Journal of Experimental Marine Biology and Ecology

295 (2003) 245 255

www.elsevier.com/locate/jembe

Comparison of ovarian functions for keeping

embryos by measurement of dissolved
oxygen concentrations in ovaries of copulatory
and non-copulatory oviparous fishes
and viviparous fishes
Youichi Hayakawa a,b,*, Hiroyuki Munehara b
b

a
Natural Science, International Christian University, 3-10-2, Osawa, Mitaka, Tokyo 181-8585, Japan
Usujiri Fisheries Laboratory, Field Science Center for Northern Biosphere, Hokkaido University, 152, Usujiri,
Minamikayabe-cho, Hokkaido 041-1613, Japan

Received 27 February 2003; received in revised form 3 May 2003; accepted 10 June 2003

Abstract
To investigate causes of anomalous development of embryos facultatively fertilized in the ovary
of a non-copulatory oviparous fish Hemilepidotus gilberti (Scorpaeniformes: Cottdae), dissolved
oxygen (DO) concentrations were measured in ovaries of copulatory oviparous (Alcichthys
alcicornis, Bero elegans), non-copulatory oviparous (H. gilberti, Hexagrammus otakii), and
viviparous (Sebastes taczanowskii, Zoarces elongatus) fishes. DO concentrations changed during
vitellogenesis and ovulation cycles, and also before and after ovulation. DO concentrations in the
ovary of H. gilberti and H. otakii at ovulation were 0.27 F 0.03 and 0.15 F 0.03 mg O2 l 1,
respectively, whereas in A. alcicornis and B. elegans, the concentrations were 0.47 F 0.08 and
0.20 F 0.06 mg O2 l 1, respectively. In the ovaries of intralumenal gestation viviparous fishes, S.
taczanowskii and Z. elongatus, DO concentration was from 0.01 to 0.11 mg O2 l 1. The average
DO concentration during the artificial pregnancy of A. alcicornis was 0.97 F 0.19 mg O2 l 1, but
all embryos showed deformity. DO concentrations recorded in oviparous fishes in this study were
lower than the oxygen level at which most oviparous fish embryos exhibit retardation or death, and
it probably caused the anomalous embryonic development. In contrast, the normal development of
viviparous fish embryos at low oxygen level was attributed to the specialized structure of ovary,

* Corresponding author. c/o Dr. Kobayashi Lab., Natural Science Department, International Christian
University, 3-10-2, Osawa, Mitaka, Tokyo 181-8585, Japan. Tel.: +81-422-33-3132; fax: +81-422-33-1449.
E-mail addresses: hyouichi@icu.ac.jp (Y. Hayakawa), hm@fish.hokudai.ac.jp (H. Munehara).
0022-0981/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0022-0981(03)00297-1

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Y. Hayakawa, H. Munehara / J. Exp. Mar. Biol. Ecol. 295 (2003) 245255

e.g. the dual arterial system to supply the developing embryos with the respiratory demands in
Sebastes.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Deformity; Embryonic development; Ovary; Oviparity; Oxygen; Concentration; Viviparity

