- Email: [email protected]
Relationship between egg specific gravity and egg quality in the Japanese eel, Anguilla japonica
Aquaculture 246 (2005) 493 – 500 www.elsevier.com/locate/aqua-online
Short communication
Relationship between egg specific gravity and egg quality in the Japanese eel, Anguilla japonica Tatsuya UnumaT, Shigenori Kondo1, Hideki Tanaka, Hirohiko Kagawa2, Kazuharu Nomura, Hiromi Ohta3 National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 516-0193, Japan Received 6 July 2004; received in revised form 21 December 2004; accepted 19 January 2005
Abstract Eggs from maturation-induced Japanese eels, Anguilla japonica, often sink in seawater immediately after artificial insemination and do not hatch. In the present study, the specific gravity of unfertilized eggs from individual females was measured in isotonic (about 310 mOsm/kg) and hypertonic (about 875 mOsm/kg) saline solutions and the relation of specific gravity to egg quality was examined. Egg specific gravity under isotonic conditions showed a significant negative correlation with egg fertility, hatchability and water content, suggesting that inadequate hydration of oocytes during final maturation, which leads to insufficient egg buoyancy, is one of the causes of poor egg quality. Some of the eggs that showed lower specific gravity than seawater under isotonic conditions exhibited higher specific gravity than seawater under hypertonic conditions, indicating that the buoyancy acquired by the eggs is sometimes lost after their transfer to seawater. Only eggs that retained low specific gravity under hypertonic as well as isotonic conditions exhibited high fertility and hatchability. Taken together with data from other studies, these results suggest that the poor quality of eggs that sink immediately after artificial insemination is attributable to at least two causes: the failure of oocytes to acquire sufficient buoyancy during maturation and the loss of buoyancy in seawater because of the inability of egg osmoregulation. D 2005 Elsevier B.V. All rights reserved. Keywords: Anguilla japonica; Eel; Egg; Specific gravity; Bouyancy; Egg quality
T Corresponding author. Tel.: +81 599 66 1830; fax: +81 599 66 1962. E-mail address: [email protected] (T. Unuma). 1 Present address: Osaka College of Communication Arts, Osaka 550-0013, Japan. 2 Present address: Division of Fisheries Science, Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan. 3 Present address: Department of Fisheries, School of Agriculture, Kinki University, Nara 631-8505, Japan. 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.01.011
1. Introduction The Japanese eel, Anguilla japonica, is one of the most widely cultivated fish in Japan; the seed stock used for aquaculture is juvenile glass eels caught in the wild. The shortage of seed for cultivation has recently become a crucial issue, leading to extensive studies on techniques for breeding the eels artificially
494
T. Unuma et al. / Aquaculture 246 (2005) 493–500
(Ohta et al., 1997). Production of leptocephali larvae (Tanaka et al., 2001) and their subsequent metamorphosis into glass eels (Tanaka et al., 2003) have recently been accomplished using a newly developed feed for eel larvae; however, production of cultured glass eels and leptocephali has remained very low. One of the causes for such low productivity is the unstable quality of eggs obtained from females induced to undergo maturation by hormone injection (maturation-induced) (Kagawa et al., 2001). Eggs stripped from the ovulating females often sink in seawater immediately after artificial insemination and do not hatch, although eel eggs normally are pelagic. To enhance the production of eel larvae, buoyant eggs of better quality need to be produced in quantity. The buoyancy of eggs has often been used as an indicator in the assessment of egg quality especially in studies of marine teleosts that spawn pelagic eggs, such as red sea bream Pagrus major (Watanabe et al., 1984a, 1984b), sea bass Dicentrarchus labrax (Carrillo et al., 1989), yellowtail Seriola quiqueradiata (Mushiake et al., 1994), Japanese flounder Paralichthys olivaceus (Furuita et al., 2000, 2002), Japanese whiting Sillago japonica (Kondo et al., 2001), and the Japanese eel (Seoka et al., 2003). In these species, the ratio of buoyant eggs to total eggs spawned, measured several hours after fertilization, correlates positively with egg hatchability, which is based on the fact that most of the unfertilized eggs and the fertilized eggs that have ceased developing do not remain buoyant for very long. There are few reports, however, on the relationship between egg quality and buoyancy or specific gravity of eggs before egg insemination and transfer to seawater. This study was conducted to reveal the relation between the specific gravity of unfertilized eggs and egg quality and to investigate the causes of the reduced buoyancy of poor quality eggs from the maturation-induced Japanese eel. We first measured the specific gravity of the eggs in isotonic saline solution of an osmolality close to that of the ovoplasm and the coelomic fluid. As our preliminary results raised the possibility that the specific gravity of eggs in isotonic saline solution does not necessarily reflect their buoyancy in full-strength seawater, we then measured the specific gravity of eggs in hypertonic saline solution of an osmolality similar to that of seawater. Comparison of the data from both media
(isotonic and hypertonic) revealed some possible causes of poor egg buoyancy.
2. Materials and methods 2.1. Broodstock and hormonal treatment Cultured eels purchased from a commercial culture pond or provided by the Aichi Fisheries Research Institute, Aichi, Japan were acclimated to seawater in the National Research Institute of Aquaculture, Mie, Japan and then induced to mature by hormonal treatment in flow-through tanks holding 400 L of the sand-filtered seawater at 20 8C. Female eels (500–1000 g) were repeatedly injected with salmon pituitary extract then with 17,20h-dihydroxy4-pregnene-3-one (Kagawa et al., 1997). Male eels (300–350 g) were repeatedly injected with human chorionic gonadotropin (Ohta et al., 1996). Gametes were obtained by gently stripping eggs from ovulating females and semen from spermiating males. The eggs were then immediately assessed for fertilization, specific gravity and osmolarity. Some of the eggs were stored for the quantitation of their water and lipid content as described below. The number of females used for each experiment is shown in Results. 2.2. Osmotic measurement To determine the osmotic conditions of the saline solutions used to measure the specific gravity of the eggs, the osmolality of the ovoplasm, the coelomic fluid, and the seawater used for artificial insemination was analyzed. Ovoplasm was prepared as described by Lønning and Davenport (1980) with slight modifications. About 0.2 g of the unfertilized eggs was placed into a 1 ml syringe, forced by the plunger and squashed through the needle (0.5 mm inner diameter) into a vial and centrifuged at 11,000 g for 10 min at 4 8C; the supernatant was then collected as ovoplasm. Coelomic fluid was collected by centrifuging 30 g of the unfertilized eggs at 760 g for 5 min at 4 8C. The osmolality of the ovoplasm, the coelomic fluid, the seawater as well as of the media for measuring the specific gravity described below was
T. Unuma et al. / Aquaculture 246 (2005) 493–500
