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Induced ovulation in coho salmon (Oncorhynchus kisutch). I. Further studies on the use of salmon pituitary preparations
26 (1981/1982) 11’7-127 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Aq~ec~lfure,
INDUCED OVULATION IN COHO SALMON (O~CO~~Y~CHUS I. FURLER STUDIES ON THE USE OF SALMON PITU~A~Y TIONS
117
KISUTCH). PREPARA-
GEORGE A. HUNTER, EDWARD M. DONALDSON and HELEN M. DYE
West Vuncouuer Laboratory, Resource Services Branch, Repartment of Fisheries and Oceans, 4160 Marine Drive, West Vancouver, B.C. V7V IN6 ~Canada} (Accepted
25 February 1981)
ABSTRACT Hunter, G.A., Donaldson, E.M. and Dye, H-M., 1981. Induced oblation in coho sahnon (Oneor~ync~us kisutch). I. Further studies on the use of salmon pituitary preparations.
Aquaculture,
26: 117-127.
The work presented extends previous studies on the use of pituitary preparations to induce ovulation in coho salmon. It has been demonstrated that coho salmon may be successfully induced to ovulate up to 2 months prior to their normal peak spawning period and that the use of a single injection of salmon pituitary extract (SPE) to induce ovulation is possible. In November 1977, the experimental fish were divided into six groups. Three groups were administered injections of 0.1 mg/kg partially purified salmon gonadotropin (SGGlOO) followed 3 days later by either 2.0 mg/kg SG-GlOO, 25.0 SPE or 50.0 SPE. A fourth group was administered 10.0 mg/kg SPE followed 3 days later by 50.0 mg/kg. The fifth group received a single injection of X00.0 mg/kg SPE. A control group was injected with saline. All treated groups ovulated significantly earlier than the control. Egg survival and mean egg diameter were within the normal range for coho from Big Qua&urn. In September and October 1978, the 10.0 mg/kg SPE primer followed by a 50.0 mg/kg secondary SPE injection were compared to a saline control. In addition, in September a second test group was administered the same primer but was followed by three daily 25.0 m&kg SPE injections. All treatment groups ovulated significantly earlier than their controls. However, only the lo-50 SPE treatment groups reached cumulative ovulation levels comparable to the controls. Further, these levels increased with closer proximity to the normal spawning period from app~ximately 40.0% in September to approximately 80% in October. Egg survival and mean degree days to the eyed stage and to hatching for the October experiment fell within the normal range for the Big Qualicum stock. In November 1978, four groups of seven fish received single intraperitoneal or intramuscular injections of either 33.0 mg/kg or 66.0 mg/kg SPE. A control group was injected with saline. All groups ovulated significantly earlier than the control: No significant differences were found between test groups related to dosage level or route of injection. Egg survival and mean degree days to the eyed stage and to hatching fell within the normal range for this stock.
0044-8486l81/0000~000~$02.50
0 1981 Elsevier Scientific Publishing Company
1
118 INTRODUCTION
In earlier publications we have outlined the potential benefits of using induced ovulation techniques to reduce prespa~ing mortality, synchronize hatchery production and obtain fertilized ova at an earlier date (Jalabert et al., 1978; Hunter et al., 1978,1979). In the first of these studies (Jalabert et al., 1978) final maturation and ovulation of coho salmon (Oncorhynchus kisutch) ova were successfully induced 3-5 weeks ahead of the normal time. The most effective treatment used in this experiment consisted of an intrape~tone~ primer (ip) injection of 0.1 mg/kg body weight partially purified salmon gonadotropin (SG-GlOO) followed within 2-3 days by an ip injection of either 1 mg/kg SG-GlOO or 3 mg/kg 17a-200 dihydroprogesterone. Later work on chinook salmon (0. tshawytscha) (Hunter et al., 1978) and coho salmon (Hunter et al., 1979) demonstrated that a crude salmon pituitary extract (SPE) could also be used to effectively induce final maturation and ovulation. The purpose of the present investigation has been to extend these initial studies with salmon pituitary gonadotropin with the intention of simplifying the injection regime, establishing the most effective route of administration, comparing the crude salmon pituitary extract with SG-GlOO, and determining how long prior to normal spawning coho salmon can be ovulated. In addition, this ~vestigation with salmon pituitary materials was conducted to provide a basis against which we could compare the effectiveness of synthetic hormones such as gonadotropin releasing hormones and an antiestrogen. MATERIALS AND METHODS
The four experiments contained in this study were conducted at the West Vancouver Laboratory of the Resource Services Branch in the fall of 1977 and 1978. Origin and transportation of fish All experiments were conducted with adult 3-year-old female coho salmon (0. ~is~tc~) which o~~nated from the Big Qualicum hatchery, V~couver Island. The normal spawning period for coho salmon from Big Qua&urn is 20 October-5 January with a peak spawning period 20-25 November. The fish used in the 1977 experiment were transported by truck directly from the Big Qualicum hatchery. Water in the holding tank was cooled with ice (6-7” C) to tranquilize the fish during transport. In 1978, the test fish were transported by truck from the Capilano hatchery, North Vancouver, B.C. These fish were Big Qu~icum stock which had been reared, fin clipped for identification and released from the Capilano facility.