1. Introduction
Reproduction of more than 20,000 teleostean species (Wourms, 1991) includes oviparity
and viviparity. In oviparity that most species adopt, males and females release sperm and
eggs in the aquatic environment almost at once. The longevity of gametes is commonly
short and fertilization need to occur immediately. Even if some eggs are left in the ovary,
they degenerate without fertilization. On the other hand, viviparity has been found in
approximately 510 species (Wourms, 1991). In those species, females copulate with males
and eggs are fertilized internally with sperm. Thus, embryos at various embryonic stages
are usually found in the ovaries among collected fishes.
Although the reproductive mode of cottid fishes is oviparous, copulatory and noncopulatory species are present (Breder and Rosen, 1966; Munehara et al., 1989; Hayakawa
and Munehara, 1996). Females of copulatory cottid fishes have no fertilized eggs in their
ovaries, because their eggs only associate with sperm in the ovary and fertilization occurs
externally after spawning (Munehara et al., 1989, 1991, 1997). In contrast, however,
females of a non-copulatory cottid fish Hemilepidotus gilberti, often have fertilized eggs in
their ovaries (Hayakawa and Munehara, 2001). Internal fertilization of H. gilberti is most
likely to be caused by sperm that invaded the ovary through the ovarian fluid which
surrounds eggs during a long spawning period (Hayakawa and Munehara, 2001). Embryos
in an ovary were found at various developmental stages, however, interestingly, all were
deformed and enveloped by unhardened chorions (Hayakawa and Munehara, 2001).
It is well known that teleost embryos need sufficient ionic materials, e.g. calcium and
magnesium, for successful development (Blaxter, 1969; Davis, 1975; Lnning et al., 1994).
Oviparous species receive those materials from the external medium, and viviparous
species obtain them from the secretions in the ovarian cavity (Hoar, 1969; Wourms et al.,
1988). Dissolved oxygen (DO) in the medium surrounding embryos is also one of the
important elements for embryonic development (Hishida and Nakano, 1954; Blaxter,
1969). Sensitivity to low oxygen tensions or anoxia varies both with species and stages of
development (Blaxter, 1969 and citations therein), but oxygen limitation often causes
retardation of development (Walsh and Lund, 1989). In addition, body deformities are a
common response of fish embryos to an environmental oxygen deficit (Keckeis et al.,
1996). Thus, anomalous embryos in the ovary of H. gilberti may have been caused either by
lack of certain chemicals or insufficiency of oxygen in the ovarian fluid, because it is
oviparous and its ovary is not adapted to supply them to embryos.
In the present study, we investigated ovarian functions in relation to oxygen supply. The
reproductive mode of Scorpaeniformes varies from oviparity including non-copulatory and
copulatory to viviparity (Breder and Rosen, 1966; Munehara et al., 1989; Wourms, 1991;
Hayakawa and Munehara, 1996). We considered that this order is suitable for comparative

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247

studies of different ovarian functions among teleosts. We measured DO concentrations

directly in ovaries of fishes belonging to the order Scorpaeniformes.

2. Materials and methods

Fishes of Scorpaeniformes used for measurement of DO concentrations were H. gilberti,
Alcichthys alcicornis, Bero elegans, Hexagrammous otakii and Senastes taczanowskii. A
viviparous eelpout Zoarces elongatus belonging to Perciformes was also used for
measurement. Fish were collected from the coastal waters of Usujiri and Akkeshi, in
Hokkaido, Japan. Reproductive characteristics of those fishes are as follows; a female H.
gilberti spawns a single clutch of eggs during the breeding season (October December)
each year (Hayakawa and Munehara, 1996). A. alcicornis and B. elegans are copulatory
cottid fishes (Munehara et al., 1989; Koya et al., 1993b) whose females repeat ovulation
and spawning several times during the breeding season (April May), and copulation is
performed after spawning in each mating (Munehara and Shimazaki, 1989; Koya et al.,
1994). Vitellogenesis is repeated before each ovulation (Koya et al., 1995). Eggs after
copulation in these cottid fishes only associate with sperm in the ovary. Internal fertilization
is inhibited by a deficiency of calcium ions in the ovarian fluid, and fertilization is initiated
by the rise in calcium concentrations following contact with seawater after egg deposition
(Munehara et al., 1989, 1994). H. otakii is oviparous, and females ovulate and spawn
frequently in one breeding season (October December). In contrast to copulatory cottid
fishes, vitellogenesis is completed before the breeding season (Munehara and Shimazaki,
1989). Sebastes taczanowskii is a viviparous fish showing intralumenal gestation that is
embryonic development progresses in the ovarian cavity (Takemura et al., 1987; Wourms et
al., 1988). They copulate more than 4 months before fertilization, and females release
neonates following a 2-month gestation period (Takemura et al., 1987; Takahashi et al.,
1991). Z. elongatus is viviparous with intralumenal gestation. Embryos are retained in the
ovarian cavity for more than 5 months after fertilization (Koya et al., 1993c).
DO concentrations in the ovarian fluid were measured with a YSI model 5300 DO meter.
The YSI model 5300 gives the relative value for the oxygen concentrations in a certain
solution rather than an absolute value. Prior to every measurement, the oxygen concentration (mg O2 l 1) of seawater was measured by using a YSI model 58 DO meter. The scale
of YSI model 5300 was calibrated using the oxygen concentration in seawater measured by
YSI model 58 as 100%. Measurement of DO using the YSI model 58 and calibration of the
YSI model 5300 were undertaken with seawater in the aquarium that fishes were kept
during experiments. The temperature was about 10 jC in accordance with the temperature
in the area they inhabit. The probe (about 4 mm in tip diameter  25 mm in length) of YSI
model 5300 was directly inserted in the ovary from the genital opening to measure the
oxygen concentrations, with gentle stirring to provide a constant current of fluid. Values
were converted into absolute values (mg O2 l 1). A small incision was made around the
genital pore of females of H. gilberti to insert the probe of YSI model 5300, because genital
opening of the fish is not opened until spawning.
DO concentrations in the ovary of H. gilberti were measured from pre-spawning period
(August September) to spawning period (October). Data for ovulated and non-ovulated