measured by analyzing freezing point depression with a One–ten osmometer (Fiske, USA). To examine the effects of seawater on the osmolality of the ovoplasm, the unfertilized eggs were immersed in the seawater for 5 min at 23 8C, transferred to an Ultrafree-MC centrifugal filtering device (pore size, 5 Am; Millipore, USA) and centrifuged at 1400 g for 1 min to remove the seawater. The osmolality of the ovoplasm of the seawater-immersed eggs was analyzed in the same manner as described above. 2.3. Measurement of specific gravity of eggs An isotonic and a hypertonic series of media, each containing 31 media of different specific gravity, were prepared for measuring the specific gravity of the eggs. For the isotonic series, 148.5 mM NaCl containing 0.02% sodium azide was mixed with 297 mM sucrose containing 0.02% sodium azide at ratios of 60:40, 59:41, 58:42, ..., 32:68, 31:69, 30:70 (weight:weight). For the hypertonic series, 448.5 mM NaCl containing 0.02% sodium azide was mixed with 897 mM sucrose containing 0.02% sodium azide at ratios of 100:0, 99:1, 98:2, ..., 72:28, 71:29, 70:30 (weight:weight). The specific gravity of the eight solutions, 60:40, 50:50, 40:60 and 30:70, in the isotonic series, and 100:0, 90:10, 80:20 and 70:30, in the hypertonic series, was determined with a hydrometer at 23 8C. The specific gravity of each of the other media was calculated by linear equations from the values of the eight media. Each medium (4 ml) was dispensed into a test tube, then 20–30 eggs were added to each tube and stood for 5 min at 23 8C (Fig. 1). The specific gravity of the medium where the eggs were most evenly dispersed (Fig. 1D) was taken as the specific gravity of the eggs. The change in specific gravity (DG) from that under isotonic conditions to that under hypertonic conditions was calculated by the formula below: DG (%) = [100 (specific gravity under hypertonic conditions) / (specific gravity under isotonic conditions)] 100. The average osmolality of the media prepared was 310.0F8.7 mOsm/kg in the isotonic series and 875.1F30.8 mOsm/kg in the hypertonic series. The osmolality of the hypertonic series could not be raised above this value, since under higher osmolality the
A
Low
B
495
C
D
E
Specific gravity
F
G
High
Fig. 1. Method for determining the specific gravity of the eel eggs. Twenty to thirty eggs were placed in tubes filled with a series of isotonic (about 310 mOsm/kg) or hypertonic (about 875 mOsm/kg) media of various specific gravity values. The specific gravity of the eggs was taken as the specific gravity of the medium in which they were most evenly dispersed (D).
specific gravity of the eggs was sometimes lower than that of the NaCl solution, the lowest end of the series. In some teleosts including eels, the unfertilized eggs are induced to form a perivitellin space by contact with the environmental water (Suzuki, 1991), which may affect egg specific gravity. In our study, however, perivitellin space was not formed to a noticeable extent during the measurement of specific gravity of eel eggs under a stereoscopic microscope. 2.4. Fertilization test Two grams of the eggs were inseminated with 1 ml of the pre-diluted milt and then dispersed in 100 ml of the seawater as described by Ohta et al. (1996). Three replicates of 2 ml of the seawater containing about 80 eggs were transferred using a glass pipet to three Petri dishes, each filled with 30 ml of filtered seawater (pore size, 0.2 Am) containing penicillin G potassium (Banyu Pharmaceutical, Tokyo) at 100,000 IU/L and streptomycin sulfate (Meiji Seika, Tokyo) at 0.1 g/L. Four hours after insemination, eggs displaying embryonic cleavage were counted under a stereoscopic microscope and the hatched larvae were counted three days after egg insemination. The rates of fertilization and hatching were calculated for each female as the average of the three replicate incubations by the following formulae: Fertilization rate (%) = 100 the number of eggs displaying cleavage / the number of eggs stocked into the Petri dish; and
T. Unuma et al. / Aquaculture 246 (2005) 493–500
Hatching (%)
75 50 25
3. Results 3.1. Osmolality and specific gravity of coelomic fluid, ovoplasm, and seawater The osmolality of the coelomic fluid and the ovoplasm was 305.4F13.3 and 298.3F11.2 mOsm/ kg (N=7), respectively. No apparent correlation was found between these parameters and fertility or hatchability. The average osmolality and the specific gravity of the seawater collected on different days were 976.1F25.6 mOsm/kg (N=7) and 1.0248F0.0003 (N=4), respectively.
25
1.028
1.024
1.026
1.020
1.022
1.026
1.022
1.028
Specific gravity
89 88 87
Lipid (% dry weight)
C
90
35
D
30 25 20
1.028
1.024
1.026
1.020
1.028
1.026
1.022
1.024
Specific gravity
1.022
15
85
1.020
Values are presented as the meansFstandard deviation. All statistical analyses were carried out using Statview 5.0 software (SAS Institute, USA). Spearman’s correlation coefficients (r s) were used to examine significant ( Pb0.05) correlation by rank between two parameters. The Wilcoxon signed-ranks test was used to examine significant ( Pb0.05) differences between values.
50
Specific gravity 91
86
2.6. Statistical analysis
B
75
0
0
1.024
To remove coelomic fluid, the eggs were washed with 150 mM NaCl solution, centrifuged at 760 g for 5 min at 4 8C to remove the wash solution, and then stored at 80 8C until analysis. The water content of the eggs was measured by drying the sample at 110 8C for 8 h. The lipid content was determined by the gravimetric method using a Soxhlet device with diethyl ether as the solvent (Yamamoto et al., 2000) and calculated as dry matter.
100
A
1.020
2.5. Water and lipid contents
100
Fertilization (%)
Hatching rate (%) = 100 the number of hatched larvae / the number of eggs stocked into the Petri dish.
Water (%)
496
Specific gravity
Fig. 2. Relation between the specific gravity under isotonic conditions and some properties of eel eggs. A) Fertilization rate; B) hatching rate; C) water content; and D) lipid content. The vertical dashed line in each panel represents the average specific gravity of the seawater (1.0248).
ranged from 1.0200 to 1.0273; eggs from 3 females exhibited higher specific gravity than that of the seawater. Significant negative correlations were observed between egg specific gravity and fertilization rate (r s= 0.54; Pb0.001; Fig. 2A) and hatching rate (r s= 0.72; Pb0.0001; Fig. 2B). Most eggs of higher specific gravity showed very low fertility and hatchability, whereas eggs of lower specific gravity were not always of good quality, indicating that low specific gravity is a necessary but insufficient condition for good egg quality. A significant negative correlation was observed between egg specific gravity and water content (r s= 0.40; Pb0.01; Fig. 2C), whereas no apparent correlation was observed between egg specific gravity and lipid content (Fig. 2D), suggesting that water content contributes more to the buoyancy of eel eggs than does lipid content.
3.2. Specific gravity of eggs under isotonic conditions The specific gravity of eggs from 44 females in the isotonic media, with osmolality similar to that of the coelomic fluid and the ovoplasm, was analyzed and related to fertility, hatchability and the water and lipid content of the eggs (Fig. 2). The specific gravity
3.3. Specific gravity of eggs under hypertonic and isotonic conditions The specific gravity of eggs from 28 females was measured under hypertonic as well as under isotonic conditions (Figs. 3–6). The relation
Hatching < 30% 1.030
1.020
400 300
Fig. 5. Relation between the specific gravity of eel eggs under hypertonic conditions and the osmolality of the ovoplasm after immersion of the eggs in seawater.
