AN ABSTRACT OF THE THESIS OF
for the degree of
Martin Stone Fitzpatrick
Fisheries and Wildlife
Master of Science
in
presented on
2 April 1985
Title:
ENDOCRINE MEDIATION OF REPRODUCTION IN COHO SALMON
(ONCORHYNCHtJS KISUTCH).
Abstract approved:
Redacted for privacy
Carl B. Schreck
The plasma concentrations of estradiol-17, l7cthydroxy-2O-dihydroprogesterone, progesterone, 11ketotestosterone, testosterone, and gonadotropin were
measured in adult female and male coho salmon (Oncorhynchus
kisutch) concomitantly with the determination of gonad
maturation during the spawning season. In females with
eggs showing premigrating (central) germinal vesicles,
migrating germinal vesicles, or peripheral germinal
vesicles, estradiol-17 concentrations were higher than
those measured in females in which the eggs had undergone
germinal vesicle breakdown or ovulation. Conversely,
plasma levels of 17ct-hydroxy-2O-dihydroprogesterone were
low at the three earliest stages of oocyte development and
then increased dramatically at germinal vesicle breakdown
and ovulation. Testosterone, li-ketotestosterone, and
gonadotropin levels generally increased with maturity of
the eggs. In males, the levels of the hormones were stable
relative to those measured in females with the exception of
an increase in l7a-hydroxy-2O-dihydroprogesterone concentrations in the plasma during milt production.
Endocrine
profiles were also sketched according to date.
In females,
plasma estradiol-17
was high in fish returning from the
ocean to a seawater holding facility before dropping off
precipitously in females sampled at the end of the run.
Plasma 17a-hydroxy-2O-dihydroprogesterone followed the
opposite pattern, being low in fish sampled early and high
in fish sampled at the end of the run.
No easily
discernable patterns emerged from the profiles of the other
steroids and gonadotropin, although some significant
variation in the concentrations occurred.
In males, the
plasma concentrations of li-ketotestosterone, testosterone,
and l7c-hydroxy-2O-dihydroprogesterone increased during
thecourseof the runwiththe latterhormone being high
during milt production.
Utilizing the data for hormone
concentrations and date, a model can be constructed for
predicting the maturation stage (i.e. status of the ovary)
of the female.
When adult hatchery coho salmon return from the sea to
their natal hatcheries, they often remain in raceways for
extended periods of time before ripening.
In an attempt to
circumvent the significant mortality often associated with
this condition, as well as to obtain eggs when needed for
various rearing strategies, adult female coho salmon were
injected intraperitoneally with the luteinizing hormonereleasing hormone analogue (LH-RHa) des-G1y10[D-A1a6JLH-RHethylamide at various dosages to induce precocious final
maturation.
The mean number of days to ovulation in all
groups receiving LH-RHa was significantly lower than that
of saline-injected controls.
Within 13 days of the initial
injection, at least 84% of the fish receiving two
injections spaced 3 days apart of LH-RHa at various dosages
had ovulated, at least 67% of the fish receiving single
injections of LH-RHa at two dosages had ovulated, whereas
less than 50% of the controls had ovulated.
In all groups
receiving LH-RHa, the percent fertilization of the eggs was
slightly less than that of the control group.
Endocrine Mediation of Reproduction in
Coho Salmon (Oncorhynchus kisutch)
by
Martin S. Fitzpatrick
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed 2 April 1985
Commencement June 1985
APPROVED:
Redacted for privacy
Pr6fessdr of Fisheries and Wildlife in charge of major
Redacted for privacy
Head ofrDepartmenrtThf Fisheries and Wildlife
Redacted for privacy
Dean of
SchoQJ
Date thesis ispresented
2 April 1985
Acknowledgements
I acknowlege my major professor, Dr. Carl B. Schreck,
for his support, advice, and guidance throughout the
duration of my research.
I wish to thank him for having
faith in me.
Special appreciation goes to all the people of the
Oregon Cooperative Fishery Research Unit including Pat
Hulett, Rhine Messmer, Corey Hutchinson, and Amy Crook
for their assistance in the field and in the lab.
I wish to
thank Dave Oberbillig for the many hours he spent assisting
me with my part of the overall project.
He appears as a co-
author in Chapter III.
I wish to acknowledge the assistance and support of
Bruce Suzumoto, Dr. William McNeil, Frank Ratti, and Rob
Chitwood of Oregon Aqua Foods, Inc. in Springfield, Oregon;
Vern Jackson and Mike Bauman of Oregon Aqua Foods, Inc. in
Newport, Oregon; and John Hoskins and his staff at Fall
Creek Hatchery, Fall Creek, Oregon.
For their efforts in
collecting data, Bruce Suzumoto (Chapter III), Frank Ratti
(Chapter V), and Rob Chitwood (Chapter V) appear as coauthors in the designated chapters.
I wish to make a special acknowledgement to Dr. Edward
M. Donaldson, Glen van der Kraak, and Helen M. Dye of the
Department of Fisheries and Oceans, West Vancouver, B.C.,
Canada, for generously providing their technical expertise
in performing the gonadotropin assay.
A special thanks goes to Hillary S. Egna for providing
much needed morale and spiritual support, and for assisting
in the laboratory.
I thank all my friends who supported me
throughout my endeavors.
TABLE OF CONTENTS
Page
I.
II.
General Introduction
Profiles of plasma sex steroids and gonadotropin
in coho salmon during final maturation.
Introduction .................................
MaterialsandMethods ........................
Results ......................................
Discussion ...................................
III. LH-RHa induces precocious sexual maturation and
ovulation in coho salmon.
1
3
4
7
16
36
48
Introduction .................................
Materials andMethods .......................
Results ......................................
Discussion ..................................
49
52
59
IV.
Bibliography ......................................
64
V.
Appendix
71
Viabilities of eggs allowed to remain in the
body cavity after ovulation in coho salmon.
72
Introduction ...............
MaterialsandMethods ........................
Results and Discussion .......................
Bibliography .................................
61
73
75
79
82
LIST OF FIGURES
Page
Figure
Plasma concentrations of estradiol-17
(E2) and l7ct-dihydroxy-2O-dihydroprogesterone (DHP) during the final
sexual maturation of female coho salmon.
14
2
Plasma concentrations of 11-ketotestosterone (11-KT) and testosterone
(T) during the final sexual maturation
of female coho salmon.
18
3
Plasma concentrations of progesterone
(P4) and gonadotropin (GtH) during the
final sexual maturation of female coho
22
salmon.
4
Plasma concentrations of estradiol-17
(E2), li-ketotestosterone (11-KT), 17ahydroxy-2O-dihydroprogesterone (DHP),
testosterone (T), and gonadotropin (GtH)
during the final sexual maturation of
female coho salmon held in freshwater.
25
5
Plasma concentrations of estradiol-17
(E2) and 17ct-hydroxy-2O-dihydroprogesterone (DHP) during the final
sexual maturation of male coho salmon.
27
6
Plasma concentrations of 11-ketotestosterone (11-KT) and testosterone
(T) during the final sexual maturation
of male coho salmon.
30
7
Plasma concentrations of progesterone
(P4) and gonadotropin (GtH) during the
final sexual maturation of male coho
33
salmon.
8
Cumulated percent ovulation of female
coho salmon treated with single
injections of either saline, SPE and
LH-RHa, or LH-RHa.
55
9
Cumulated percent ovulation of female
coho salmone treated with two
injections of LH-RHa or with a single
injection of saline.
57
10
Percentage of viable eggs from females
stripped at various times after
ovuat ion.
77
LIST OF TABLES
Page
Table
I
Ovulatory response of coho salmon after
treatment with SPE and LH-RHa, various
doses of LH-RHa, or saline.
51
ENDOCRINE MEDIATION OF REPRODUCTION
IN COHO SALMON (ONCORHYNCHUS KISUTCH)
I.
GENERAL INTRODUCTION
Coho salmon, Oncorhynchus kisutch, are economically
important.
The success of a hatchery depends upon the
establishment and maintenance of a broodstock.
In order to
meet the demand for salmon, hatcheries must develop flexible
production strategies.
Attempts have been made to
understand and control various aspects of salmon physiology.
The endocrinology of reproduction is one area of research
offering potential tools for the use in hatchery practices.
A variety of hormones are considered to exert control
over the intricate physiology of reproduction in fish
(Donaldson 1973; Schreck 1974).
In maturing fish,
high concentrations of gonadotropin(s) have been found in
the blood (Crim etal. 1973, 1975; Billard et aj. 1977;
Fostier et al. 1978; Hontela and Peter 1978).
In vitro
studies have demonstrated the importance of gonadotropin(s)
in stimulating steroidogenesis (Kagawa et al. 1982; Nagahama
and Kagawa 1982) and also promoting final maturation of the
ovary (Jalabert 1976; Jalabert et al. 1978a; Sower and
Schreck 1982a).
The hypothalamus is presumed to have some
control over the secretion of gonadotropin(s) as has been
demonstrated in mammals (Schally et
],.
1973).
2
The gonads of teleosts produce steroids (Arai and
Tamaoki 1967; Idler 1969; Hoar and Nagahama 1978; van
Bohemen and Lambert 1978; Nagahama et al. 1982) and many of
these steroids are found in the plasma (Campbell et
1980).
j.
By convention, the determination of the plasma
profiles of various steroids and gonadotropin(s) constitutes
the best way to assess in vivo the dynamics and progress of
sexual maturation in teleosts.
Initially,
I described some of the endocrine changes
during sexual maturation in coho salmon and correlated
these changes with histological development of the gonads.