119
Holding facilities Fish were held under natural photoperiod in five 3-m diameter fiberglass tanks supplied with aerated well water (60 l/min) to a depth of 0.8 m. The water temperature range was lO.O-10.5”C for all experiments. Handling and injection procedure The procedures used for the anaesthetization, weighing and tagging of fish have been described by Hunter et al. (1978). Intraperitoneal injections (ip) were directed anteriorly from a point just posterior of the pelvic fins (Hunter et al., 1978). The intramuscular injections (im) were administered at a point just ventral of the posterior limit of the dorsal fin and on the side opposing the identification tag. Following treatment fish were examined at intervals of not less than 2 days. Test solutions Physiological saline (0.65%) was used as the vehicle for all pituitary preparations tested. The partially purified salmon gonadotropin SG-GlOO used was prepared according to procedures described by Donaldson et al. (1972) and Donaldson (1973) from chinook salmon (0. tshawytscha) pituitaries obtained from mature fish at the Spring Creek National Fish Hatchery, Washington, U.S.A. The SG-GlOO powder was dissolved in saline at the required concentration. Chinook salmon pituitaries collected in 1973 at the Spring Creek hatchery were used in the preparation of the salmon pituitary extract SPE according to the method described by Hunter et al. (1978). Determination
of egg maturity
and ovulatory
responses
Egg samples were taken on each sampling day and cleared formaldehyde, acetic acid and Cortland’s salt solution (5 : 4 1976). The stage of egg maturation was determined according of Jalabert et al. (1976). The days to maturation determined vesicle breakdown (GVBD), and ovulation were recorded for Spawning
of ripe females and determination
in a solution
of
: 100) (Goetz,
to the criteria by germinal each fish.
of egg size and survival
Fish showing a strong ovulatory response were normally spawned within 24 h. Where possible the eggs from each female were fertilized with milt from at least four males. Only one male was available for the fertilization of the September 1978 SPE lo-50 fish. Excess ovarian fluid was removed prior to fertilization. Eggs and milt were allowed to stand together for 5-15 min. before contact with water. The eggs were incubated in Heath trays (Heath
7 7
October 1 1978 (Day 0 =Oct .13) 2
1 2 3 4 5
aReceived injections on Days 3, 6 and 9. bReceived injections on Days 3,4 and 5. ‘Received injections intermuscularly. dSignificant at 01< 0.05.
November 1978 (Day O=Nov.l)
15 18 17
September 1 1978 (Day O=Sept. 13) i
i 4 5 6
12 13 13 13 11 9
1
November 1977 (Day O=Nov. 4)
Number of females per group
Group number
Experiment
saline 33 SPE 33 SPEC 66 SPE 66 SPEC
saline 10.0 SPE
saline 10.0 SPE 10.0 SPE
0.1 SG-GlOO 0.1 SGGlOO 10.0 SPE 100.0 SPE
0.1 SG-GlOO
Day 0
-
salineb -
saIineb 50.0 SPE
salinea 50.0 SPE 25.0 SPEa
saline 2.0 SGGlOO 25.0 SPE 50.0 SPE 50.0 SPE
Day 3
Injection protocol dosage mg/kg body weight
f * + t + +
0.71 1.14 0.72 0.55 0.66 1.15
2.77 2.26 2.41 2.30 2.46
f f f f *
0.72 0.32 0.52 0.38 0.72
2.30 f 0.79 2.27 f 0.64
2.50 + 0.69 2.51 * 0.64 2.56 f 0.50
3.70 3.73 3.50 3.02 3.16 3.55
Mean weight of females + S.D. (kg)
100.0 100.0 100.0 100.0 100.0
100.0 100.0
60.0 100.0 100.0
75.0 100.0 100.0 100.0 100.0 100.0
Cumulative percent GVBD
7 7 6 7
9
6 5
6 8 2
7 11 13 13 11 9
Number of females reaching ovulation
100.0 100.0 100.0 85.7 100.0
85.7 71.4
40.0 44.4 11.8
58.3 84.6 100.0 100.0 100.0 100.0
Cumulative percent ovulation
+ + + f + ?
O.Od 0.9d 3.1d
o.od
6.8 l.5d
11.4 7.0 7.3 7.7 7.6
+ 4.8 * O.Od + 1.8d f 1.6d f O.gd
26.0 + 9.9 12.8 + 3.0d
64.3 + 13.2 17.1 + 12.1d 14.0 + o.od
16.9 11.1 10.0 10.0 10.3 11.6
Mean days to ovulation + S.D.