Fig. 3. Relation between the specific gravity of the eel eggs under isotonic and hypertonic conditions. Circles and crosses represent high (N30%) and low (b30%) rates of hatching, respectively. The dashed lines represent the average specific gravity of the seawater (1.0248).
between the two measurements, with their respective hatchability divided into two grades of high (N30%; N=6) versus low (b30%; N=22) hatchability, is shown in Fig. 3. The specific gravity of eggs under isotonic conditions ranged from 1.0205 to 1.0248, whereas that under hypertonic conditions ranged from 1.0206 to 1.0404. Eggs from some females showed similar values under both conditions (lower left side of Fig. 3), with a larger number of such eggs demonstrating high hatching rates. On the other hand, eggs from some females showed lower specific gravity under isotonic conditions but higher values under hypertonic conditions (upper left side of Fig. 3), with most of such eggs demonstrating low hatching rates. Fig. 4 shows the relation between the change of specific gravity,
Hatching (%)
A
75 50 25 0
B
75
∆G (%)
90% sink
1.020 90% float 1.010 1.000
50 25 0
0.0 0.5 1.0 1.5 2.0
1.030
0.0 0.5 1.0 1.5 2.0 ∆G (%)
Fig. 4. Relation between the changes in specific gravity of eel eggs from isotonic to hypertonic conditions (DG) and the corresponding rates of fertilization (A) and hatching (B).
Hypertonic
100
1.040
Isotonic
100
after the transfer of eggs from isotonic to hypertonic conditions, and the corresponding rates of fertilization and hatching. A significant negative correlation was observed between the change of specific gravity and fertilization rate (r s= 0.52; Pb0.01; Fig. 4A) and hatching rate (r s= 0.52; Pb0.01; Fig. 4B). Most of the eggs with marked change of specific gravity under hypertonic conditions exhibited very low fertility and hatchability, whereas eggs with little change of specific gravity were not always of good quality, indicating that maintaining specific gravity after the transfer of eggs to a hypertonic medium is a necessary but insufficient condition for good egg quality. Fig. 5 shows the relation between the specific gravity under hypertonic conditions and the osmolality of the ovoplasm after immersion of the eggs in seawater for 5 min. A significant positive correlation was observed
Specific gravity
1.040
1.035
1.030
1.025
1.020
Specific gravity (hypertonic)
Specific gravity (isotonic)
Fertilization (%)
500
1.020
1.025
600
1.035
Hatching > 30%
700
1.040
1.035
800
1.025
1.040
497
1.030
Osmolality (mOsm/kg)
Specific gravity (hypertonic)
T. Unuma et al. / Aquaculture 246 (2005) 493–500
Fig. 6. Specific gravity at which about 90% of eggs sank (Fig. 1B) or floated (Fig. 1F) under isotonic and hypertonic conditions. Values represent the meansFstandard deviation (N=28). *Significantly different from the value where 90% of eggs floated under isotonic conditions ( pb0.0001).
498
T. Unuma et al. / Aquaculture 246 (2005) 493–500
between these parameters (r s=0.73; Pb0.001), suggesting that the eggs that lose buoyancy in seawater are incapable of maintaining the osmolality of the ovoplasm in seawater. The unevenness of the specific gravity of individual eggs from the same female was assessed by measuring the specific gravity of the media in which about 90% of the eggs sank (Fig. 1B) or floated (Fig. 1F), which represents the range of specific gravity covering about 80% of the eggs from the same female (Fig. 6). The values were 1.0202F0.0010 (90% sank) and 1.0231F0.0015 (90% floated) under isotonic conditions and 1.0193F0.0017 (90% sank) and 1.0286F0.0076 (90% floated) under hypertonic conditions. The lower values (90% sank) were very similar under both conditions, whereas the higher values (90% floated) were significantly different between isotonic and hypertonic conditions ( Pb0.0001). These results indicate that the specific gravity of individual eggs from the same female is almost uniform under isotonic conditions but highly variable under hypertonic conditions and that among the poor quality eggs from the same female, not all of the eggs always lose buoyancy under hypertonic conditions, with a number of eggs sometimes maintaining buoyancy.
4. Discussion In marine teleosts spawning pelagic eggs, egg buoyancy is acquired during the final maturation of oocytes through a reduction in ovoplasm density attributed mainly to massive influx of water (Craik and Harvey, 1987). At first, we assumed that failure to acquire sufficient buoyancy during maturation was the main cause of the poor quality of eel eggs that sink immediately in seawater after insemination. Therefore, we measured the specific gravity of eel eggs under isotonic conditions (Fig. 2). Consistent with our assumption, significant negative correlations were found between the specific gravity of eggs under isotonic conditions and egg fertility, hatchability, and water content. These correlations suggest that attaining low specific gravity by adequate hydration is a prerequisite for good egg quality in the Japanese eel. Eggs that exhibited poor buoyancy in an isotonic medium were, however, unexpectedly rare, despite the frequently observed
sinking of eggs immediately after insemination and transfer to seawater. These observations raised the possibility that the specific gravity of eggs measured under isotonic conditions does not necessarily reflect their eventual buoyancy in seawater. Determination of the specific gravity of eggs under hypertonic conditions (Figs. 3–6) revealed that eggs that acquired buoyancy sometimes lost it after transfer to the hypertonic medium. Only eggs that were able to maintain low specific gravity in the hypertonic medium as well as in the isotonic medium exhibited high fertility and hatchability. The change of egg specific gravity after transfer to the hypertonic medium was highly variable among batches of eggs from different females and also among individual eggs from the same female. The ability to maintain buoyancy in a hypertonic medium probably varies among individual eggs, whereas the initial acquisition of buoyancy probably progresses evenly in batches of eggs from the same female. Based on these data, the low buoyancy of poor quality eggs appears to be attributable to at least two causes. One is the failure of acquiring sufficient buoyancy because of inadequate hydration of the oocytes during final maturation, although this may not occur very often during the artificial induction of eel maturation. In marine teleosts that spawn pelagic eggs, vitellogenin-derived yolk proteins, such as lipovitellins and phosvitin, are proteolyzed into free amino acids (FAA) that act as osmotic effectors to induce water influx into maturing oocytes (Craik and Harvey, 1987; Thorsen et al., 1996; Matsubara and Koya, 1997). Seoka et al. (2003) have demonstrated that in eel eggs showing development of embryos several hours after insemination, FAA content is higher in buoyant eggs than in nonbuoyant ones, indicating that FAA content contributes to the buoyancy of eel eggs in seawater. Some disruption of the supply of FAA arising from yolk proteolysis during oocyte maturation may give rise to nonbuoyant eel eggs that are poorly hydrated. Another possible cause of nonbuoyant eggs is the inability of eggs to maintain specific gravity in seawater, and this occurs more often than problems with oocyte hydration during maturation. Dehydration and/or influx of salt probably occur in eggs that lose buoyancy in seawater. The system of osmoregulation through the plasma membrane (Alderdice,
T. Unuma et al. / Aquaculture 246 (2005) 493–500
1988) may be damaged in such eggs, probably to varying degrees in individual eggs from the same female. In the maturation-induced Japanese eel, deterioration of egg quality due to over-ripening of the eggs occurs in the ovarian cavity as a result of delayed stripping of the eggs after ovulation (Ohta et al., 1996). Over-ripening in the Japanese eel progresses relatively faster than in other teleosts (Ohta et al., 1996; Adachi, 2000). A decline in egg quality also ensues from the deterioration of oocytes in ovarian follicles if hormonal induction of oocyte maturation is not undertaken at the proper time before follicles become atretic (Adachi, 2000). Delayed hormonal induction of oocyte maturation and/or delayed stripping of the ovulated eggs may be associated with problems in the osmoregulatory system. In conclusion, both acquisition of egg buoyancy before ovulation (oocyte hydration) and maintenance of egg buoyancy after transfer to seawater are prerequisites for good egg quality in the Japanese eel. To reduce the incidence of nonbuoyant, poor quality eggs, insufficient hydration of the oocytes during final maturation needs to be eliminated and the inability of eggs to osmoregulate properly in seawater needs to be corrected. Further studies focusing on detailed clarification of the physiological mechanisms underlying these processes will lead to improvement of regimes for the artificial induction of female maturation, eliminating these obstacles to reliable production of seed stock for eel aquaculture.
