Ž .
reviewed by Swanson, 1991 . GtH-I and GtH-II are produced by different gonadotropin Ž
. cell-types Nozaki et al., 1990a; Naito et al., 1993 . During vitellogenesis and spermato-
genesis, large numbers of GtH-I cells were observed relative to low numbers of GtH-II cells and blood GtH-I levels increased and peaked prior to spawning while GtH-II levels
remained around or below assay detection limits. In contrast, the numbers of GtH-II cells exceeded the number of GtH-I cells during spawning, a time when circulating
Ž GtH-I declined and GtH-II displayed a rapid increase Suzuki et al., 1988; Nozaki et al.,
. 1990b; Swanson, 1991; Naito et al., 1993 . GtH-I and GtH-II are equipotent in
Ž .
stimulating 11-ketotestosterone 11KT and 17,20bP production during spermatogenesis Ž
. although 11KT production exceeded 17,20bP production Planas et al., 1991 . Later,
however, GtH-II was more potent than GtH-I in stimulating 17,20bP synthesis. Thus, early GnRHa treatment may have had a relatively greater impact on GtH-I cell activity
and GtH-I production while the effectiveness of GnRHa injection at the height of the spawning season is consistent with increased impact on GtH-II production and 17,20bP
synthesis at that stage of the reproductive cycle.
One of the primary factors limiting the early synthesis of 17,20bP may also be the Ž
lack in availability of its precursor 17a-hydroxyprogesterone 17P; Sakai et al., 1989; .
Schulz et al., 1991, 1992 due to the dominance of the androgen biosynthetic pathway. A number of authors have demonstrated increased 17,20bP production and increased
Ž spermiation in response to exogenous 17P both in vitro and in vivo Ueda et al., 1983,
. 1984, 1985; Sakai et al., 1989; Miura et al., 1991; Pankhurst, 1994 . Thus, the variation
in the efficacy of GnRHa treatment as a stimulant of spermiation during different stages of reproductive development may also be related to 17P availability. However, the roles
of GtH-I and GtH-II in the production of 17P, relative to androgens during spermatogen-
Ž .
esis, remain to be assessed Swanson, 1991 . We hypothesised that providing substrate for 17,20bP synthesis would enhance the
effectiveness of the GnRHa treatment. Three experiments were conducted immediately Ž
prior to the commencement of the 1993 and 1994 spawning seasons late April in .
Tasmania in order to investigate the effect of exogenous 17,20bP, 17P and GnRHa, alone or in combination, on the spermiation response of mature non-spermiating male
Atlantic salmon.
2. Materials and methods
2.1. Stock Ž
. Experiments were conducted over two years 1993 and 1994 . Each year, maturing
Ž 2 q Atlantic salmon broodstock were transferred from Saltas Marine Operations Dover,
. Ž
. Tasmania to Saltas Freshwater Operations Wayatinah, Tasmania in March in order to
facilitate final maturation in freshwater. Prior to transfer, fish were maintained in a single 65-m circumference polar circle-type cage at a maximum stocking density not
greater than 10 kg P m
y3
. Fish were fed to appetite on a commercial steam-pelleted Ž
. ration Gibsons, Cambridge, Tasmania and experienced ambient seawater temperatures
Ž .
; 13–158C at time of transfer . Following transfer, fish were starved and held in
3
Ž .
40 m fibreglass raceways maximum 200 fish per raceway supplied with river water Ž
y1
. ;
15 l P s at ambient temperature. During the experiments, water temperatures
ranged from 12.28C–7.38C and 14.08C–7.58C in 1993 and 1994, respectively, such that mean water temperature declined from 11.38C to 7.68C in 1993 and from 13.08C to
8.58C in 1994. Ž
Prior to the commencement of each experiment, non-spermiating male fish mean wt. .
Ž approx. 6.5 kg were selected from the broodstock and anaesthetised in benzocaine 25
. Ž
. mgrl . Fish were individually weighed 0.1 kg and marked by placing visible
Ž .
implant tags VI Tags, Northwest Marine Technology, Shaw Island, WA in the adipose Ž
. eyelid according to the methods described by Bergman et al. 1992 and Kincaid and
Ž .
Calkins 1992 . Thereafter, individual fish were randomly assigned to treatment groups. As all treatment groups were housed in a single raceway, treatment groups were further
Ž .
distinguished by implantation of a colour-coded nylon dart tag Floy Tag, Seattle, WA at the base of the dorsal fin to facilitate future treatment and sampling.
2.2. Hormone solutions Ž
A commercial salmon GnRHardomperidone preparation Ovaprim, Syndel Laborato- ries, Vancouver, BC; 20 mg
D
-Arg
6
, Pro
9
-Net sGnRHa q 10 mg domperidone per ml .