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females were compared in October. DO was measured in the ovary of A. alcicornis and B.
elegans before and after ovulation. The ovulation of females in those fishes was confirmed
by appearance of eggs from the ovary, because we can obtain eggs from the genital opening
by pressing the females belly when those fishes mature. After DO measurement, the
remaining eggs were squeezed instead of spawning. Following 2 3 days, DO was
remeasured to provide data for non-ovulated females. Furthermore, when females were
confirmed to ovulate again, DO in the ovary was remeasured. For H. otakii, measurement of
DO was carried out in the same way as for A. alcicornis and B. elegans, and fishes in the
pre-spawning period (September) were included in the measurements. The change of
oxygen level in the ovary of A. alcicornis which was artificially pregnant was measured as
embryonic development progressed. As stated above, A. alcicornis is a copulatory cottid
fish whose eggs are only associated with sperm in the ovary, and fertilization is initiated by
contact with seawater (Munehara et al., 1989, 1994). Thus, for an artificial pregnancy
experiment, internal fertilization was induced by pouring boiled seawater into the ovarian
cavity from the genital opening.
For S. taczanowskii, measurements were done on females in the late phase of the
gestation, and for females in the early phase of the vitellogenic period. Pregnant females of
Z. elongatus were used for measurement of DO. After measurement, vaseline was applied
to the genital opening of all fishes to prevent seawater from entering into the ovary.

3. Results
DO concentrations in the ovary of H. gilberti and H. otakii were higher in the prespawning period than in spawning period (Fig. 1). That of H. gilberti in August and

Fig. 1. Monthly changes of dissolved oxygen concentrations in ovaries of non-copulatory oviparous fish, H. gilberti
(a) and H. otakii (b). Data in October was obtained from combined data of non-ovulated and ovulated females.
Scales of vertical bar are different between two graphs. *Indicates data with significant difference ( p < 0.0001) from
other data in Schaffers ANOVA. **Indicates data with significant difference ( p < 0.05) in t-test.

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249

Fig. 2. Dissolved oxygen concentrations in the ovaries of non-copulatory oviparous fish, H. gilberti (a) and H. otakii
(b), before and after ovulation. Scales of vertical bar are different between two graphs. *Indicates data with
significant difference ( p < 0.05) in t-test.

September was 2.14 F 0.33 mg O2 l 1 (mean F S.D., n = 6) and 2.60 F 0.27 mg O2 l 1

(n = 8), respectively. It significantly declined in October to 0.27 F 0.03 mg O2 l 1 (n = 43,
Schaffers ANOVA, p < 0.0001; Fig. 1a). DO concentrations in the ovary of H. otakii in the
pre-spawning period (1.15 F 0.17 mg O2 l 1, n = 13) were significantly higher than that in
the spawning period (0.15 F 0.03 mg O2 l 1, n = 33, t-test, p < 0.05; Fig. 1b). Those
concentrations were even lower than those of H. gilberti.
DO concentration in the ovary of H. gilberti rose significantly after ovulation (t-test,
p < 0.05; Fig. 2a). The average value increased from 0.24 F 0.03 mg O2 l 1 (n = 36) to
0.47 F 0.08 mg O2 l 1 (n = 7). This tendency was also found in H. otakii, although not

Fig. 3. Dissolved oxygen concentrations in the ovaries of copulatory oviparous fish, A. alcicornis (a) and B.
elegans (b), before and after ovulation. Scales of vertical bar are different between two graphs. *Indicates data
with significant difference ( p < 0.05) in t-test.