Acknowledgements We are grateful to the staff of the Freshwater Resources Research Center, Aichi Fisheries Research Institute for providing the broodstock of the eel. We also thank Ryoko Okamoto at the National Research Institute of Aquaculture for technical assistance throughout the study. This study was supported, in part, by a grant-in-aid (Development of seed production and releasing techniques for stock enhancement of marine resources considering the conservation of ecosystem) from the Ministry of Agriculture, Forestry and Fisheries, Government of Japan.
499
References Adachi, S., 2000. Artificial control of maturation and spawning, and its relation to egg quality. Kaiyo Monthly 132, 120 – 126 (In Japanese). Alderdice, D.F., 1988. Osmotic and ionic regulation in teleost eggs and larvae. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, vol. XIA. Academic Press, San Diego, pp. 163 – 251. Carrillo, M., Bromage, N., Zanuy, S., Serrano, R., Prat, F., 1989. The effect of modifications in photoperiod on spawning time, ovarian development and egg quality in the sea bass (Dicentrarchus labrax L.). Aquaculture 81, 351 – 365. Craik, J.C.A., Harvey, S.M., 1987. The causes of buoyancy in eggs of marine teleosts. J. Mar. Biol. Ass. U. K. 67, 169 – 182. Furuita, H., Tanaka, H., Yamamoto, T., Shiraishi, M., Takeuchi, T., 2000. Effects of n-3 HUFA levels in broodstock diet on the reproductive performance and egg and larval quality of the Japanese flounder, Paralichthys olivaceus. Aquaculture 187, 387 – 398. Furuita, H., Tanaka, H., Yamamoto, T., Suzuki, N., Takeuchi, T., 2002. Effect of high levels of n-3 HUFA in broodstock diet on egg quality and egg fatty acid composition of the Japanese flounder Paralichthys olivaceus. Aquaculture 210, 323 – 333. Kagawa, H., Tanaka, H., Ohta, H., Okuzawa, K., Iinuma, N., 1997. Induced ovulation by injection of 17,20h-dihydroxy-4-pregnen3-one in the artificially matured Japanese eel, with special reference to ovulation time. Fish. Sci. 63, 365 – 367. Kagawa, H., Ohta, H., Tanaka, H., 2001. Recent progress of research on larvae production of Japanese eel, Anguilla japonica. Suisan Zoshoku 49, 127 – 132. Kondo, S., Yoshioka, M., Kashiwagi, M., 2001. Changes in specific gravity and osmolarity of eggs of Japanese whiting Sillago japonica during the embryonic development. Nippon Suisan Gakkaishi 67, 743 – 744 (In Japanese). Lønning, S., Davenport, J., 1980. The swelling egg of the long rough dab, Hippoglossoides platessoides limandoides (Bloch). J. Fish Biol. 17, 359 – 378. Matsubara, T., Koya, Y., 1997. Course of proteolytic cleavage in three classes of yolk proteins during oocyte maturation in barfin flounder Verasper moseri, a marine teleost spawning pelagic eggs. J. Exp. Zool. 278, 189 – 200. Mushiake, K., Kawano, K., Sakamoto, W., Hasegawa, I., 1994. Effects of extended daylength on ovarian maturation and HCGinduced spawning in yellowtail fed moist pellets. Fish. Sci. 60, 647 – 651. Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Hirose, K., 1996. Changes in fertilization and hatching rates with time after ovulation induced by 17,20h-dihydroxy-4-pregnen-3-one in the Japanese eel, Anguilla japonica. Aquaculture 139, 291 – 301. Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Iinuma, N., Hirose, K., 1997. Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica. Fish Physiol. Biochem. 17, 163 – 169. Seoka, M., Yamada, S., Iwata, Y., Yanagisawa, T., Nakagawa, T., Kumai, H., 2003. Differences in the biochemical content of buoyant and non-buoyant eggs of the Japanese eel, Anguilla japonica. Aquaculture 216, 355 – 362.
500
T. Unuma et al. / Aquaculture 246 (2005) 493–500
Suzuki, R., 1991. Developmental physiology and biotechnology. In: Itazawa, Y., Hanyu, I. (Eds.), Fish Physiology. KoseishaKoseikaku, Tokyo, pp. 585 – 609 (In Japanese). Tanaka, H., Kagawa, H., Ohta, H., 2001. Production of leptocephali of Japanese eel (Anguilla japonica) in captivity. Aquaculture 201, 51 – 60. Tanaka, H., Kagawa, H., Ohta, H., Unuma, T., Nomura, K., 2003. The first production of glass eel in captivity: fish reproductive physiology facilitates great progress in aquaculture. Fish Physiol. Biochem. 28, 493 – 497. Thorsen, A., Kjesbu, O.S., Fyhn, H.J., Solemdal, P., 1996. Physiological mechanism of bouyancy in eggs from brackish water cod. J. Fish Biol. 48, 457 – 477.
Watanabe, T., Itoh, A., Kitajima, C., Fujita, S., 1984a. Effect of dietary protein levels on reproduction of red sea bream. Bull. Japan. Soc. Sci. Fish. 50, 1015 – 1022. Watanabe, T., Itoh, A., Murakami, A., Tsukashima, Y., Kitajima, C., Fujita, S., 1984b. Effect of nutritional quality of diets given to broodstock on the verge of spawning on reproduction of red sea bream. Bull. Japan. Soc. Sci. Fish. 50, 1023 – 1028. Yamamoto, T., Shima, T., Unuma, T., Shiraishi, M., Akiyama, T., Tabata, M., 2000. Voluntary intake of diets with varying digestible energy contents and energy sources, by juvenile rainbow trout Oncorhynchus mykiss, using self-feeders. Fish. Sci. 66, 528 – 534.