I measured estradiol-17, progesterone, 17ct-hydroxy-20-dihydroprogesterone, testosterone,
1 1-ketotestosterone, and
gonadotropin in coho salmon throughout the period of final
maturation.
I also developed a model that would allow the
prediction of the maturational state of female coho salmon.
A successful model would permit the determination of
ripeness based on physiological characteristics rather than
on secondary sexual characteristics.
Finally, I investigated the possibility of inducing
precocious final sexual maturation in females by treatment
with an analogue of a mammalian hypothalamic hormone.
'3
II.
Profiles of sex steroids and gonadotropin in the
plasma of coho salmon, Oncorhynchus kisutch,
during final maturation'
Martin S. Fitzpatrick
Carl B. Schreck2
Oregon Cooperative Fishery Research Unit3
Oregon State University
Corvallis, OR
97331
'Oregon State University, Agricultural Experiment Station
Technical Paper No.
whom reprint requests should be sent.
3Cooperators are Oregon State University, Oregon Department
df Fish and Wildlife, and U.S. Fish and Wildlife Service.
4
II.
Profiles of sex steroids and gonadotropin in the
plasma of coho salmon, Oncorhynchus kisutch, during
final maturation.
INTRODUCTION
A variety of hormones are considered to exert control
over the intricate physiology of reproduction in fish
(Donaldson 1973; Schreck 1974).
Many investigators have
found high concentrations of gonadotropin(s) from the
pituitary in the blood of maturing fish (Crim
.
1973,
1975; Billard et al. 1977; Fostier et al. 1978; Hontela and
Peter 1978).
In vitro studies have demonstrated the
importance of gonadotropin(s) in stimulating steroidogenesis
(Kagawa et al. 1982; Nagaharna and Kagawa 1982) and also
promoting final maturation in females (Jalabert 1976;
Jalabert et al. 1978a; Sower and Schreck 1982a).
The
hypothalamus is presumed to have some control over the
secretion of gonadotropin as has been demonstrated in
mammals (Schally et al. 1973).
King and Millar (1980) found
a hypothalamic substance from fish which was immunoreactive
with antisera raised against mammalian gonadotropinreleasing hormone and Sherwood et
j. (1983) isolated and
sequenced salmon gonadotropin-releasing hormone.
Van der
Kraak et al. (1984) recently showed that mammalian
gonadotropin-releasing hormone and superactive analogues
5
were potent stimulators of gonadotropin secretion in coho
salmon.
The gonads of teleosts produce steroids (Arai and
Tamaoki 1967; Idler 1969; Hoar and Nagahama 1978; van
Bohemen and Lambert 1978; Nagahama et al. 1982) and many of
these steroids are found in the plasma (Campbell et
1980).
j.
There is some indication that the interrenal may
also produce steroids which are important in sexual
maturation in some teleosts (Sundararaj and Goswami
1966a,b).
By convention, the determination of the plasma
profiles of various steroids and gonadotropin(s) constitutes
the best way to assess in vivo the dynamics and progress of
sexual maturation in teleosts.
The first objective of this study was to describe the
changes in circulating levels of specific hormones during
sexual maturation in coho salmon and to correlate these
changes to histological development in the gonads.
Sower
and Schreck (1982b) reported on the circulating levels of
estradiol-17, progesterone, and total androgens in coho
salmon during the spawning season--the
current study set
out to elaborate upon this earlier work by measuring not
only the concentrations of estradiol-17
and progesterone,
but also the levels of 17ct-dihydroxy-20dihydroprogesterone (17ct,20-dihydroxy--4-pregnen-3-one) and
two androgens: testosterone and 11-ketotestosterone.
In
addition, the plasma levels of gonadotropin were measured.
Sower et al. (1982a) attempted to induce ovulation in coho
salmon held in seawater using exogenous hormone treatment
with little success and Sower and Schreck (1982b) found
that the circulating levels of the various steroids
differedincoho thatwereheldjnseawaterfromthosethat
were held in freshwater; thus, it may be that the
susceptibility of coho salmon to hormonally-induced
ovulation may be in part compromised in seawater by an
alteration in the internal endocrine milieu of each fish.
The levels of the hormones described in the current study
were measured in coho salmon held in both seawater and
freshwater in order to determine if any differences
occurred in the endocrine profiles of fish held in either
environment.
A model was developed to predict the maturational
state of female coho salmon.
This model is based on the
information gathered about the circulating concentrations of
the hormones and also the concomitant determination of
oocyte development during the final phases of sexual
maturation. In addition, testicular development was measured
in the males in order to compare the hormone levels between
males at various stages of final maturation.
7
MATERIALS AND METHODS
The studies were conducted at the seawater facility of
Oregon-Aqua Foods, Inc. (OreAqua), Newport, Oregon during
1981 and 1982.
One group of fish was sampled at the
freshwater facility of OreAqua in Springfield, Oregon in
1981.
In addition, coho salmon returning to the State of
Oregon's freshwater hatchery at Fall Creek in 1982 were also
used in the study.
Seawater Studies.
From late September (24th and 30th
respectively) until late November (20th and 30th
respectively) in 1981 and 1982, groups of about 20 (10
females and 10 males) coho salmon on each sampling day were
killed and bled.
On seven of the 10 sampling days in 1981
and eight of the nine sampling days in 1982, gonads were
removed from the fish and fixed in buffered formalin
(females 1981; females and males 1982) or Bouin's solution
(1981 males).
Blood from the caudal vessel was collected
in lithium heparinized vacutainers and centrifuged; plasma
was drawn off and stored at _200 C until analyzed for
progesterone (P4), estradiol-17
dihydroprogesterone (DHP),
testosterone (T),
(E2), 17a-hydroxy-20-
11-ketotestosterone (11-KT),
and gonadotropin (GtH).
The gonads were
removed from the formalin or Bouin's fixative, washed with
water, held in 50Z ethanol (EtOH) overnight, and then
stored in 70Z EtOH.
Stages of oocyte maturation were
The
determined by histological examination of the ovaries.
stages of oocyte maturation in adult coho salmon, as
described by Sower and Schreck (1982b), were defined as
follows: (I) premigrating or central germinal vesicle
(CGV), (II) migrating germinal vesicle (MGV), (III)
peripheral germinal vesicle (PGV), i.e. germinal vesicle
against the chorion, (IV) germinal vesicle breakdown and
coalescence of lipid drops (GVBD), and (V) ovulation.
The
testes were also examined histologically and rated
according to the following criteria: (1) predominance of 1°
and 2° spermatocytes;
(2) predominance of spermatocytes and
spermatids; (3) spermatocytes, spermatids, and spermatozoa
present equally; (4) predominance of spermatocytes and
spermatozoa; (5) predominance of spermatids and
spermatozoa;
(6) predominance of spermatozoa;
(7)
production of milt, i.e. males gave milt when squeezed
gently on the abdomen.
Freshwater Studies.
On 24 November 1981, a group of 10
adult females and 10 adult males were sampled at spawning at
the Springfield facility of OreAqua.
These fish had been
transported by truck from the OreAqua seawater facility.
The
methods of plasma collection were the same as those
described for the seawater studies.
In 1982, a study was initiated to monitor the dynamics
of the endocrine system in individual females held
throughout final maturation.
A group of 10 female coho
salmon which had just entered the hatchery trap at Fall
Creek were anesthetized with CO2 gas, tagged in each
operculum, and then an aliquot of blood was collected from
the caudal vein of each fish in vacutainers which contained
heparin.
The fish were then returned to the hatchery
raceway for approximately three days when they were checked
for ripeness.
An aliquot of blood was collected and if a
fish had not ovulated, itwas returned to the raceway and
the process was repeated until ovulation.
The blood
samples were centrifuged; the plasma was drawn off and
stored at _200 C until analysis.
Assays.
The plasma levels of P4 and E2 from the fish
sampled in 1981 were measured by radioimmunoassay (RIA) as
described by Sower and Schreck (1982b) except that in the E2
assays, no y-globulin was added to assay tubes.
For the
1982 samples, the E2 assay was modified slightly to increase
the sensitiyity at higher concentrations.
The plasma levels
of DHP, 11-KT, and T were determined by RIAs set up for the
first time in our laboratory using the same generalized
procedure as the RIAs for the E2 and P4 (Sower and Schreck
1982b).
Each blood sample was extracted twice with diethyl
ether for E2, DHP, 11-KT, and T or hexane:benzene (2:1) for
P4.
Extraction recovery for each steroid was determined by
spiking 10 tubes containing plasma at the respective assay
10
volume with 5000 or 10000 dpms (disintegrations per minute)
of the appropriate [3H]steroid in phosphate buffered salinegelatin (PG) and putting each tube through the extraction
procedure.
The procedure entailed adding 1.5 ml of
extraction solvent to the plasma, vortexing, freezing the
aqueous phase in liquid N2, decanting and combining the two
extracts, and drying under N2 gas or air.
For P4, E2, and
DHP the replicate extracts were reconstituted each with 0.2
ml of PG (p11=7.0).
For extraction recovery efficiency, 0.05
ml PG-extract solution was removed to a scintillation vial
and counted.
1 ml of PG was added
For 11-KT and T assays,
to each extract tube and two 0.1 ml aliquots were removed
for the 11-KT assay (with 0.1 ml of PG added) and two 0.2 ml
aliquots were removed for the T assay.
For all assays, each
tube of extract and each standard tube received 0.1 ml of
the appropriate antiserum in PG (at the proper dilution)-the standard tubes contained steroid in a range of
concentrations from 3.8 to 2000 pg.