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Y. Hayakawa, H. Munehara / J. Exp. Mar. Biol. Ecol. 295 (2003) 245255

Fig. 4. Change of oxygen concentrations in ovaries of A. alcicornis during artificial gestation.

significantly (t-test, p>0.05; Fig. 2b), DO rose from 0.12 F 0.02 mg O2 l 1 (n = 21) to
0.20 F 0.06 mg O2 l 1 (n = 12). On the other hand, DO in the ovary of A. alcicornis
significantly differed (t-test, p < 0.05) between non-ovulated (1.10 F 0.12 mg O2 l 1,
n = 85) and ovulated females (0.54 F 0.06 mg O2 l 1, n = 47), indicating a decline at
ovulation (Fig. 3a). That of B. elegans was also significantly different depending on the
conditions of females (non-ovulated; 0.51 F 0.06 mg O2 l 1 (n = 19), ovulated; 0.47 F 0.04
mg O2 l 1 (n = 13), p < 0.05; Fig. 3b). DO concentrations in the ovaries of nonpregnant and
pregnant females of S. taczanowskii were 0.11 mg O2 l 1 (n = 1) and 0.01 mg O2 l 1 (n = 1),
respectively. DO in the ovary of two pregnant Z. elongatus were 0.081 and 0.021 mg O2 l 1.
Fertilization induced by internal insemination with seawater was confirmed for 82% of
eggs in the ovary of A. alcicornis. DO concentrations in the ovary of A. alcicornis decreased
to the initial level after having risen (Fig. 4). The average DO concentration during artificial
pregnancy was 0.97 F 0.19 mg O2 l 1 (n = 10), which was not significantly different from
that of non-ovulated females ( p < 0.05). Most embryos reached morula or blastula stage
within 10 days, but they showed deformities such as loose cell aggregations with
unhardened chorions.

4. Discussion
4.1. Do concentrations in H. gilberti and H. otakii
DO concentrations in the ovaries of non-copulatory species declined as they approached
maturation. Values of DO concentrations in the ovaries of H. gilberti in the pre-spawning
period were 8 9.6 times as high as those in the spawning period.
Eggs of fish are located in the center of the follicle, and are surrounded by follicle cells
(Redding and Patino, 1993). During vitellogenesis, nutrients are accumulated as yolk
granules in the oocytes through follicular fluid in the follicle with hepatic synthesis