Short communication
Relationship between egg specific gravity and egg quality in the Japanese eel, Anguilla japonica Tatsuya UnumaT, Shigenori Kondo1, Hideki Tanaka, Hirohiko Kagawa2, Kazuharu Nomura, Hiromi Ohta3 National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 516-0193, Japan Received 6 July 2004; received in revised form 21 December 2004; accepted 19 January 2005
Abstract Eggs from maturation-induced Japanese eels, Anguilla japonica, often sink in seawater immediately after artificial insemination and do not hatch. In the present study, the specific gravity of unfertilized eggs from individual females was measured in isotonic (about 310 mOsm/kg) and hypertonic (about 875 mOsm/kg) saline solutions and the relation of specific gravity to egg quality was examined. Egg specific gravity under isotonic conditions showed a significant negative correlation with egg fertility, hatchability and water content, suggesting that inadequate hydration of oocytes during final maturation, which leads to insufficient egg buoyancy, is one of the causes of poor egg quality. Some of the eggs that showed lower specific gravity than seawater under isotonic conditions exhibited higher specific gravity than seawater under hypertonic conditions, indicating that the buoyancy acquired by the eggs is sometimes lost after their transfer to seawater. Only eggs that retained low specific gravity under hypertonic as well as isotonic conditions exhibited high fertility and hatchability. Taken together with data from other studies, these results suggest that the poor quality of eggs that sink immediately after artificial insemination is attributable to at least two causes: the failure of oocytes to acquire sufficient buoyancy during maturation and the loss of buoyancy in seawater because of the inability of egg osmoregulation. D 2005 Elsevier B.V. All rights reserved. Keywords: Anguilla japonica; Eel; Egg; Specific gravity; Bouyancy; Egg quality
T Corresponding author. Tel.: +81 599 66 1830; fax: +81 599 66 1962. E-mail address: [email protected] (T. Unuma). 1 Present address: Osaka College of Communication Arts, Osaka 550-0013, Japan. 2 Present address: Division of Fisheries Science, Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan. 3 Present address: Department of Fisheries, School of Agriculture, Kinki University, Nara 631-8505, Japan. 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.01.011
1. Introduction The Japanese eel, Anguilla japonica, is one of the most widely cultivated fish in Japan; the seed stock used for aquaculture is juvenile glass eels caught in the wild. The shortage of seed for cultivation has recently become a crucial issue, leading to extensive studies on techniques for breeding the eels artificially
494
T. Unuma et al. / Aquaculture 246 (2005) 493–500
(Ohta et al., 1997). Production of leptocephali larvae (Tanaka et al., 2001) and their subsequent metamorphosis into glass eels (Tanaka et al., 2003) have recently been accomplished using a newly developed feed for eel larvae; however, production of cultured glass eels and leptocephali has remained very low. One of the causes for such low productivity is the unstable quality of eggs obtained from females induced to undergo maturation by hormone injection (maturation-induced) (Kagawa et al., 2001). Eggs stripped from the ovulating females often sink in seawater immediately after artificial insemination and do not hatch, although eel eggs normally are pelagic. To enhance the production of eel larvae, buoyant eggs of better quality need to be produced in quantity. The buoyancy of eggs has often been used as an indicator in the assessment of egg quality especially in studies of marine teleosts that spawn pelagic eggs, such as red sea bream Pagrus major (Watanabe et al., 1984a, 1984b), sea bass Dicentrarchus labrax (Carrillo et al., 1989), yellowtail Seriola quiqueradiata (Mushiake et al., 1994), Japanese flounder Paralichthys olivaceus (Furuita et al., 2000, 2002), Japanese whiting Sillago japonica (Kondo et al., 2001), and the Japanese eel (Seoka et al., 2003). In these species, the ratio of buoyant eggs to total eggs spawned, measured several hours after fertilization, correlates positively with egg hatchability, which is based on the fact that most of the unfertilized eggs and the fertilized eggs that have ceased developing do not remain buoyant for very long. There are few reports, however, on the relationship between egg quality and buoyancy or specific gravity of eggs before egg insemination and transfer to seawater. This study was conducted to reveal the relation between the specific gravity of unfertilized eggs and egg quality and to investigate the causes of the reduced buoyancy of poor quality eggs from the maturation-induced Japanese eel. We first measured the specific gravity of the eggs in isotonic saline solution of an osmolality close to that of the ovoplasm and the coelomic fluid. As our preliminary results raised the possibility that the specific gravity of eggs in isotonic saline solution does not necessarily reflect their buoyancy in full-strength seawater, we then measured the specific gravity of eggs in hypertonic saline solution of an osmolality similar to that of seawater. Comparison of the data from both media
(isotonic and hypertonic) revealed some possible causes of poor egg buoyancy.
2. Materials and methods 2.1. Broodstock and hormonal treatment Cultured eels purchased from a commercial culture pond or provided by the Aichi Fisheries Research Institute, Aichi, Japan were acclimated to seawater in the National Research Institute of Aquaculture, Mie, Japan and then induced to mature by hormonal treatment in flow-through tanks holding 400 L of the sand-filtered seawater at 20 8C. Female eels (500–1000 g) were repeatedly injected with salmon pituitary extract then with 17,20h-dihydroxy4-pregnene-3-one (Kagawa et al., 1997). Male eels (300–350 g) were repeatedly injected with human chorionic gonadotropin (Ohta et al., 1996). Gametes were obtained by gently stripping eggs from ovulating females and semen from spermiating males. The eggs were then immediately assessed for fertilization, specific gravity and osmolarity. Some of the eggs were stored for the quantitation of their water and lipid content as described below. The number of females used for each experiment is shown in Results. 2.2. Osmotic measurement To determine the osmotic conditions of the saline solutions used to measure the specific gravity of the eggs, the osmolality of the ovoplasm, the coelomic fluid, and the seawater used for artificial insemination was analyzed. Ovoplasm was prepared as described by Lønning and Davenport (1980) with slight modifications. About 0.2 g of the unfertilized eggs was placed into a 1 ml syringe, forced by the plunger and squashed through the needle (0.5 mm inner diameter) into a vial and centrifuged at 11,000 g for 10 min at 4 8C; the supernatant was then collected as ovoplasm. Coelomic fluid was collected by centrifuging 30 g of the unfertilized eggs at 760 g for 5 min at 4 8C. The osmolality of the ovoplasm, the coelomic fluid, the seawater as well as of the media for measuring the specific gravity described below was
T. Unuma et al. / Aquaculture 246 (2005) 493–500