To each tube was added
0.1 ml of the appropriate [3Hjsteroid (10000 dpms in PG).
The tubes were incubated at room temperature for 90 minutes,
placed in an ice bath for 5 minutes, and then 1.0 ml of
dextran charcoal suspension (2.5 g charcoal and 250 mg
Dextran in 1.0 liter of phosphate buffer saline) was added
to each tube in the P4, DHP, 11-KT, and T assays or 0.5 ml
of dextran charcoal suspension (6.25 g charcoal and 4.00 g
11
Dextran in 1.0 liter PG) was added to each tube in the E2
assay.
The tubes were incubated for 15 minutes in an ice
bath, centrifuged at 2200 g for 20 mm, and then a 0.5
aliquot was decanted into Budget-Solve® (Research Products
International Corp., Mount Prospect, IL) scintillation fluid
and counted in either a Packard liquid scintillation
spectrophotometer or a Beckman LS 1800 liquid scintillation
system.
Progesterone was determined in 200 p1 of plasma or
serum.
from Dr.
Antiprogesterone antisera (11-BSA) was obtained
G. Niswender (Colorado State University, Fort
Collins, Colorado) and diluted to 1:2500 in PG.
The
interassay coefficient of variation for the P4 RIA of coho
plasma was 16.3%.
The lower limit of detection was about
7.8 pg per tube.
Estradiol-17
was determined in 100 p1 of plasma from
all fish in 1981 and males in 1982, and 25 p1 of plasma from
females in 1982.
Antiestradiol-17
antisera (S-244) was
obtained from Dr. G. Niswender and diluted to either
1:60,000 (1981) or 1:7500 (1982) in PG.
The antisera used
on the 1981 samples was from a batch obtained in 1979.
The
intra-assay and interassay coefficients of variation for
the E2 RIA were 8.1% (n=5) and 19.8% (n=5) respectively.
The lower limit of detection was about 3.9 pg per tube.
The following assays were set up for the first time in
our laboratory--each assay was verified using thin-layer
12
chromatography.
17ct-hydroxy-20-dihydroprogesterone was
determined in 25 p1 (or 10 p1 for expected high values) of
plasma.
Anti-17ct-hydroxy-20-dihydroxprogesterone antisera
was obtained as a gift from Dr. A.P. Scott (Ministry of
Agriculture, Fisheries Laboratory, Lowestoft, Suffolk,
The intra-
United Kingdom) and diluted 1:100,000 in PG.
assay and interassay coefficients of variation for the DHP
assay were 5.9% (n=6) and 12.9% (n=8) respectively.
The
cross-reactivity with cortisol, 17a-hydroxyprogesterone and
P4 was less than 0.1% (Scott et al. 1982).
il-ketotestosterone was determined in an equivalent of
2.5 p1 of plasma (diluted from 25 jil plasma).
Anti-il-
ketotestosterone antisera was obtained as a gift from Dr.
Y. Nagahama (Laboratory of Reproductive Biology, National
Institute for Basic Biology, Okazaki, Japan) and diluted
1:50,000.
The intra-assay and interassay coefficients of
variation for the i1-KT assay were 6.8% (n=7) and 10.9%
(n=7) respectively.
The crossreactivity with 11-hydroxy-
testosterone was 5.5% (Y. Nagahama, pers. comm.) and with T
was 1.8%.
Testosterone was determined in an equivalent of 5.0 jil
(diluted from 25 p1 of plasma) of plasma.
Antitestosterone
antisera (T3-125) was obtained from Endocrine Sciences
(Tarzana, California) and diluted 1:1,020.
The intra-assay
and interassay coefficients of variation for the T assay
13
were 7.8% (n=8) and 13.7% (n=5) respectively.
The
crossreactivity with 11-KT was about 4.7%.
Gonadotropin was determined by RIA with the assistance
of Glen van der Kraak, Helen Dye, and Dr. Edward Donaldson
(West Vancouver Laboratory, Fisheries and Oceans, West
Vancouver, British Columbia).
The assay used an antiserum
directed against chinook salmon GtH (see van der Kraak et
al. 1983).
Statistics.
Data for hormone concentrations were
analyzed by a Student-Newman-Keuls test, which computes the
significances of differences for samples of unequal sizes
after preliminary analysis of variance.
level of significance was P'(O..05.
In all tests, the
The model for determining
maturity from date and hormone concentrations was
constructed with a multiple regression statistical computer
package (Nie et al.).
A stepwise regression procedure in which
variables were added in the order of significance was
employed to construct the final models.
A lack of fit test
was used to determine the significance (p<O.O5) of the
addition of each variable to the model (Snedecor and
Cochran 1980).
14
Figure 1.
Plasma concentrations (ng/ml) of estradiol17
(E2) and 17-hydroxy-2O-dihydroprogesterone
(DHP) during final sexual maturation in female
coho salmon sampled in seawater (1981 and 1982)
Each value represents
and in freshwater (1981).
the mean and standard error of the indicated
number of samples. Plasma levels are
presented in relation to both date sampled (a,c)
and maturation stage (I=premigrating germinal
vesicle; II=migrating germinal vesicle; 111=
peripheral germinal vesicle; IV=germinal vesicle
breakdown; and V=ovulation) of the ovary (b,d).
Figure 1.
I
0
0.
w
C
E
1982
1981
SW
SEP
24
a
9
3058
30
I
A 1961 FW,
iaei
I
lie
-
20 25
1922 282
OCT
14
-
01982 8W
NOV
12 17 2024
510
I
-4 %
F I'
30
FEMALES
I
UI
IV
1981
MATURATiON STAGE
U
Jj
V
16
RESULTS
Females.
The circulating levels of E2 tended to
decrease during the spawning season.
The plasma
concentrations of E2 in most of the female fish that entered
the seawater recapture facility between 24 September and 12
November in 1981 were elevated in comparison to the
concentrations found in the fish that entered later on 17
and 20 November and in the fish which were spawned on 24
November in freshwater (at Springfield).
In 1982, the
plasma concentrations of E2 in females entering the facility
remained elevated throughout the sampling period until 30
November when the levels dropped precipitously (Fig. la).
Although a downward trend in the concentrations of the
steroid is apparent in Figure la, the variation between
individuals is pronounced.
In 1981, only the mean
concentrations from females sampled on 24 and 30 September
and 19 and 29 October were significantly different
(statistically) from the mean concentrations of fish sampled
on 17,
20, and 24 November.
If the data for 1981 are
examined with regard to maturation state of the ovary
instead of the date upon which the fish were sampled,
females in which the eggs had migrating germinal vesicles
(MGVs) or peripheral germinal vesicles (PGVs) had
significantly higher plasma concentrations of E2 than those
17
of fish in which the eggs showed germinal vesicle breakdown
(GVBD) or in which ovulation had occurred.
Similarly in
1982, females in which the eggs had central germinal
vesicles (CGVs), MGVs, or PGVs had plasma concentrations of
E2 higher than those of fish in which the eggs had undergone
GVBD or ovulation (Fig.
lb).
In contrast to E2, the circulating levels of DHP
increased during the spawning season.
The plasma
concentrations of DHP were low in females sampled in
September and October (1981) and were very high in females
sampled on 17 and 2ONovember in seawater and on 24November
in freshwater at Springfield (Fig.
lc).
The plasma
concentrations of DHP started to increase in females sampled
in midNovember in 1981, although the variability in levels
on some sampling dates was considerable.
The 10 fish
sampled on 2 November had a wide range of plasma DHP values
(2.8 to 76.6 ng/ml), whereas the three females sampled on 12
November all had low plasma levels of the. steroid.
All of
the females sampled in freshwater at Springfield (24
November 1981) had high plasma concentrations of DHP.
From
the end of September until 20 October in 1982, the plasma
concentrations of DHP remained low in females entering the
facility.
Individual females began showing elevated plasma
titers of the hormone between 25 October and 10 November.
By the end of November, the mean plasma level of DHP were
18
Figure 2.
Plasma concentrations (ng/ml) of li-ketotestosterone (11-KT) and testosterone (T)
during final sexual maturation in female
coho salmon sampled in seawater (1981 and 1982)
and in freshwater (1981). Each value represents
the mean and standard error of the indicated
number of samples. Plasma levels are
presented in relation to both date sampled (a,c)
and maturation stage (I=premigrating germinal
vesicle; II=migrating germinal vesicle; 111=
peripheral germinal vesicle; IV=germinal vesicle
breakdown; and V=ovulation) of the ovary (b,d).
19
w
C,
C,)
z
E
I-
=
Cl,
w
-J
0
'U,
p.
lU
IQ-
IL
--
'4
0
N
>
0
Z
p.
,-
N
0
N
0
N
N
0
0
0
N0
ON
0
I-
V
C-)
0
0
00
.0
.4 D
'U
0
N
OW/6U) .LN-L I.
U
N
6
0.
N
N
'
a.
LU
U)
00
0G
(IW/5U) I
0'
U.
20
significantly higher than any earlier group (Fig.
ic).
The
mean concentrations from females in which GVBD or ovulation
had occurred were significantly higher than those from
females in which the eggs had CGVs, MGVs, or PGVs in both
1981 and 1982 (Fig.
ld).
In 1982, females with PGVs had
significantly higher levels of DHP than did females with
oocytes containing CGVs or MGVs.
The levels of 11-KT tended to be variable throughout the
run.
The plasma concentrations of 11-KT in females ranged
from 0.6 to 21.7 ng/ml.