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251

regulated by hormonal controls. After vitellogenesis, oocytes are released from the follicle
to the ovarian cavity (reviewed in, e.g. Moyle and Cech, 2000). The period from August to
September when DO concentrations are very high corresponds to the period when
histological observations show yolk globules are accumulating in H. gilberti (Hayakawa,
unpublished data). After that, DO concentrations declined. At ovulation, the environment
surrounding oocytes changes from follicular fluid to ovarian fluid in the ovarian cavity
where the eggs remain until spawning. The ovarian fluid of cottid and hexagramid fishes is
intensively secreted from epithelia lining of the ovarian wall and from the ovigerous
lamellae as ovulation approaches (Koya et al., 1993a, 1995). These factors indicate that
decrease of DO concentrations may be related to approaching ovulation and secretion of
ovarian fluid. On the other hand, the female H. otakii repeats ovulation and spawn several
times in a breeding season. It is known that vitellogenesis of the congenetic species H.
octogrammus is completed within 1 month prior to spawning period (Munehara and
Shimazaki, 1989). Thus, the decrease of oxygen level must have occurred in the period that
corresponded to maturation. The exact reason why DO concentrations change of requires
further investigation. It is possible that newly secreted ovarian fluid has a low DO
concentration, and therefore the changes observed may reflect the secretory activity of
the epithelium. However, it is certain that the initiation of the secretion of ovarian fluid is
observed after the period of vitellogenesis. Thus, decrease of DO concentrations in ovaries
may represent a shift of ovarian function from vitellogenesis to egg storage.
4.2. DO concentrations before and after ovulation
Changes in the patterns of DO concentrations in the ovary before and after ovulation
differed between copulatory cottid fishes and non-copulatory species. DO concentrations
in the ovaries of the former decreased after every ovulation whereas those of the latter
increased after ovulation. Increase of DO concentrations in the ovary of non-copulatory
species is considered to be an adaptation to keep eggs until spawning, because the ovary
does not participate in vitellogenesis until next year. On the other hand, the change in
oxygen level in the ovary of copulatory species may be related to vitellogenesis and
ovulation. In copulatory cottid fish, ovulation and spawning is repeated several times in a
breeding season, and vitellogenesis takes place before each ovulation, thus differing from
H. otakii (Koya et al., 1995). It is known that, among multiple spawning fishes, the
secretion of oestradiol-17h from the ovarian follicle which induces vitellogenin synthesis,
and the rapid intensive secretion of GTH-II from the pituitary which stimulates ovulation
are repeated, and the latter leads to subsequent secretion of steroid act as oocytes
maturation inducing substances (MIS) (Nagahama, 1991; Kagawa and Asahina, 1991;
Tanaka, 1991; Redding and Patino, 1993). In those species, oestradiol-17h and MIS are
not secreted in large quantities simultaneously, and GTH-II is effective only for follicles
that contain oocytes close to ovulation (Nagahama, 1991; Redding and Patino, 1993). This
situation must apply to copulatory cottid fishes, and functional changes of ovary occur in
each cycle of oogenesis, although remarkable distinction between vitellogenesis and
ovulation periods are not present. In addition, Korsgaard (1994) demonstrated that
secretion of ovarian fluid in the viviparous fish Zoarces viviparous follows the secretion
of follicular fluid, and there is little contribution to store ovulated eggs and embryos even

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Y. Hayakawa, H. Munehara / J. Exp. Mar. Biol. Ecol. 295 (2003) 245255

if some secretion of follicular fluid continued after ovulation. Thus, the ovarian fluid of
multiple spawning fishes must be actively secreted at each ovulation while vitellogenesis
of the next clutch of oocytes is occurring. Therefore, the decrease in DO concentrations in
the ovary at ovulation may be related to intensive secretion of ovarian fluid, similarly to
that seen in non-copulatory species, a reflecting repeat cycles of vitellogenesis and
ovulation within a short period.
4.3. Oxygen levels and survival of embryos
The average DO concentration in the ovary of H. gilberti and H. otakii before and after
ovulation was less than 0.47 and 0.20 mg O2 l 1, respectively. In copulatory cottid fishes, it
was below 1.10 mg O2 l 1 in A. alcicornis and 0.51 mg O2 l 1 in B. elegans. Eggs
artificially fertilized in the ovary of A. alcicornis showed deformities at 0.97 F 0.19 mg O2
l 1. Several experiments have demonstrated responses to low oxygen levels near natural
conditions (reviewed in, e.g. Davis, 1975). For example, pacific cod embryos (fertilization
to hatching) decrease hatching rate at 2.0 3.0 mg O2 l 1 (authors cited in Davis (1975)).
The critical level of dissolved oxygen of the steelhead rises from 1.0 mg O2 l 1 at
fertilization to 7.5 9.7 mg O2 l 1 at stages close to hatching (Rombough, 1987). The early
embryos of the lumpfish, Cycropterus lumps, within 6 days of age are capable of
maintaining high oxygen uptake rates at about 1.2 mg O2 l 1, whereas embryos close to
hatching (about 36-day-old) require oxygen more than 2 times DO level. On the other hand,
DO in ovarian fluid measured in ovulated females of C. lumpus is between 2.77 and 7.13
mg l 1 (all values for this species are converted by the authors from Davenport, 1983). DO
levels that fish embryos exhibit critical response differ among species and developmental
stages, and are influenced by environmental factors such as temperature. However,
according to those data, eggs of oviparous species used in the present study are thought
to be placed under extremely hypoxic conditions in the ovary until being released,
conditions that do not support the survival or successful development of embryos.
Eggs of demersal fishes like cottid fish and lumpfish, which are initially sticky but
afterward adhere strongly to each other, have relative thick chorions require a water
current for their respiration. Under natural conditions, eggs of these fishes are commonly
spawned in areas where water movements are pronounced, e.g. among rocks and
vegetation in shallow waters (Alderdice and Velsen, 1971; DeMartini, 1978). Giorgi
and Congleton (1984) demonstrated that a water current is necessary for early embryos to
take up oxygen even though enough oxygen is dissolved in the water. The main reason
why early embryos need a water current is that respiration is cutaneous since branchial
function is not yet established (Pelster, 1999). Thus, residual eggs in the ovary are unable
to take up sufficient oxygen, because little current occurs in the ovary. On the other hand,
the eggs of the Pacific herring Clupea pallasi sustained different osmolarities between the
perivitelline fluid and the external medium (Alderdice et al., 1979 referenced in
Davenport, 1981). It has been revealed that Na+ enters the eggs from the environment
via diffusion across the chorion into the perivitelline fluid (Barrett et al., 2001). This
observation suggests that a difference of osmotic pressure between the inside and outside
of the chorion is important for embryos to absorb ionic materials. In addition, wastes from
embryos may affect embryonic development (Giorgi and Congleton, 1984). Therefore, it