measured by analyzing freezing point depression with a One–ten osmometer (Fiske, USA). To examine the effects of seawater on the osmolality of the ovoplasm, the unfertilized eggs were immersed in the seawater for 5 min at 23 8C, transferred to an Ultrafree-MC centrifugal filtering device (pore size, 5 Am; Millipore, USA) and centrifuged at 1400 g for 1 min to remove the seawater. The osmolality of the ovoplasm of the seawater-immersed eggs was analyzed in the same manner as described above. 2.3. Measurement of specific gravity of eggs An isotonic and a hypertonic series of media, each containing 31 media of different specific gravity, were prepared for measuring the specific gravity of the eggs. For the isotonic series, 148.5 mM NaCl containing 0.02% sodium azide was mixed with 297 mM sucrose containing 0.02% sodium azide at ratios of 60:40, 59:41, 58:42, ..., 32:68, 31:69, 30:70 (weight:weight). For the hypertonic series, 448.5 mM NaCl containing 0.02% sodium azide was mixed with 897 mM sucrose containing 0.02% sodium azide at ratios of 100:0, 99:1, 98:2, ..., 72:28, 71:29, 70:30 (weight:weight). The specific gravity of the eight solutions, 60:40, 50:50, 40:60 and 30:70, in the isotonic series, and 100:0, 90:10, 80:20 and 70:30, in the hypertonic series, was determined with a hydrometer at 23 8C. The specific gravity of each of the other media was calculated by linear equations from the values of the eight media. Each medium (4 ml) was dispensed into a test tube, then 20–30 eggs were added to each tube and stood for 5 min at 23 8C (Fig. 1). The specific gravity of the medium where the eggs were most evenly dispersed (Fig. 1D) was taken as the specific gravity of the eggs. The change in specific gravity (DG) from that under isotonic conditions to that under hypertonic conditions was calculated by the formula below: DG (%) = [100 (specific gravity under hypertonic conditions) / (specific gravity under isotonic conditions)] 100. The average osmolality of the media prepared was 310.0F8.7 mOsm/kg in the isotonic series and 875.1F30.8 mOsm/kg in the hypertonic series. The osmolality of the hypertonic series could not be raised above this value, since under higher osmolality the
A
Low
B
495
C
D
E
Specific gravity
F
G
High
Fig. 1. Method for determining the specific gravity of the eel eggs. Twenty to thirty eggs were placed in tubes filled with a series of isotonic (about 310 mOsm/kg) or hypertonic (about 875 mOsm/kg) media of various specific gravity values. The specific gravity of the eggs was taken as the specific gravity of the medium in which they were most evenly dispersed (D).
specific gravity of the eggs was sometimes lower than that of the NaCl solution, the lowest end of the series. In some teleosts including eels, the unfertilized eggs are induced to form a perivitellin space by contact with the environmental water (Suzuki, 1991), which may affect egg specific gravity. In our study, however, perivitellin space was not formed to a noticeable extent during the measurement of specific gravity of eel eggs under a stereoscopic microscope. 2.4. Fertilization test Two grams of the eggs were inseminated with 1 ml of the pre-diluted milt and then dispersed in 100 ml of the seawater as described by Ohta et al. (1996). Three replicates of 2 ml of the seawater containing about 80 eggs were transferred using a glass pipet to three Petri dishes, each filled with 30 ml of filtered seawater (pore size, 0.2 Am) containing penicillin G potassium (Banyu Pharmaceutical, Tokyo) at 100,000 IU/L and streptomycin sulfate (Meiji Seika, Tokyo) at 0.1 g/L. Four hours after insemination, eggs displaying embryonic cleavage were counted under a stereoscopic microscope and the hatched larvae were counted three days after egg insemination. The rates of fertilization and hatching were calculated for each female as the average of the three replicate incubations by the following formulae: Fertilization rate (%) = 100 the number of eggs displaying cleavage / the number of eggs stocked into the Petri dish; and
T. Unuma et al. / Aquaculture 246 (2005) 493–500
Hatching (%)
75 50 25
3. Results 3.1. Osmolality and specific gravity of coelomic fluid, ovoplasm, and seawater The osmolality of the coelomic fluid and the ovoplasm was 305.4F13.3 and 298.3F11.2 mOsm/ kg (N=7), respectively. No apparent correlation was found between these parameters and fertility or hatchability. The average osmolality and the specific gravity of the seawater collected on different days were 976.1F25.6 mOsm/kg (N=7) and 1.0248F0.0003 (N=4), respectively.
25
1.028
1.024
1.026
1.020
1.022
1.026
1.022
1.028
Specific gravity
89 88 87
Lipid (% dry weight)
C
90
35
D
30 25 20
1.028
1.024
1.026
1.020
1.028
1.026
1.022
1.024
Specific gravity
1.022
15
85
1.020
Values are presented as the meansFstandard deviation. All statistical analyses were carried out using Statview 5.0 software (SAS Institute, USA). Spearman’s correlation coefficients (r s) were used to examine significant ( Pb0.05) correlation by rank between two parameters. The Wilcoxon signed-ranks test was used to examine significant ( Pb0.05) differences between values.
50
Specific gravity 91
86
2.6. Statistical analysis
B
75
0
0
1.024
To remove coelomic fluid, the eggs were washed with 150 mM NaCl solution, centrifuged at 760 g for 5 min at 4 8C to remove the wash solution, and then stored at 80 8C until analysis. The water content of the eggs was measured by drying the sample at 110 8C for 8 h. The lipid content was determined by the gravimetric method using a Soxhlet device with diethyl ether as the solvent (Yamamoto et al., 2000) and calculated as dry matter.
100
A
1.020
2.5. Water and lipid contents
100
Fertilization (%)
Hatching rate (%) = 100 the number of hatched larvae / the number of eggs stocked into the Petri dish.
Water (%)
496
Specific gravity
Fig. 2. Relation between the specific gravity under isotonic conditions and some properties of eel eggs. A) Fertilization rate; B) hatching rate; C) water content; and D) lipid content. The vertical dashed line in each panel represents the average specific gravity of the seawater (1.0248).
ranged from 1.0200 to 1.0273; eggs from 3 females exhibited higher specific gravity than that of the seawater. Significant negative correlations were observed between egg specific gravity and fertilization rate (r s= 0.54; Pb0.001; Fig. 2A) and hatching rate (r s= 0.72; Pb0.0001; Fig. 2B). Most eggs of higher specific gravity showed very low fertility and hatchability, whereas eggs of lower specific gravity were not always of good quality, indicating that low specific gravity is a necessary but insufficient condition for good egg quality. A significant negative correlation was observed between egg specific gravity and water content (r s= 0.40; Pb0.01; Fig. 2C), whereas no apparent correlation was observed between egg specific gravity and lipid content (Fig. 2D), suggesting that water content contributes more to the buoyancy of eel eggs than does lipid content.
3.2. Specific gravity of eggs under isotonic conditions The specific gravity of eggs from 44 females in the isotonic media, with osmolality similar to that of the coelomic fluid and the ovoplasm, was analyzed and related to fertility, hatchability and the water and lipid content of the eggs (Fig. 2). The specific gravity
3.3. Specific gravity of eggs under hypertonic and isotonic conditions The specific gravity of eggs from 28 females was measured under hypertonic as well as under isotonic conditions (Figs. 3–6). The relation
Hatching < 30% 1.030
1.020
400 300
Fig. 5. Relation between the specific gravity of eel eggs under hypertonic conditions and the osmolality of the ovoplasm after immersion of the eggs in seawater.