No readily discernable pattern
emerged from viewing the hormone concentrations relative to
date in 1981 except that the females which were spawned in
freshwater on 24 November had levels of 11-KT significantly
higher than those of females sampled in September, on two
dates in October, and on two dates in November (Fig. 2a).
In 1982, the mean plasma levels of 11-KT did not differ
significantly between females sampled on the various dates,
although the data hint of a trend towards higher levels as
the season progressed.
degree of maturation.
11-KT appears to increase with
In 1981, this trend is apparent as
ovarian development progressed, although not statistically
significant.
In 1982, the mean concentration of 11-KT from
females containing ovulated eggs was significantly higher
than the mean levels found in females with CGVs or MGVs
(Fig.
2b).
In general, the plasma levels of T tended to increase
21
in females (although not statistically significant) over the
period of the run and were an order of magnitude higher than
the concentrations of I1-KT.
The concentrations were low
in females sampled in late September (1981) and showed a
trend of higher levels thereafter (except for the three
females sampled on 20 November, all of which had low levels
of T).
The variation between individuals was pronounced and
only the levels of T from females that were sampled in
freshwater on 24 November were judged significantly higher
than the levels found in fish sampled in September (Fig.
2c).
A slight upward trend can be seen in the mean plasma
levels of T from females sampled on the various dates in
1982, although no significant differences were found.
In
1981, each successive stage of maturation showed higher mean
plasma levels of T and in fish which had ovulated, the mean
plasma concentration of T was significantly higher than that
of females in which the eggs had MGVs.
In 1982, females in
which the eggs contained CGVs had significantly lower levels
of T than females with PGVs (Fig. 2d).
In spite of
individual variation which precluded the identification of
significant differences between other maturity levels, it is
apparent that the females in which the eggs had PGVs, GVBD,
or in which ovulation had occurred had higher mean plasma
concentrations of T than the means from females with CGVs or
MGVs.
The mean plasma levels of P4 did not differ
22
Figure 3.
Plasma concentrations (ng/ml) of
progesterone (P4) and gonadotropin (GtH)
during final sexual maturation in female
coho salmon sampled in seawater (1981 and 1982)
and in freshwater (1981). Each value represents
the mean and standard error of the indicated
number of samples. Plasma levels are
presented in relation to both date sampled (a,c)
and maturation stage (I=premigrating germinal
vesicle; II=migrating germinal vesicle; 111=
peripheral germinal vesicle; IV=germinal vesicle
breakdown; and V=ovulation) of the ovary (b,d).
FIgure 3.
a.
E
1982
1981
:
50
19 22
SEP
OCT
29 2
01982 SW
3068 142025
24 30
?OIC
0.1
0.3
0.7
09
1981 FW
.1981 SW
I
I
NOV
510
12 172024
I,
-
Io
30
FEMALES
I
II
IV
MATURATION STAGE
H
1981
V
L.)
24
significantly between sampling dates or between maturation
stages in females (Figs. 3a,b).
The plasma concentrations
ranged from 0.1 to 1.1 ng/ml and remained constant
throughout the season and also through oocyte development.
The plasma concentrations of GtH were extremely
variable in females sampled in seawater in 1982.
Although
the mean levels of GtH on 10 and 30 November were much
higher than those from the other dates (Fig. 3c), the mean
concentrations did not differ significantly between sampling
dates.
The mean plasma concentrations of GtH were higher
for females in which the oocytes had PGVs, GVBD, or ovulated
than those of females with CGVs or MGVs; however, only the
PGV group had GtH levels significantly higher than the MGV
group (Fig. 3d).
No significant differences were found in the means of
any hormones from females at the same maturation stage which
had entered the seawater facility at various times
throughout the run.
In other words, a female with MCVs
entering in September had similar plasma levels of the
various steroids and GtH as a female with MGVs entering in
October or November.
The plasma concentrations of E2, DHP, 11-KT, T, and GtH
in females sampled in freshwater at Fall Creek hatchery were
similar to those found in the more mature (i.e. GVBDs or
ovulated) females sampled in seawater.
The mean plasma
concentrations of E2, DHP, 11-KT, T, and GtH in females
25
Figure 4.
Plasma concentrations (ng/ml) of: a)
estradio1-17 (E2), li-ketotestosterone (11-KT),
and gonadotropin (GtH); and b) 17ct-hydroxy-2Odihydroprogesterone (DHP) and testosterone (T);
in female coho salmon sampled in freshwater at
Each value
Fall Creek Hatchery in 1982.
represents the mean and standard error of the
indicated number of samples.
FEMALES
(freshwater)
I
a
E
1
a)
z
0
I4
I
I-
z
w
C)
z
0
C.)
4
U)
4
-J
a-
DHP T
7-14
Figure 4.
3-4
0
7-14
DAYS BEFORE OVULATION
DHP T
3-4
DHP T
0
0'
27
Figure 5.
Plasma concentrations (nglml) of estradiol17
(E2) and 17-hydroxy-2O-dihydroprogesterone
(DHP) during final sexual maturation in male
coho salmon sampled in seawater (1981 and 1982)
and in freshwater (1981). Each value represents
the mean and standard error of the indicated
number of samples. Plasma levels are
presented in relation to both date sampled (a,c)
and maturation stage (I=predominance of 10 and
20 spermatocytes; II=predominance of spermatocytes and spermatids; III=spermatocytes, spermatids, and spermatozoa present equally; IV=predominance of spermatocytes and spermatozoa;
V=predominance of spermatids and spermatozoa;
VI=predotninance of spermatozoa; and VII=milt
production, i.e. males gave milt when sqeezed
gently on the abdomen) of the testes (b,d).
Figure 5.
1901
1982
0
a.
C
I
01982 aw
SEP
OCT
3058 142025
24 30
1081 SW
510
NOV
MALES
MATURATION STAGE
1981
sampled in freshwater at the Fall Creek hatchery are shown
in Figure 4.
By noting the time of ovulation, the days
prior to ovulation were back-calculated and thus, the
hormone dynamics in the circulation over the two weeks
before ovulation were followed.
Although none of the mean
plasma levels of the hormones showed any significant
changes, some trends did emerge in the individual fish.
In
nine of 10 females, plasma E2 levels were lower and GtH
levels were higher at ovulation than prior to ovulation.
Plasma concentrations of DHP, 11-I(T, and T fluctuated before
ovulation, but not in any readily discernable pattern.
Males.
The plasma levels of E2 in males were low in
comparison to those measured in females.
The mean
concentrations remained constant in males sampled in
seawater and in males sampled in freshwater at Springfield
in 1981. (Fig. 5a).
Similarly, the mean levels of E2 in
males sampled in 1981 did not differ significantly when
viewed according to maturation state of the testes,
although a slight drop occurred in males with testes
predominated by spermatozoa and in males which were
producing milt.
In 1982, males which were taken at the end
of the run and males which were producint milt had mean
plasma E2 levels lower than the mean concentrations from
any other date or from any other maturity group (Fig. 5b).
The concentration of DHP increased in males as the
spawning run progressed.
The mean plasma concentrations of
Figure 6.
Plasma concentrations (ng/ml) of il-ketotestosterone (11-KT) and testosterone (T) during
final sexual maturation in male coho salmon
sampled in seawater (1981 and 1982) and in
freshwater (1981).
Each value represents
the mean and standard error of the indicated
number of samples. Plasma levels are presented
in relation to both date sampled (a,c) and
maturation stage (I=predominance of 10 and 20
spermatocytes; II=predominance of spermatocytes
and spermatids; III=spermatocytes, spermatids,
and spermatozoa present equally; IV=predominance
of spermatocytes and spermatozoa; V=predominance
of spermatids and spermatozoa; VI=predominance of
spermatozoa; and VII=milt production, i.e. males
gave milt when sqeezed gently on the abdomen) of
the testes (b,d).
31
I0
I-
w
C,
IC,)
z
0
I-
I-
WI-6N
0
CO
-9;:--
-i-'
>
0
Z
p.
N
10
J2
10
10
10
I-
9
S
32
DHP inmales entering the seawater facility on 17 and 20
November and in males spawned in freshwater on 24 November
were significantly higher than those of males sampled on any
other date during the 1981 run.
In 1982, males sampled on 5
and 30 of November had mean levels significantly higher than
those of males sampled in September or October (Fig. 5c).
In both 1981 and 1982, males which were producing milt had
a mean concentration higher (although not significantly for
1981) than that of any other group of males.
Males with
testes containing predominantly spermatozoa had mean levels
of DHP higher than those of males with any less developed
testes in both years (Fig. Sd).
In general, the mean plasma concentrations of 11-KT in
males showed an upward trend as time progressed.
In 1981,
the mean plasma 11-KT level was significantly higher in
males sampled on 17 November in seawater and 24 November in
freshwater at Springfield than those of males sampled in
late September.
In 1982, males sampled in November and the
latter part of October had higher mean levels than those
found in males sampled between 30 September and 8 October
(Fig. 6a).
Plasma concentrations of 11-KT were higher in
males than in females and tended to increase with time (in
1981 and 1982) and maturation state (in 1982) of the testes
(Fig. 6b).
In both 1981 and 1982, males sampled at some point
before the end of the run in the seawater facility had the
33
Figure 7.
Plasma concentrations (ng/ml) of progesterone
(P4) and gonadotropin (GtH) during final sexual
maturation in male coho salmon sampled in
seawater (1981 and 1982) and in freshwater
(1981).
