3 groups, respectively. The marks with the different alphabetical letters at the same sampling time were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

Fig. 32 Schematic representation of acute changes in physical condition during rearing at excessive density (>40 kg/m3). The four parameters designated in this figure were not influenced by the duration of the experiment in the 10 kg/m3 group, which appeared to maintain the best physical condition during the experiment. The first sign of an aggravated physical condition was an increase in plasma cortisol concentration and an increase in somatic ATP content and decrease in ATP synthase (AST) transcription levels. Thereafter, the ATP content and lysozyme activity decreased. Modified from Aquaculture Science, 58, Mizuno et al., Physiological impacts of high rearing density on chum salmon Oncorhynchus keta fry, 387–399, © 2010, Japanese Society for Aquaculture Research.

Fig. 33 The somatic adenosine triphosphate (ATP) contents (left side) and ATP synthase transcription (AST) levels (right side) of hatchery-reared and wild chum salmon fry. The numbers I to IX represent the different hatcheries the chum salmon fry were collected at. Yellow areas indicate the range considered standard healthy values for each parameter.

Fig. 34 Relationships between rearing density, dissolved oxygen concentration (DO) and somatic ATP content (A), and between rearing density, DO and somatic ATP synthase transcription levels (B). X- and Y-axes show rearing density and DO respectively. Contours represent somatic ATP content in the A graph and carcass ATP synthase transcription levels in the B graph. Modified from Aquaculture Science, 58, Mizuno et al., Relationship between rearing conditions and health in chum salmon (Oncorhynchus keta) fry, 529–531, © 2010, Japanese Society for Aquaculture Research.

Fig. 35 Osmerid egg (A) and jar incubators for culturing the eggs (B). Panel A shows a Japanese smelt egg cultured at 10°C for one day. Arrowhead shows the inverted adhesive membrane, a part of egg membrane. Scale bar shows 1.00 mm. Panel B shows 6l-jar incubators in Mukawa hatchery, southern Hokkaido.

Fig. 36 Observation of the eggs after non-treatment (A and D), tannic acid treatment (B and E) and kaolin treatment (C and F). The upper and lower halves show eggs on November 21, 2003 and March 23, 2004, respectively. The arrowheads indicate borderline between the egg membrane and the inverted adhesive membrane. Scale bar indicates 1.00 mm. Reprinted from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

Fig. 37 Effects of the kaolin suspension treatment on elimination of egg adhesiveness in the first experiment. The letters 'a' and 'b' indicate significant differences in the value compared to the non-treatment and the tannic acid treatment, respectively (P < 0.05; Chi-square test for independence). Reprinted from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

Fig. 38 Changes in total iron concentration in the river water of the Mukawa hatchery and the Salmon and Freshwater Fisheries Research Institute during this experiment. Modified from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

Fig. 39 Changes in the total amount of iron on the egg surface in the non-treated, tannic acid-treated and kaolin-treated groups during the experiment. Asterisks express significant differences compared to the initial value of each group (P < 0.05; One way ANOVA). Cross marks show significant differences compared to the value of non-treatment group at the same sampling time (P < 0.05; One way ANOVA). The letter 'a' indicates significant differences with the value of the kaolin-treated group at the same sampling time (P < 0.05; One way ANOVA). Modified from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

Fig. 40 Hatching rate (A), mortality during hatching (B) and survival rate of larvae after the seawater transfer (C) in non-treated, tannic acid-treated and kaolin-treated eggs. Columns with different alphabetical letters were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

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Fig. 41 Egg pressure in the non-treated, tannic acid-treated and kaolin-treated groups. Columns with different alphabetical letter were statistically different (P < 0.05; One-way ANOVA). Modified from Aquaculture, 242, Mizuno et al., Elimination of adhesiveness in the eggs of shishamo smelt Spirinchus lanceolatus using kaolin treatment to achieve high hatching rate in an environment with a high iron concentration, 713–726, © 2004, with permission from Elsevier.

Fig. 42 Egg adhesiveness elimination rate in Japanese smelt eggs treated with freshwater (control), kaolin suspension or SSP suspensions in the first experiment. Different alphabetical letters showed significant differences in the hatching rate between groups (P < 0.05; Chi-square test for independence). Modified from Aquaculture Science, 58, Mizuno et al., Effects of treatment using unbaked scallop shell powder suspension on eliminating egg adhesiveness, hatching rate and larval quality in Japanese smelt (Hypomesus nipponensis) eggs, 97–104, © 2010, Japanese Society for Aquaculture Research.