Fig. 3. Relation between the specific gravity of the eel eggs under isotonic and hypertonic conditions. Circles and crosses represent high (N30%) and low (b30%) rates of hatching, respectively. The dashed lines represent the average specific gravity of the seawater (1.0248).
between the two measurements, with their respective hatchability divided into two grades of high (N30%; N=6) versus low (b30%; N=22) hatchability, is shown in Fig. 3. The specific gravity of eggs under isotonic conditions ranged from 1.0205 to 1.0248, whereas that under hypertonic conditions ranged from 1.0206 to 1.0404. Eggs from some females showed similar values under both conditions (lower left side of Fig. 3), with a larger number of such eggs demonstrating high hatching rates. On the other hand, eggs from some females showed lower specific gravity under isotonic conditions but higher values under hypertonic conditions (upper left side of Fig. 3), with most of such eggs demonstrating low hatching rates. Fig. 4 shows the relation between the change of specific gravity,
Hatching (%)
A
75 50 25 0
B
75
∆G (%)
90% sink
1.020 90% float 1.010 1.000
50 25 0
0.0 0.5 1.0 1.5 2.0
1.030
0.0 0.5 1.0 1.5 2.0 ∆G (%)
Fig. 4. Relation between the changes in specific gravity of eel eggs from isotonic to hypertonic conditions (DG) and the corresponding rates of fertilization (A) and hatching (B).
Hypertonic
100
1.040
Isotonic
100
after the transfer of eggs from isotonic to hypertonic conditions, and the corresponding rates of fertilization and hatching. A significant negative correlation was observed between the change of specific gravity and fertilization rate (r s= 0.52; Pb0.01; Fig. 4A) and hatching rate (r s= 0.52; Pb0.01; Fig. 4B). Most of the eggs with marked change of specific gravity under hypertonic conditions exhibited very low fertility and hatchability, whereas eggs with little change of specific gravity were not always of good quality, indicating that maintaining specific gravity after the transfer of eggs to a hypertonic medium is a necessary but insufficient condition for good egg quality. Fig. 5 shows the relation between the specific gravity under hypertonic conditions and the osmolality of the ovoplasm after immersion of the eggs in seawater for 5 min. A significant positive correlation was observed
Specific gravity
1.040
1.035
1.030
1.025
1.020
Specific gravity (hypertonic)
Specific gravity (isotonic)
Fertilization (%)
500
1.020
1.025
600
1.035
Hatching > 30%
700
1.040
1.035
800
1.025
1.040
497
1.030
Osmolality (mOsm/kg)
Specific gravity (hypertonic)
T. Unuma et al. / Aquaculture 246 (2005) 493–500
Fig. 6. Specific gravity at which about 90% of eggs sank (Fig. 1B) or floated (Fig. 1F) under isotonic and hypertonic conditions. Values represent the meansFstandard deviation (N=28). *Significantly different from the value where 90% of eggs floated under isotonic conditions ( pb0.0001).
498
T. Unuma et al. / Aquaculture 246 (2005) 493–500
between these parameters (r s=0.73; Pb0.001), suggesting that the eggs that lose buoyancy in seawater are incapable of maintaining the osmolality of the ovoplasm in seawater. The unevenness of the specific gravity of individual eggs from the same female was assessed by measuring the specific gravity of the media in which about 90% of the eggs sank (Fig. 1B) or floated (Fig. 1F), which represents the range of specific gravity covering about 80% of the eggs from the same female (Fig. 6). The values were 1.0202F0.0010 (90% sank) and 1.0231F0.0015 (90% floated) under isotonic conditions and 1.0193F0.0017 (90% sank) and 1.0286F0.0076 (90% floated) under hypertonic conditions. The lower values (90% sank) were very similar under both conditions, whereas the higher values (90% floated) were significantly different between isotonic and hypertonic conditions ( Pb0.0001). These results indicate that the specific gravity of individual eggs from the same female is almost uniform under isotonic conditions but highly variable under hypertonic conditions and that among the poor quality eggs from the same female, not all of the eggs always lose buoyancy under hypertonic conditions, with a number of eggs sometimes maintaining buoyancy.
4. Discussion In marine teleosts spawning pelagic eggs, egg buoyancy is acquired during the final maturation of oocytes through a reduction in ovoplasm density attributed mainly to massive influx of water (Craik and Harvey, 1987). At first, we assumed that failure to acquire sufficient buoyancy during maturation was the main cause of the poor quality of eel eggs that sink immediately in seawater after insemination. Therefore, we measured the specific gravity of eel eggs under isotonic conditions (Fig. 2). Consistent with our assumption, significant negative correlations were found between the specific gravity of eggs under isotonic conditions and egg fertility, hatchability, and water content. These correlations suggest that attaining low specific gravity by adequate hydration is a prerequisite for good egg quality in the Japanese eel. Eggs that exhibited poor buoyancy in an isotonic medium were, however, unexpectedly rare, despite the frequently observed
sinking of eggs immediately after insemination and transfer to seawater. These observations raised the possibility that the specific gravity of eggs measured under isotonic conditions does not necessarily reflect their eventual buoyancy in seawater. Determination of the specific gravity of eggs under hypertonic conditions (Figs. 3–6) revealed that eggs that acquired buoyancy sometimes lost it after transfer to the hypertonic medium. Only eggs that were able to maintain low specific gravity in the hypertonic medium as well as in the isotonic medium exhibited high fertility and hatchability. The change of egg specific gravity after transfer to the hypertonic medium was highly variable among batches of eggs from different females and also among individual eggs from the same female. The ability to maintain buoyancy in a hypertonic medium probably varies among individual eggs, whereas the initial acquisition of buoyancy probably progresses evenly in batches of eggs from the same female. Based on these data, the low buoyancy of poor quality eggs appears to be attributable to at least two causes. One is the failure of acquiring sufficient buoyancy because of inadequate hydration of the oocytes during final maturation, although this may not occur very often during the artificial induction of eel maturation. In marine teleosts that spawn pelagic eggs, vitellogenin-derived yolk proteins, such as lipovitellins and phosvitin, are proteolyzed into free amino acids (FAA) that act as osmotic effectors to induce water influx into maturing oocytes (Craik and Harvey, 1987; Thorsen et al., 1996; Matsubara and Koya, 1997). Seoka et al. (2003) have demonstrated that in eel eggs showing development of embryos several hours after insemination, FAA content is higher in buoyant eggs than in nonbuoyant ones, indicating that FAA content contributes to the buoyancy of eel eggs in seawater. Some disruption of the supply of FAA arising from yolk proteolysis during oocyte maturation may give rise to nonbuoyant eel eggs that are poorly hydrated. Another possible cause of nonbuoyant eggs is the inability of eggs to maintain specific gravity in seawater, and this occurs more often than problems with oocyte hydration during maturation. Dehydration and/or influx of salt probably occur in eggs that lose buoyancy in seawater. The system of osmoregulation through the plasma membrane (Alderdice,
T. Unuma et al. / Aquaculture 246 (2005) 493–500
1988) may be damaged in such eggs, probably to varying degrees in individual eggs from the same female. In the maturation-induced Japanese eel, deterioration of egg quality due to over-ripening of the eggs occurs in the ovarian cavity as a result of delayed stripping of the eggs after ovulation (Ohta et al., 1996). Over-ripening in the Japanese eel progresses relatively faster than in other teleosts (Ohta et al., 1996; Adachi, 2000). A decline in egg quality also ensues from the deterioration of oocytes in ovarian follicles if hormonal induction of oocyte maturation is not undertaken at the proper time before follicles become atretic (Adachi, 2000). Delayed hormonal induction of oocyte maturation and/or delayed stripping of the ovulated eggs may be associated with problems in the osmoregulatory system. In conclusion, both acquisition of egg buoyancy before ovulation (oocyte hydration) and maintenance of egg buoyancy after transfer to seawater are prerequisites for good egg quality in the Japanese eel. To reduce the incidence of nonbuoyant, poor quality eggs, insufficient hydration of the oocytes during final maturation needs to be eliminated and the inability of eggs to osmoregulate properly in seawater needs to be corrected. Further studies focusing on detailed clarification of the physiological mechanisms underlying these processes will lead to improvement of regimes for the artificial induction of female maturation, eliminating these obstacles to reliable production of seed stock for eel aquaculture.