Each value represents the mean and
standard error of the indicated number of
samples. Plasma levels are presented in relation
to both date sampled (a,c) and maturation stage
(Ipredominance of 10 and 20 spermatocytes;
IIpredominance of spermatocytes and spermatids;
III=spermatocytes, spermatids, and spermatozoa
present equally; IV=predominance of spermatocytes
and spermatozoa; V=predominance of spermatids and
spermatozoa; VI=predominance of spermatozoa; and
VII=milt production, i.e. males gave milt when
sqeezed gently on the abdomen) of the testes
(b,d).
Figure 7.
1901
1982
::
15.0
30
OCT
12 1
NOV
5 10
4rt
19 22
4
1°9
01982 SW
3058 142025
SEP
24
21.0 C
0.1
0.3
-Ia
0.7-I
0.5
1981 SW
Ioiei FW
30
2
MALES
MATURATION STAGE
35
highest mean concentrations of plasma T.
In 1981, the males
sampled on 22 October in seawater and sampled in freshwater
on 24 November had mean levels significantly higher than
those of any other group.
In 1982, males sampled on 5
October had a mean concentration significantly higher than
the means from six of the eight other groups sampled in
seawater (Fig. 6c).
The plasma levels of T in males were
generally lower than those found in females and were always
lower than the levels of il-ICT found in the same males.
No
significant differences were found in the means from the
males of the various maturity groups in either 1981 or 1982
(Fig. 6d).
The mean plasma levels of P4 did not differ
significantly between groups of males differentiated by
either date or testes maturity (Figs. 7a and 7b) in 1981.
Neither groups sampled on different dates nor groups with
the various stages of maturity had mean plasma
concentrations of GtH which were different from other
respective groups of males (Figs. 7c and 7d).
However, the
plasma levels of GtH were generally higher in males
throughout the run than those in all but the latest entering
females.
36
DISCUSSION
From the endocrine profiles, a model was
Females.
The
constructed to predict maturity state of the females.
general form of the model is:
Y
=
B0 + B11ogDHP + B2D
-
B3logE2
where Y is the maturity state (1=CGV, 2=MGV, 3PGV, 4GVBD,
and 5=ovulation), DHP and E2 are the plasma concentrations
of DHP and E2 respectively, D is the number of days from 1
January to the sampling date, and B0, B1, B2, and B3 are
the multiple regression coefficients.
For the 1981 data,
the addition of B4logT (where T is the plasma concentration
of T) significantly (P<O.05) increased the
model.
fit of the
Separate multiple regression models were con-
structed for each year.
The coefficients of determination
for 1981 and 1982 were 0.77 and 0.84 respectively--in other
words, 77%and84%of thetotal variationobservedinthe
maturity state of females could be accounted for by the
respective models.
Specifically, the models constructed for each spawning
run were:
(1981) Y = -6.59 +
0.721ogDHP
+
O.02D
- O.72logE2
+
O.02D
- O.62logE2
0.83logT
(1982) Y
=
-3.67
1.O9logDHP
+
37
Examination of the regression coefficients reveals that
logDHP, D, and logT are positively correlated with maturity
state whereas logE2 is negatively correlated.
This model depicts the strong correlation between the
endocrine environment, as reflected by the plasma
concentrations of the various hormones, and the development
of the ovary, as reflected by the distinct histological
stages of the eggs.
The model may have commercial
importance as the ability to manipulate the physiology of
reproduction in females (e.g. accelerate maturation, induce
ovulation) depends on the sensitivity of the females to
treatment, i.e. successful induction of ovulation using
exogenous hormones may depend not only on the dosage and
number of treatments, but also on the endocrine status and
ovarian development of the females.
Thus, determining the
levels of specific steroids in the plasma from a population
may allow for a prediction of the sensitivity of that
population to treatment with maturation-inducing hormones.
Sower and Schreck (1982b) found that the gonadosomatic
index (GSI),
i.e.
the percentage of the gonad weight in
relation to the somatic weight, in coho salmon increased
over the course of the spawning run.
In rainbow trout,
Salmo gairdneri, increasing GSI has been positively
correlated to an increase in plasma vitellogenin (van
Bohemen et al. 1981)--the yolk protein--which in turn has
been correlated to elevated E2 concentrations in the plasma
(van Bohemen and Lambert 1981; Bromage et al. 1982).
Rising
levels of plasma E2 and vitellogenin have been reported to
parallel increases in ovarian yolk lipophosphoprotein in
female brown trout S. trutta (Crim and Idler 1978).
The
elevated plasma titers of E2 in female coho salmon early in
the run and in fish with egg maturity stages of CGV, MGV,
and PGV suggest that these fish are undergoing
vitellogenesis.
Several steroids have been reported to bind
to specific regions of the brain (Kim et al. 1978; Demski
and Hornby 1982).
The high concentrations of E2 in the
plasma generally correspond to low levels of plasma GtH,
supporting the contention that in mature teleosts E2
inhibits the secretion of GtH through negative feedback on
the hypothalamus and/or pituitary (Billard and Peter 1977).
With the end of vitellogenesis comes the onset of GVBD and
finally ovulation--events which do take place in coho salmon
held in seawater, but evidence suggests that fertility may
be impaired through dysfunction in the regulation of ions
and osmolality (Sower and Schreck 1982b).
The low plasma
levels of E2 at these final stages of oocyte maturation
indicate that steroid production has been shifted away from
the aromatization pathway and the high GtH levels suggest
that the negative feedback of E2 to the brain has been
curtailed or that low levels of E2 exert a positive
influence upon GtH secretion.
39
Since the isolation of DHP from the plasma of sockeye
salmon, Q. nerka, (Idler et al. 1960), this hormone has been
found in high concentrations in the plasma of rainbow trout
(Campbell et al. 1980; Scott et al. 1983), amago salmon, Q.
rhodurus, (Young et al. 1983, Ueda et al. 1983), and chum
salmon, 0. keta, (Ueda et al. 1984).
In vitro studies have
shown that this steroid is a potent inducer of final
maturation in a number of teleosts (Jalabert 1976; Jalabert
et al. 1978a,b; Suzuki et al. 1981; Sower and Schreck 1982a;
Nagahama et al. 1983).
The high levels of DHP in the plasma
of coho salmon at GVBD and ovulation suggest that this
steroid is important during these two phases of egg
development, although in vitro research has shown that it
cannot induce ovulation without the addition of GtH
(Jalabert et al. 1978a).
In fact, plasma concentrations of GtH were elevated in
females which had ovulated and in which the eggs had PGV or
GVBD; however, the individual variation in concentrations
was pronounced--suggesting that maximal secretion of GtH may
not be required for final maturation.
In addition, the
extent of the variation in the capacity of individual
females to produce and secrete GtH is unknown.
Despite the
variability between concentrations in individuals, the
plasma levels of GtH in females sampled over the period of
final maturation in fresh water did follow a pattern,
GtI-I concentrations were higher at ovulation than before
i.e.
40
ovulation in 9 of 10 fish.
Although the plasma concentrations of DHP increase
dramatically during final maturation, this steroid may not
be the final inducer of maturation.
The oocyte maturation
of amphibians is also stimulated by a progestin--P4--which
acts by binding to the surface of the egg (Masui. and Markert
1971) and stimulating the production of a maturationpromoting factor within the egg (Masui and Clarke 1979;
Masul and Markert 1971; Mailer and Krebs 1977; Kanatani and
Nagahama 1980).
The process in teleosts may be analogous.
In addition, prostaglandins have been implicated as
inducers of ovulation in teleosts (Jalabert 1976; Cetta and
Goetz 1982; Stacey and Goetz 1982).
The high plasma
concentrations of DHP in coho salmon may indicate that this
steroid has more influence than just on the maturation of
oocytes, but it is unknown if and how DHP affects, for
instance, the central nervous system.
The plasma dynamics of T and 11-KT seem to "anticipate"
the increase in plasma DHP concentrations during the final
stages of oocyte development.
Despite the concentrations of
T being about 10-fold higher than those of 11-KT, both
androgens show the same profile of rising throughout oocyte
maturation.
Scott and Sumpter (1983) suggest that the high
T levels in female rainbow trout are due to superactivity of
the ovarian steroidogenic cells during oocyte maturation.
They postulate that the rapid rise in 17c-hydroxylated
41
progestagens (i.e.
DHP and l7cL-hydroxyprogesterone) just
before ovulation in trout occurs through the blockage of
ovarian androgen synthesis.
However, in coho salmon,
apparently no blockage occurs as both T and 11-KT
concentrations continue to rise in the plasma through
ovulation.
Testosterone production by the thecal layer of
the ovarian follicle has been reported to be a necessary
step in estrogen synthesis by the granulosa cells (Kagawa et
al. 1982; Nagahama et al. 1982; Young et al. 1982); however,
T levels in the plasma are 10-fold higher than those of E2
and T levels in the plasma are high throughout the
maturation period whereas E2 concentrations fall off
precipitously as ovulation approaches.
Testosterone may
perform other roles besides that of serving as a reservoir
for the production of other steroids.
Crim and Evans (1979)
demonstrated that treatment of juvenile rainbow trout with T
resulted in increased concentrations of pituitary GtH,
Soivio et al. (1982) showed that T was metabolized in the
skin of brown trout, Sower et al. (1983) showed that 17 methyltestosterone increased skin thickness, and Nagahama et
al. (1980) reported that although not as effective as DHP, T
could induce GVBD in amago salmon and rainbow trout.
Testosterone was particularly elevated in the plasma of
female coho salmon spawned in freshwater which may be
indicative of a function during ovulation or a role in
osmoregulation.
42
The role of 11-KT in females is not known.