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253

can be concluded that the ovary of oviparous fishes used in the present study is adequate
neither with respect to oxygen concentrations nor the physical conditions required for
successful embryonic development.
Apart from statistical reliability, DO concentration in the ovary of viviparous fishes was
from 0.01 to 0.11 mg O2 l 1. DO concentrations in the ovarian fluid of the viviparous pile
perch, Rhacochilus vacca, decrease as gestation proceeds, and the minimum value recorded
just before parturition is 1.02 mg O2 l 1 (authors converted from Webb and Brett, 1972a).
If DO concentration levels of S. taczanowskii and Z. elongatus measured in this study occur
widely, embryos of those fishes are placed under extremely low oxygen levels. Among
viviparous fishes, a maternal fetal relationship is established during pregnancy, and
specializations occur in both maternal and fetal structures (Wourms, 1981; Wourms et
al., 1988). Females of Sebastes have a specialized dual arterial system which meets the
respiratory demands of the developing embryos, and the post-ovulatory follicle is
considered to participate in this process (Moser, 1969; Wourms et al., 1988). In Z.
viviparus, the female has ovigerous processes which extend into the lumen during
oogenesis. They increase of the surface area of the lining of the ovarian lumen and
promote the secretion of ovarian fluid which contains nutrients (Wourms et al., 1988). On
the other hand, in Embiotocids, embryos in late gestation develop enlarged vertical fins to
increase their absorptive surface area. The fins also become highly vascularized to assist in
gas exchange (Webb and Brett, 1972b; Wourms et al., 1988). In addition, it has been
suggested that convection of ovarian fluid in the ovary during gestation is caused by
maternal muscle activity (Webb and Brett, 1972b). Thus, the normal embryonic development of viviparous fish in hypoxic conditions is facilitated by specialized ovarian functions,
the properties of the ovarian fluid, and the physiological and structural characteristics of the
embryos. Therefore, low oxygen levels and the paucity of water (ovarian fluid) currents in
the ovary are possible causes, for the anomalous development of H. gilberti.

Acknowledgements
We would like to thank the staff of Usujiri Fisheries Laboratory, Hokkaodo University,
and the Usujiri Fisheries Cooperative Society, for collecting the specimens. We appreciate
Prof. Richard E. Williamson, Australian National University for critically reading this
manuscript. We are also indebted to Mr. Nobuyoshi Ozawa, Meridian cooperation for
operation of YSI model 5300 DO meter. This study was supported in part by a Research
Fellowship of Japan Society for Promotion of Science for Young Scientists. [SS]

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