Acknowledgements We are grateful to the staff of the Freshwater Resources Research Center, Aichi Fisheries Research Institute for providing the broodstock of the eel. We also thank Ryoko Okamoto at the National Research Institute of Aquaculture for technical assistance throughout the study. This study was supported, in part, by a grant-in-aid (Development of seed production and releasing techniques for stock enhancement of marine resources considering the conservation of ecosystem) from the Ministry of Agriculture, Forestry and Fisheries, Government of Japan.
499
References Adachi, S., 2000. Artificial control of maturation and spawning, and its relation to egg quality. Kaiyo Monthly 132, 120 – 126 (In Japanese). Alderdice, D.F., 1988. Osmotic and ionic regulation in teleost eggs and larvae. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, vol. XIA. Academic Press, San Diego, pp. 163 – 251. Carrillo, M., Bromage, N., Zanuy, S., Serrano, R., Prat, F., 1989. The effect of modifications in photoperiod on spawning time, ovarian development and egg quality in the sea bass (Dicentrarchus labrax L.). Aquaculture 81, 351 – 365. Craik, J.C.A., Harvey, S.M., 1987. The causes of buoyancy in eggs of marine teleosts. J. Mar. Biol. Ass. U. K. 67, 169 – 182. Furuita, H., Tanaka, H., Yamamoto, T., Shiraishi, M., Takeuchi, T., 2000. Effects of n-3 HUFA levels in broodstock diet on the reproductive performance and egg and larval quality of the Japanese flounder, Paralichthys olivaceus. Aquaculture 187, 387 – 398. Furuita, H., Tanaka, H., Yamamoto, T., Suzuki, N., Takeuchi, T., 2002. Effect of high levels of n-3 HUFA in broodstock diet on egg quality and egg fatty acid composition of the Japanese flounder Paralichthys olivaceus. Aquaculture 210, 323 – 333. Kagawa, H., Tanaka, H., Ohta, H., Okuzawa, K., Iinuma, N., 1997. Induced ovulation by injection of 17,20h-dihydroxy-4-pregnen3-one in the artificially matured Japanese eel, with special reference to ovulation time. Fish. Sci. 63, 365 – 367. Kagawa, H., Ohta, H., Tanaka, H., 2001. Recent progress of research on larvae production of Japanese eel, Anguilla japonica. Suisan Zoshoku 49, 127 – 132. Kondo, S., Yoshioka, M., Kashiwagi, M., 2001. Changes in specific gravity and osmolarity of eggs of Japanese whiting Sillago japonica during the embryonic development. Nippon Suisan Gakkaishi 67, 743 – 744 (In Japanese). Lønning, S., Davenport, J., 1980. The swelling egg of the long rough dab, Hippoglossoides platessoides limandoides (Bloch). J. Fish Biol. 17, 359 – 378. Matsubara, T., Koya, Y., 1997. Course of proteolytic cleavage in three classes of yolk proteins during oocyte maturation in barfin flounder Verasper moseri, a marine teleost spawning pelagic eggs. J. Exp. Zool. 278, 189 – 200. Mushiake, K., Kawano, K., Sakamoto, W., Hasegawa, I., 1994. Effects of extended daylength on ovarian maturation and HCGinduced spawning in yellowtail fed moist pellets. Fish. Sci. 60, 647 – 651. Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Hirose, K., 1996. Changes in fertilization and hatching rates with time after ovulation induced by 17,20h-dihydroxy-4-pregnen-3-one in the Japanese eel, Anguilla japonica. Aquaculture 139, 291 – 301. Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Iinuma, N., Hirose, K., 1997. Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica. Fish Physiol. Biochem. 17, 163 – 169. Seoka, M., Yamada, S., Iwata, Y., Yanagisawa, T., Nakagawa, T., Kumai, H., 2003. Differences in the biochemical content of buoyant and non-buoyant eggs of the Japanese eel, Anguilla japonica. Aquaculture 216, 355 – 362.
500
T. Unuma et al. / Aquaculture 246 (2005) 493–500
Suzuki, R., 1991. Developmental physiology and biotechnology. In: Itazawa, Y., Hanyu, I. (Eds.), Fish Physiology. KoseishaKoseikaku, Tokyo, pp. 585 – 609 (In Japanese). Tanaka, H., Kagawa, H., Ohta, H., 2001. Production of leptocephali of Japanese eel (Anguilla japonica) in captivity. Aquaculture 201, 51 – 60. Tanaka, H., Kagawa, H., Ohta, H., Unuma, T., Nomura, K., 2003. The first production of glass eel in captivity: fish reproductive physiology facilitates great progress in aquaculture. Fish Physiol. Biochem. 28, 493 – 497. Thorsen, A., Kjesbu, O.S., Fyhn, H.J., Solemdal, P., 1996. Physiological mechanism of bouyancy in eggs from brackish water cod. J. Fish Biol. 48, 457 – 477.
Watanabe, T., Itoh, A., Kitajima, C., Fujita, S., 1984a. Effect of dietary protein levels on reproduction of red sea bream. Bull. Japan. Soc. Sci. Fish. 50, 1015 – 1022. Watanabe, T., Itoh, A., Murakami, A., Tsukashima, Y., Kitajima, C., Fujita, S., 1984b. Effect of nutritional quality of diets given to broodstock on the verge of spawning on reproduction of red sea bream. Bull. Japan. Soc. Sci. Fish. 50, 1023 – 1028. Yamamoto, T., Shima, T., Unuma, T., Shiraishi, M., Akiyama, T., Tabata, M., 2000. Voluntary intake of diets with varying digestible energy contents and energy sources, by juvenile rainbow trout Oncorhynchus mykiss, using self-feeders. Fish. Sci. 66, 528 – 534.