The
importance of 11-KT during the final maturation of females
has been discounted because of low or non-detectable levels
in the plasma of rainbow trout (Scott et al. 1980a) and the
winter flounder, Pseudopleuronectes americanus (Campbell et
al. 1976); however, the levels of this steroid in the plasma
of Pacific salmon reported previously (Idler et al. 1961)
and here, suggest that its function may be largely
uninvestigated rather than unimportant.
Idler et al. (1961)
found that injections of 11-KT increased skin thickness and
coloration in female sockeye salmon.
Testosterone and 11-KT
may work synergistically with the other sex steroids to
regulate the complex physiological and behavioral events
which comprise final maturation and spawning in coho salmon.
The question of whether one or two GtHs exist makes
interpretation of the GtH data difficult.
Schulz
aJ.
(1981) reported that GtH acts by increasing cyclic
adenosinemonophosphate (cAMP) concentrations in the gonads
of rainbow trout, and Fontaine-Bertrand et al. (1978) showed
that incubating pieces of ovaries of eel, Anguilla anguilla,
with carp GtH increased the levels of cAMP in the media.
It
may be through the production of cAMP that the elevated
concentrations of GtH observed in maturing coho salmon
females act to increase the ovarian synthesis and release of
steroids, prostaglandins, and/or the maturation-promoting
43
factor.
The mean levels of the various hormones in females held
in freshwater at Fall Creek throughout the 2 week period
ending at ovulation corresponded to the mean levels found in
females which entered the seawater facility at Newport late
in the run, i.e. mid to late November.
This suggests that
by the time the Fall Creek females were sampled (late
October), vitellogenesis was complete and GVBD had
commenced.
The magnitude of individual variation in hormone
levels suggests that absolute plasma concentrations may not
be exclusively important in the determination of maturation
status, which may explain the prominent position date has in
the model presented above.
Males.
Since all the males that underwent histological
examination of the testes had spermatozoa present and the
majority of males sampled in both years were producint milt
or had spermatozoa as the most prevalent cell type in the
testes, it appears that males entering the seawater
facility were near the end of spermiogenesis.
This
reproductive "strategy" means that the reproductive
physiology of the male must essentially "wait" for that of
the female to undergo final maturation.
This situation is
evident in salmonids in general where the males typically
ripen before the females.
It is not surprising that most
of the hormone levels showed stability relative to the
profound changes observed in those of the females.
44
Of all the steroids measured, DHP showed the most
prominent changes during maturation in the males.
The high
concentrations late in the run and in males which were
producing milt supports the contention that DHP plays an
important role in the process of milt production in
salmonids (Scott and Sumpter 1983; Ueda
j. 1983).
The levels of 11-KT in the plasma were higher than
those of T in the males which is consistent with
observations made in other salmonids (Campbell et al. 1976,
1980; Idler et a].. 1971; Scott et a].. 198Ob; Hunt et a]..
1982).
Scott et a].. (1980b) suggested that 11-KT played a
major role in spermiation (i.e. milt production) in rainbow
trout; however, no major increase in the plasma
concentrations of this steroid occurred in coho salmon
which were producing milt.
The concentrations in males
were elevated throughout the run--generally higher at the
end than at the beginning--implying that 11-KT may play
some role in the development of spermatocytes into
spermatozoa and perhaps the maintenance of spermatozoa.
The influence of 11-KT may not be limited to testicular
development.
Idler et a].. (1961) reported that treatment
of male sockeye salmon with 11-KT increased skin thickness,
elongation of the snout, and redness of the skin.
The mid-run peak in the mean plasma concentrations of T
did not correspond to any particular maturity level in males
45
although the levels appeared higher prior to spermiation.
Scott et al. (1980b) suggested the possibility that the
presence of T in the plasma of male trout may be incidental
because it is one of the intermediate products in the
synthesis of il-ICT; however, T has been shown to have a
variety of other effects including stimulation of GUT
synthesis in juvenile rainbow trout (Crim and Evans 1979)
and it is known to be metabolized by the skin of brown
trout (Soivio et al. 1982).
The high levels of T observed
in males held in freshwater during the production of milt
relative to those in males of the same maturity held in
seawater may indicate an osinoregulatory role for this
steroid in freshwater or endocrine dysfunction in fish
retained in seawater.
Billard et al. (1977) demonstrated
that castration of maturing rainbow trout resulted in
increased GUT levels in the plasma, thus suggesting the
existence of negative feedback by either T or 11-KT, and
Demski and Hornby (1982) reported that teleosts concentrate
tritiated T in areas of the brain.
Thus the role of T in
physiology other than sperrniogenesis may not be
insignificant.
In the literature, the plasma levels of E2 in males is
largely ignored due to the lack of prominence of this
steroid relative to other steroids in males and relative to
levels found in females.
Despite the low concentrations and
the change is assay protocol (which affected sensitivity of
46
the assay), males which were producing milt showed a
decrease in the plasma levels of E2 in comparison to males
at other stages of spermiogenesis for both years.
Whether
this finding is coincidental or important is unknown.
Throughout the run, males had higher plasma levels of
GtH than did the females early in the run.
The males
entering the seawater facility were already in a mature
state and therefore, baseline levels of GtH are probably
unobserved.
The steady secretion over the course of the run
and at all the various maturity stages suggests that a
general stimulation by GtH of steroid production in the
testes is occurring.
The lack of a peak in plasma GtH
corresponding to milt production once again raises
questions of the existence of a second GtH and of the need
for maximal secretion to influence milt production.
Crirn
(1975) in a review article reported that the
pituitary is necessary for mitotic division of
spermatogonia, the transformation of spermatogonia into
primary spermatocytes, and spermiation in salnionids.
Most of the endocrine profiles of fish reported in the
literature present concentrations of hormones relative to
date of sampling.
Such a practice carries the implication
that the populations under study were synchronous in
maturation--this is not the case with the coho salmon
sampled in this study, especially the females.
The model
47
developed here had a high degree of success in predicting
the maturational status of the oocytes through knowledge of
specific endocrine parameters and date.
It appears that
the significance of certain changes in the plasma
concentrations of hormones can be better understood by
viewing these changes relative to the development of the
gonads rather than simply by date.
III.
LH-RHa induces precocious sexual maturation
and ovulation in coho salmon1
Martin S. Fitzpatrick
Bruce K. Suzumoto2
Carl B. Schreck3
David Oberbillig
Oregon Cooperative Fishery Research Unit4
Oregon State University
Corvallis, OR.
97331
1Oregon State University Agricultural Experiment Station
Report Number
7008.
2Oregon Aqua Foods, Inc., 88700 Marcola Road, Springfield,
Oregon
97477.
Present address:
Prince William Sound
Regional Aquaculture Corporation, Cordova, Alaska.
3To whom reprint requests should be sent.
4Cooperators are Oregon State University, Oregon Department
of Fish and Wildlife, and U.S. Fish and Wildlife Service.
III.
LH-RHa induces precocious sexual maturation and
ovulation in coho salmon.
INTRODUCTION
The acceleration of the final processes of sexual
maturation in salmonids is desirable for various hatchery
practices, and exogenous hormone treatments are effective
for this purpose (Jalabert et al. 1978b, Hunter et al.
1981, Sower et al. 1982).
The most successful treatment to
date consists of an injection of partially purified salmon
gonadotropin (SG-G100) or salmon pituitary extract (SPE)
followed by an injection of the luteinizing hormonereleasing hormone analogue (LH-RHa) des-G1y10{D-A1a6}LH--RHethylamide.
Although this treatment results in a
significant acceleration of final maturation, the cost and
difficulty of obtaining the salmon pituitary products make
this treatment very expensive for use on a production scale.
In this study, we determined whether LH-RHa could be
used alone to induce ovulation in coho salmon, thereby
obviating the need for exogenously administered salmon
pituitary products.
We evaluated the effects of LH-RHa at
various doses when administered in single or in two serial
injections.
Since the technique of administering SG-G100 or
SPE followed by LH-RHa requires handling the fish several
times, which causes stress, we also investigated the
50
possibility of using a single combined injection of SPE and
LH-RHa to accelerate maturation.
TABLE I.
Ovulatory response of coho salmon after treatment with lyophilized pituitary extract (SPE) and luteinizing
Injection protocol,
hormone-releasing hormone analogue (LH-RHa), different doses of LH-RHa, or saline.
mean number of days to ovulation, and mean percent fertilization of the eggs.
Treatment
group
No. of
females
Injetion protocol
dosage 1jg/kg body weight
Day 0
Day 3
Days to
Ovulation
Percent
fertilization
Mean
S.E.
Mean
S.E.
1
19
Saline
--
21.1
2.4
91.2
3.1
2
20
SPE(500.0) and
LH-RHa(5.0)
--
20.1
2.8
85.3
3.5
3
21
LH-RHa(50.0)
--
14.4
1.9
73.4
5.8
4
22
LH-RHa(50.0)
LH-RHa(5.0)
124
1.2
82.9
4.0
5
21
LH-RHa(5.0)
--
13.9
1.8
80.3
4.2
6
25
LH-RMa(5,O)
LH-RBa(5.0)
13.0
1.4
85.0
3,3
7
25
LIL-RHa(O.5)
LIi-Rlla(O.5)
13.0
1.4
86.9
2.3
U,
52
MATERIALS AND METHODS
Adult coho salmon from the seawater facility of Oregon
Aqua Foods, Inc. at South Beach, Oregon, were transported by
truck to the freshwater hatchery at Springfield, Oregon.
On
October 15, 1982, adult female coho salmon exhibiting
coloration indicative of advanced sexual maturity were
sorted from the hatchery broodstock population.