Ž .
propylene glycol was used. Stock 10 wrv absolute ethanol 17,20bP and 17P Ž
. Steraloids, Wilton, NH solutions were prepared and diluted with propylene glycol to
achieve concentrations which gave a dose of 1 mgrkg body wt. at a final injection volume of 0.5 mlrkg body wt.
Ž
y1
. Ž
. Fig. 1. Milt volume mlPkg body wt.
in Atlantic salmon injected with vehicle control , GnRHa, 17P, Ž
. 17,20bP or GnRHaq17HP. Values are meanqs.e.
ns 4 . Columns with the same superscript are not Ž
. significantly different P - 0.05 .
2.3. Experiment 1 19–27 April 1993 On day 0, 20 non-spermiating male Atlantic salmon were anaesthetised and given an
Ž .
Ž .
intra-muscular i.m. injection of the commercial salmon GnRHa 10 mgrkg prepara- Ž
. Ž
. tion n s 8 or vehicle alone n s 12 . On both days 6 and 7, fish received a further i.m.
Ž .
injection of 17P, 17,20bP both 1 mgrkg or vehicle alone in a format resulting in the Ž
. Ž
. following five experimental groups
n s 4 : control vehicle only ; GnRHa; 17P;
17,20bP; GnRHa q 17P. As the manufacturer of the GnRHa preparation indicated that a maximal response could be expected 7–10 days post treatment, fish were anaesthetised
on day 8 and milt was collected from each fish using a catheterisation technique similar
Ž
y1
. Fig. 2. Day 8 blood plasma 11KT, 17,20bP and 17P ngPml
in Atlantic salmon injected with vehicle Ž
. Ž
. control , GnRHa, 17P, 17,20bP or GnRHaq17P. Values are meanqs.e. ns 4 . Columns with the same
Ž .
superscript are not significantly different P - 0.05 .
Ž .
to that described by Erdahl 1982 . Milt volume was recorded and spermatocrit
Ž .
determined by the method of Miura et al. 1991 . One-millilitre blood samples from Ž
. each fish were collected in heparinized lithium heparin syringes by puncture of the
Ž .
duct of Cuvier Lied et al., 1975 . After centrifugation, the resulting plasma was stored at y208C prior to analysis for steroid levels. Seminal plasma samples were also retained
for steroid analysis.
2.4. Experiment 2 5–13 April 1994 Thirty non-spermiating male Atlantic salmon were handled as before except that a
blood sample was collected on day 0 before they received an i.m. injection of the
Ž
y1
. Fig. 3. Day 8 seminal plasma 11KT, 17,20bP and 17P ngPml
in Atlantic salmon injected with vehicle Ž
. Ž
. control , GnRHa, 17P, 17,20bP or GnRHaq17P. Values are meanqs.e. ns 4 . Columns with the same
Ž .
superscript are not significantly different P - 0.05 .
Ž .
Ž .
Ž .
commercial salmon GnRHa 10 mgrkg preparation n s 15 or vehicle alone n s 15 . Ž
Again, on both days 6 and 7, they received an i.m. injection of 17P, 17,20bP both 1 .
Ž .
Ž mgrkg or vehicle alone resulting in six experimental groups n s 5 : control vehicle
. only ; GnRHa; 17P; 17,20bP; GnRHa q 17P; GnRHa q 17,20bP. On day 8, milt was
collected from each fish and seminal and blood plasma samples were taken. 2.5. Experiment 3 20–28 April 1994
Twenty non-spermiating male Atlantic salmon were subjected to the same treatment and sampling protocol as for experiment 2, except that only 17P was used.
2.6. Steroid radioimmunoassays Levels of 11KT, 17,20bP and 17P were measured in blood and seminal plasma
Ž .
following diethyl-ether extraction using the methods of Ueda et al. 1984 and Young et Ž
. al. 1986 .
2.7. Statistical analysis Where appropriate, data were log-transformed in order to homogenise variances and
group differences between means for milt volume and blood and seminal plasma steroid concentrations were assessed by one-way analysis of variance followed by a Tukey HSD
test of pairwise multiple comparisons using the SYSTAT statistical analysis package
Ž
y1
. Ž
. Fig. 4. Milt volume mlPkg body wt.
in Atlantic salmon injected with vehicle control , GnRHa, 17P, Ž
. 17,20bP, GnRHaq17P or GnRHaq17,20bP. Values are meanqs.e.
ns 4-5 . Columns with the same Ž
. superscript are not significantly different P - 0.05 .
Ž .
SYSTAT, 1991 . The same package was used to derive linear correlation coefficients between parameters.
3. Results