Fig. 43 Hatching rate of Japanese smelt eggs treated with freshwater (control), kaolin suspension or SSP suspensions in the first experiment. Different alphabetical letters showed significant differences in the hatching rate between groups (P < 0.05; Chi-square test for independence). Modified from Aquaculture Science, 58, Mizuno et al., Effects of treatment using unbaked scallop shell powder suspension on eliminating egg adhesiveness, hatching rate and larval quality in Japanese smelt (Hypomesus nipponensis) eggs, 97–104, © 2010, Japanese Society for Aquaculture Research.

Fig. 44 Observation of freshwater-treated (A, control), 5 g/l kaolin-treated (B), 5 g/l (C) and 20 g/l (D) SSP-treated embryos 20 days after fertilization in the second experiment. Arrows indicate inverted adhesive membrane. Scale bars designate 1.0 mm. Control eggs (A) were unfastened from the palm tree skins before their observation. Modified from Aquaculture Science, 58, Mizuno et al., Effects of treatment using unbaked scallop shell powder suspension on eliminating egg adhesiveness, hatching rate and larval quality in Japanese smelt (Hypomesus nipponensis) eggs, 97–104, © 2010, Japanese Society for Aquaculture Research.

Fig. 45 Observation of the eyed-stage embryos used in this study. Panels (a), (b), (c), (d) and (e) show embryos at 133 (Stage 15), 140 (Stage 16), 154 (Stage 17), 188 (Stage 18) and 243°C (Stage 19) cumulative temperature, respectively. The arrowheads designate the position of the end of the tail. Scale bar shows 1.00 mm. Reprinted with permission of John Wiley & Sons, Inc. from Aquaculture Research, 36, Mizuno et al., Changes in seawater tolerance during the development of eyed-stage embryos in shishamo smelt Spirinchus lanceolatus (Hikita), 615–619, Fig. 1, © 2005, Wiley-Liss, Inc., a Wiley Company.

Fig. 46 Impact of seawater environments on the hatching rate of embryos at various embryogenetic stages. The letter 'a' shows significant differences in the hatching rate compared to 133°C (Stage 15) in each environment (P < 0.05; Chi-square test for independence). The letter 'b' designates significant differences in the rate compared to the freshwater group at each embryogenetic stage (P < 0.05; Chi-square test for independence). The letter 'c' expresses significant differences in the rate between 17 psu brackish water and 34 psu seawater at each embryogenetic stage (P < 0.05; Chi-square test for independence). Modified with permission of John Wiley & Sons, Inc. from Aquaculture Research, 36, Mizuno et al., Changes in seawater tolerance during the development of eyed-stage embryos in shishamo smelt Spirinchus lanceolatus (Hikita), 615–619, Table 1, © 2005, Wiley-Liss, Inc., a Wiley Company.

Fig. 47 Changes in egg Na+,K+-ATPase activity during the development of eyed-stage embryos. Asterisks indicate significant differences in the activity compared to 133.3°C (Stage 15) (P < 0.05; One-way ANOVA). Modified with permission of John Wiley & Sons, Inc. from Aquaculture Research, 36, Mizuno et al., Changes in seawater tolerance during the development of eyed-stage embryos in shishamo smelt Spirinchus lanceolatus (Hikita), 615–619, Fig. 2, © 2005, Wiley-Liss, Inc., a Wiley Company.

Fig. 48 Relationships between mean egg Na+,K+-ATPase activity and hatching rate in freshwater (FW), 17 psu brackish water (BW) and 34 psu seawater (SW) environments of various eyed-stage embryos. Modified with permission of John Wiley & Sons, Inc. from Aquaculture Research, 36, Mizuno et al., Changes in seawater tolerance during the development of eyed-stage embryos in shishamo smelt Spirinchus lanceolatus (Hikita), 615–619, Fig. 3, © 2005, Wiley-Liss, Inc., a Wiley Company.

Table 1 Pond conditions and water quality, and condition of the chum salmon fry. Modified from Aquaculture Science, 58, Mizuno et al., Relationship between rearing conditions and health in chum salmon (Oncorhynchus keta) fry, 529–531, © 2010, Japanese Society for Aquaculture Research. Body weight (n = 15), somatic ATP content (n = 5) and somatic ATP synthase transcription level (n = 5) shown as means ± standard errors.

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