The fish
were anesthetized with ethyl P-aminobenzoate (benzocaine)
and marked according to treatment group (25 fish each) with
colored, 2.2-cm Petersen disk tags anchored below and
slightly posterior to the dorsal fin.
injected with LH-RHa at 50.0,
5.0,
The fish were then
or 0.5 ug/kg body weight;
a combined injection of 0.5 mg/kg body weight of chum salmon
(Oncorhynchus keta) lyophilized pituitary extract (SPE) and
5.0 i.ig/kg body weight of LH-RHa; or saline.
Three days
later, some groups were injected with 5.0 or 0.5 ug/kg body
weight of LH-RHa (Table I).
The SPE and the LH-RHa were obtained from Syndel
Laboratories Ltd., Vancouver, British Columbia, Canada.
Hormone preparations were dissolved in sterile 0.6% saline
and injected in volumes of 0.5 ml.
All injections were
administered intraperitoneally on the ventral side of the
fish about midway between the vent and pelvic fins.
Experimental fish were held in a concrete raceway (20.4. x
53
6.1 m) for the duration of the study.
maintained at 0.9 m.
13.10 C (mean, 9.20 C).
Water depth was
Water temperatures ranged from 50 to
Fish were sorted and checked for
ripeness every 3 to 4 days.
After ovulation, 100 eggs were
removed from each female and fertilized in separate
containers.
Individual males were used to fertilize eggs
from an average of 5 females of several different treatment
groups.
Each group,of eggs was then incubated in Heath
Techna tray-type incubators at 10° C.
After 10 days,
percent fertilization was determined by clearing the eggs in
a solutionof l0Zaceticacidandthencounting thenumber
of eggs with developing embryos.
A total of four fish died prior to ovulation.
Two died
on day 0--probably due to handling stress--and were not
considered in the analysis.
The other two mortalities were
taken into account when determining the cumulative percent
ovulation of the population.
A total of five fish from four
different groups were unripe when spawned and were not
considered in the analysis, as it was impossible to
determine the date of ovulation.
In all other fish, the eggs
flowed freely from the body cavity when the females were
spawned.
One-way analysis of variance (Snedecor and Cochran
1980) was conducted on the data for days to ovulation and
fertility.
The data were then subjected to Duncan's
54
Multiple Range Test to determine significant differences
between specific treatments and between specific days.
55
Figure 8.
Single injection experiment. Induced ovulation
of coho salmon (Oncorhynchus kisutch). The
numbers of fish which ovulated following
injections of saline, lyophilized pituitary
extract (SPE) and luteinizing hormone-releasing
hormone (LH-RHa), or LH-RHa are expressed as
cumulative percentages of their respective
Arrow indicates day on which the
groups.
injections were administered.
Ap
100
90
Ac
80
70
/ 1"
JJ
o
i_
60
Oaise"uiuCJ..u(..,
1
50
IT
/,
40
I
/)
0 LH-RHa 50Opg/kg
A LH-RHa 5.Oig/kg
/,' .1 /
/,'.P
30
,"
20
10
/
DLPE 500.Opg/kg
I
/
+ LH-RHa 5.Opg/kg
oSaline
DAY 0
A
Figure 8.
7
10
13
17
21
24
28
3335
40
57
Figure 9.
Induced ovulation
Serial injection experiment.
of coho salmon (Oncorhynchus kisutch). The
numbers of fish which ovulated following
injections of saline or luteinizing hormonereleasing hormone analogue (LH-RHa) are expressed
as cumulative percentages of their respective
Arrows indicate the days on which the
groups.
hormone injections were administered (saline was
administered on day 0).
1o0
z
o
90
80
I,
7o
'iii'
6O
(4'
50
4o
w
Q_
I
30
* LH-RHa 50.0 -5 .Opglkg
sic
LH-RHa 5.O-5.Opg/kg
LH-RHa O.5-0.5pg/kg
I.':
20
ii.,
10
Saline
I
DAY0
3
IA
FIgure 9.
7
10
13
17
I
I
I
I
21
I
I
I
24
p
1
I
I
28
I
I
I
I
I
I
I
3335
I
I
4
1
L
40
59
RESULTS
Fish in groups treated with LH-RHa alone ovulated in
significantly (P<0.0l) fewer days than did fish in the
control group (Table I).
The mean days to ovulation for
fish treated with SPE and LH-RHa were not significantly
different from that for fish of the control group.
Within
10 days after injection, 48% to 57% of the fish treated with
LH-RHa alone--by either single or double injections and at
all doses tested--had ovulated, whereas only 10% of the
control group had ovulated (Figs. 8 and 9).
Within 13 days
after injection, 77% and 67% of the groups receiving single
injections of LH-RHa at 50.0 and 5.0 pg/kg body weight,
respectively, had ovulated; 91%, 84%, and 84% of the groups
treated with two injections spaced three days apart of LHRHa at 50.0 and 5.0, 5.0 and 5.0, and 0.5 and 0.5 pg/kg body
weight, respectively, had ovulated; whereas only 47% of the
control group had ovulated.
The mean percent fertilization of the eggs from the
seven treatment groups ranged from 73% to 91% (Table I).
The percent fertilization of eggs from individual females
ranged from 6% to 100%.
Although fertilization in the
control group was slightly higher than in any treated group,
this difference was not significant as judged by analysis of
variance.
Lower egg viabilities did not correlate directly
to specific males used for fertilization and, therefore,
viabilities were considered dependent upon the condition of
the females, although other factors may have influenced the
fertilization of the eggs.
Within a treatment group, fertilization generally
varied inversely with the number of days elapsed between
treatment and ovulation, the means ranging from 65Z to 98.
Eggs taken on day 7 had significantly (P<O.05) lower
percent fertilization than eggs taken on any other day
except day 35.
Fertilization percentages were similar in
all fish spawned on the same day, independent of treatment,
except that on day 13 the fertility of eggs from the group
receiving 50.0 pg/kg body weight of the LH-RHa was
significantly (P<0.05) lower than that of eggs from the
control group.
61
DISCUSSION
Intraperitoneal injections of LH-RHa significantly
accelerated the date of ovulation in female coho salmon.
The treatments were initiated 5 to 6 weeks prior to the
projected peak spawning time of the hatchery population.
Within two weeks, between 67% and 91% of the fish treated
with LH-RHa alone had ovulated, suggesting that the
analogue accelerated maturation up to one month prior to
the normal peak spawning time and from 2 to 3 weeks prior
to that of the control group.
The LH-RHa, a synthetic
hormone, successfully stimulated early ovulation without an
initial "priming" injection of salmon gonadotropin or
pituitary extract--an important finding due to the expense
and difficulty in using the latter two natural products.
That all of the groups treated with hormone analogue showed
similar effects in relation to mean days to ovulation
implies that LH-RHa is effective over a broad range of
doses and that a lower dose than any we tested may be
effective.
The treatment of two serial injections of LH-
RHa at 0.5 iig/kg body weight is the regime showing the
greatest potential for use at a production level due to its
cost-effectiveness, low mean days to ovulation, and the
high fertility rate of eggs.
It is apparent that this
dosage sufficiently stimulates pituitary production and
secretion of gonadotropin (van der Kraak at
1983),
which in turn mediates the final maturational process.
The
reason for the ineffectiveness of the combined SPE and LHRHa treatment is at present undetermined.
The average percent fertilization was lower in eggs
from fish treated with LH-RHa than in eggs of the control
group, and was lowest in groups receiving single injections
of the analogue.
This relationship indicates that the
serial injection technique, though offering little advantage
in the magnitude of accelerated maturation, is superior to
the single injection treatment in terms of egg fertility.
Jalabert et al. (1978a) demonstrated that the
administration of DHP to rainbow trout (Salmo gairdneri)
resulted in precocious oocyte maturation in most fish, but
only a few of the fish ovulated.
These authors suggested
that high levels of GtH were needed to complete maturation
and induce ovulation.
Perhaps the duration of elevated
titers of gonadotropin is directly related to the fertility
of the eggs.
Analysis of variance indicated that fertilization did
not differ significantly between the treatment groups and
control group; however, the average percent fertilization of
the eggs from each group treated with LH-RHa was lower than
that of the control group.
Perhaps treatment with LH-RHa
increases the incidence of extremely low egg viability in
63
certain individuals rather than causing a general decrease
in the egg viability of each fish in the population.
Only
3 fish (16%) in the control group had eggs with less than
80% fertilization, whereas the groups treated with LH-RHa
had 5 (23%) to 9 (45%) fish with less than 80% fertilization
of the eggs.
The induction of precocious maturation may
reach a point of diminishing returns due to lower
percentages of fertilizable eggs.
Fertility was lowest in
eggs from the fish taken earliest in the experiment (day
7)--all of which received LH-RHa injections.
It may be
prudent to allow early fish an extra 24-72 hours after
ovulation before taking the eggs, as the extra time might
allow further maturation of the eggs and will also guard
against human error in determining the ripeness of the fish.
64
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(Oncorhynchus kisutch) in seawater or fresh water.
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kisutch) held in seawater and fresh water. Can. J.
Fish. Aquat. Sd. 39: 627-632.
Role of prostaglandins
N.E. and F.W. Goetz (1982).
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92-98.
Stacey,
in fish reproduction.
Effects of
Sundararaj, B.I. and S.V. Goswami (1966a).
mammalian hypophysial hormones, placental
gonadotropins, gonadal hormones and adrenal corticosteroids on ovulation and spawning in hypophysectomized catfish, Heteropneustes fossilis
J. Exp. Zool. 161: 287-296.
(Bloch).
Sundararaj, B.I. and S.V. Goswami (1966b). Effect of
metopiron on luteinizing hormone and corticosteroidinduced ovulation and spawning in hypophysectomized
J. Exp.
catfish, Heteropneustes fossilis (Bloch).
Zool.
163: 49-54.
In vitro
Suzuki, K., B.I. Tamaoki, and Y. Nagahama (1981).
synthesis of an inducer for germinal vesicle breakdown
of fish oocytes, 17ct,20-dihydroxy-4-pregnen-3-one by
ovarian tissue preparation of arnago salmon
70
(Oncorhynchus rhodurus).
533-535.
Gen. Comp. Endocrinol. 45:
Ueda, H., 0. Hiroi, A. Hara, K. Yanauchi, and Y. Nagahama
Changes in serum concentrations of steroid
(1984).
hormones, thyroxine, and vitellogenin during spawning
Gen.
migration of the chum salmon, Oncorhyrichus keta.
Comp. Endocrinol. 53: 203-211.
Ueda, H., G. Young, L.W. Crim, A. Kainbegawa, and Y. Nagahama
17a,20-Dihydroxy-4-pregnen-3-one: plasma
(1983).
vitro
levels during sexual maturation and j
production by the testes of amago salmon (Oncorhynchus
Gen.
rhodurus) and rainbow trout (Salmo airdneri).
Comp. Endocrinol. 51: 106-112.
Young, G., L.W. Crim, H. Kagawa, A. Kambegawa, and Y.
Nagahama (1983). Plasma 1 7, 206-dihydroxy-4-pregnen3-one levels during sexual maturation of amago salmon
(Oncorhynchus rhodurus): correlation with plasma
gonadotropin and in vitro production by ovarian
Gen. Comp. Endocrinol. 51: 96-105.
follicles.
71
APPENDIX
72
V.
Viabilities of eggs stripped from coho salmon
at various times after ovulation'
Martin S. Fitzpatrick
Carl B. Schreck2
Frank Ratti3
Robert Chitwood3
Oregon Cooperative Fishery Research Unit4
Oregon State University
Corvallis, OR
97331
1Oregon State University, Agricultural Experiment Station
Technical Paper No.
whom reprint requests should be sent.
3Oregon Aqua Foods, Inc., 88700 Marcola Rd., Springfield,
OR.
97477.
4Cooperators are Oregon State University, Oregon Department
of Fish and Wildlife, and U.S. Fish and Wildlife Service.
73
V.
Viabilities of eggs stripped from coho salmon at
various times after ovulation.
INTRODUCTION
The use of hormones to accelerate final sexual
maturation in salmonids has met with great success
(Donaldson, et al. 1981; Hunter, et al. 1981; Sower, et al.
1982; Crim, et al. 1983, Sower,
et al. 1984).
However,
little is known about the effects of these treatments on
gamete quality.
Sower et al. (1984) found that treatment of
coho salmon, Oncorhynchus kisutch, with single injections of
partially purified chinook salmon gonadotropin followed by
single injections of the gonadotropin-releasing hormone
analogue (LH-RHa) des-G1y10[D-A1a6ILH-RH-ethylamide
accelerated maturation and that development of the eggs from
these fish to eyed stage or later to ponding appeared to be
equal to that of the control group.
The highest mean
percent viability in that study was 67.2 in eggs from
control fish.
As reported previously (see Chapter 3),
treatment of coho salmon females with two injections of
various dosages of LH-RHa accelerated final maturation, but
the means for viability of the eggs were consistently lower
for eggs from treated fish than for eggs from the control
group--although the differences were not statistically
significant.
74
In this study, females were allowed to retain their eggs
after ovulation in order to determine if varying the amount
of time between ovulation and egg-take had any effect on egg
viability.
Both LH-RHa-treated and untreated females were
used to investigate the effect of holding the fish past
ovulation had on egg viability.
75
MATERIALS AND METHODS
Adult female coho salmon from the seawater facility of
Oregon Aqua Foods, Inc. at South Beach, Oregon were
transported to the freshwater hatchery at Springfield,
Oregon.
On 17 October 1983, females exhibiting coloration
indicative of advanced sexual maturity were separated from
the rest of the broodstock population.
These fish were
anesthetized with ethyl P-aminobenzoate (benzocaine) and
injected with LH-RHa at a dose of 0.5 ug/kg body weight.
Three days later, the fish were injected with the same
dosage of LH-RHa.
Seven days after the initial injection,
the fish were checked for ripeness.
Approximately 100
females which had been designated as ripe (i.e. ovulated)
were separated from the rest of the population.
group, 10 were removed and spawned.
Of this
Approximately 100 eggs
from each female were fertilized in separate containers with
sperm pooled from four males.
incubated at 10 C.
Each group of eggs was
After 10 days, percent fertilization was
determined by clearing the eggs in a solution of 1OZ acetic
acid and then counting the number of eggs with developing
embryoes.
Two days after the initial group of eggs were taken (and
every two days thereafter), another group of ten females was
sampled from the isolated population in the manner described
76
above.
The last group of females was sampled 16 days after
ovulation,
On 12 December 1983, approximately 50 untreated
females which had ovulated were separated from the rest of
the broodstock population.
Ten fish were removed and
spawned, and the eggs were treated in the same manner as
described above.
Thereafter, groups of ten females were
sampled in this way 4, 7, 15, and 20 days after ovulation
and percent fertilization of each batch of eggs was
determined.
Oneway analysis of variance (Snedecor and Cochran 1980)
was conducted on the fertilization data with respect to days
after ovulation within treated and untreated groups.
77
Figure 10.
Mean (with standard error) and median percentages of viable eggs (i.e. with developing
embryos 10 days after fertilization) from
females stripped at various times after
ovulation. The females were either
a) treated with LH-RHa at 0.5 pg/kg body wt and
ovulation took place on 24 October 1983; or
b) untreated and ovulation took place on 12
December 1983.
a
b
Msen
Msan
o
o
C,)
C,
C,
'U
-J
I-1
z
'U
0
'U
'1'
I
0
2
4
S
I
8
I
I
10 12 14
I
16
TI
0
DAYS AFTER OVULATION
Figure 10.
I
I
4
7
15
20
79
RESULTS AND DISCUSSION
The mean and median viabilities of eggs taken from
LH-RHa--treated fish are presented in Fig. lOa.
No
significant differences were detected in the viabilities
between eggs taken on the various days after ovulation.
The
values for the median viabilities of eggs were consistently
higher than the values for the mean viabilities which
indicates that the distribution of viabilities is skewed.
In other words, the mean values have been disproportionately
influenced by a few very low viabilities.
The skewness of
the distribution is readily apparent in an examination of
the viabilities of eggs from all fish treated with LH-RHa.
The mean percent viability of the eggs was 81.5 ± 2.3,
whereas the median percent viability was 90.2.
Almost 71%
of the viabilities were greater than or equal to the mean
value.
Three incidences of overripe (i.e. water hardened) eggs
occurred in the group treated with LH-RHa--in one fish on day
12 and in two fish on day 14 after ovulation.
The
viabilities of these eggs were less than or equal to 45.9%.
No overripe eggs were observed in the 10 fish spawned 16
days after ovulation.
Eleven of the 106 females in the
treated group died during the course of the experiment,
representing 10.4% of the population.
Mortalities began
appearing 3 days after ovulation.
The mean and median viabilities of eggs taken from
untreated fish are presented in Fig. lOb.
No significant
differences in the viabilities between days were detected
and the median values were generally closer to the mean
values for untreated fish than for LH-RHa--treated fish.
The
mean viabilities of eggs remained high (over 9O7) for the
duration of the experiment, 20 days after ovulation.
Nomura et al. (1974) described 4 distinct stages of
overripeness in eggs from rainbow trout, Salmo gairdneri.
Sakai et al. (1975) showed that the percentage of eyed eggs
and percentage of hatched rainbow trout exceeded 70% in
those eggs which had been stripped by the 10th day after
ovulation; however, the viabilities at the same stages
declined rapidly in eggs stripped between 10 and 20 days
after ovulation and reached 0% for eggs stripped one month
after ovulation.
Takashima et al. (1975) described
histologically some of the profound
changes taking place in
overripe female rainbow trout and Craik and Harvey (1984),
examining some of the biochemical changes occurring with
overripening in eggs of rainbow trout, found that free,
bound, and total lipid varied significantly between ripe and
overripe eggs (i.e. eggs taken 30 or more days after
ovulation) as did protein phosphorus, lipid phosphorus, iron
and calcium.
The results of the present study on coho salmon agree
in general with that which has been reported in the
literature.
Eggs of high viability (i.e. showing embryonic
development) were obtained from females that were treated
with LH-RHa to induce precocious final maturation up to 16
days after ovulation and from untreated females up to 20
days after ovulation.
However, in hormone-treated fish, the
mortality of adult females was high, commencing three days
after ovulation, and incidences of water hardened eggs
appeared 12 days after ovulation.
Thus, although it appears
that females treated with LH-RHa can be held for long periods
of time after ovulation in order to satisfy the need fora
flexible strategy of taking eggs, some adverse effects can
occur, such as mortality and water hardened eggs.
Untreated
femalesdonotappeartosufferinthesarnemagnitude from
these maladies, although it is difficult to determine from
this study if the higher egg viabilities were due to not
receiving hormone treatments or due to some other effect,
such as later-spawning females having eggs with different
characteristics than early-spawning females.
LPI
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