Ovulatory response of coho salmon to treatment with saimon pituitary extract (SPE). Injection protocol, number and mean weight of females, cumulative percent germinal vesicle breakdown (GVBD) and ovulation, and mean days to ovulation are presented for each experimental group in the November 1977 and the September, October and November 1978 experiments
TABLE I
121
Techna Corp.) supplied with aerated well water 15 l/min at 10°C. Approximately one half of the eggs from the 1978 experiment were incubated in city water at 6°C. Degree days to the eyed stage and to hatching were determined’ for the 1978 egg samples. Mean egg diameter based on the measurement of five samples of ten eggs, and percent survival were determined at the eyed stage. The percent survivals for the 1977 experiment were determined by incubation of all of the eggs from four females from each group. In 1978, a 200 ml (approx. 1100 eggs) sample of eggs from each of the fish in the experimental groups were used to determine egg survival. Plexiglas dividers were used to separate the Heath trays into quadrants, each of which held one 200 ml sample. Experimental protocol The experimental 1978, October 1978 I. Dates of initiation 1977,13 September
protocols for the November 1977 and the September and November 1978 experiments are presented in Table (Day 0) for these four experiments were 4 November 1978,13 October 1978 and 4 November 1978.
Induction of spermiation To permit the fertilization of ova from induced females in the November 1977 experiment, 11 males which had not yet spermiated were given a 50 mg/kg body weight ip injection of SPE on Day 10. Four males in September 1978 and five males in October 1978 were given two injections of 10 and 50 mg/kg SPE on Days 0 and 3. In the November 1978 experiment, 14 males were given a 50 mg/kg injection of SPE on Day 0. Statistical analysis One way analysis of variance (MIDAS Statistical Research Laboratory, University of Michigan) was conducted on the logarithmic transformations of the days to ovulation data. Days to ovulation data were randomized within sample intervals to permit the use of analysis of variance. In addition, measure ments of egg diameter and weight of females were subjected to analysis of variance. RESULTS
The results of the November 1977 study are presented in Fig. 1 and Tables I and II. In the SPE 25 and SPE 50 groups, all fish were found ovulated in the sampling interval preceding Day 10. Statistical analysis indicated that the five groups treated with pituitary preparations ovulated significantly earlier than the control group. No significant differences were found between treated
122
90-
30-
20-
IO-
DAY NW
0 4
3 i
6 10
9 13
12 16
v
0.1
SG-GlOO.
25
SPE
a
0.1
SG-GlOO.
50
SPE
q
10 SPE,
o
100
.
0.1
l
CONTROL
15 ,P
50
SPE
SPE SG-GlOO.
2.0
SG-GlW
-Saline
18 ??
21 25
24 28
1
Fig. 1. November 1977 experiment. Induced ovulation of coho salmon (Oncorhynchus kisutch). The numbers of fish which ovulated in each experimental group are expressed as a cumulative percentage of the total number of fii in each group. Experimental groups were administered injections of either a saline control 0.1 mg/kg SG-GlOO followed by a 25 or 50 mg/kg salmon pituitary extract (SPE), 10 mg/kg SPE followed by 50 mg/kg SPE, 0.1 mg/kg SG-GlOO followed by 2.0 mg/kg SG-GlOO or a single injection of 100.0 mg/kg SPE. Arrows indicate the days on which injections were administered. The normal peak spawning period for Big Qualicum coho is 20-25 November.
groups. All of the fish in the five treated groups and 75% of the fish in the saline control group reached final maturation as determined by GVBD. With the exception of the SG-GlOO 0.1-2.0 groups (84.6%), the treated groups reached 100% cumulative ovulations. Only 58.3% of the fish in the control group ovulated. The percent survival to the eyed stage of eggs from the six groups ranged from 78 to 99%. Of the five test groups, only salmon from the SG-GlOO 0.1-2.0 group had eggs which were significantly smaller than the control. The mean egg diameters from all six groups fell well within the normal range of egg diameters for coho from Big Qualicum. There were no significant differences between the initial body weights of females in the experimental groups.
Group number
1 2 3 4 5 6
1 2 3
1 2
1 2 3 4 5
Experiment
November 1977
September 1978
October 1978
November 1978
6 7 7 6 6
3 4
1 1 2
4 4 4 4 4 4
Egg samples incubated per group
97.3 98.4 97.6 97.7 97.4
f f * f *
92.7 f 98.0 + 2.3 1.1 1.8 3.8 1.1
5.0 2.5 213.0 221.6 210.0 219.5 239.9
+ f f f f
236.2 f 233.9 +
249 279 279.0 f
-
94 5 42.5 f 24.8
-
79 84 8S 99 78
19.6 23.0 17.2 21.5 24.1
1.1 8.8
0.0
Mean degree days to eyed stage f S.D.
93
Survival to eyed stage (%) + + f f f *
0.12 0.08 0.11 0.14 0.12 0.08
7.1 6.6 7.0 6.6 6.8
f * f f +
0.48 0.43 0.21 0.24 0.30
6.6 * 0.70 6.7 f 0.31
7.0 5.5 6.5 + 1.4
7.5 7.1 7.4 7.3 7.4 7.3
Mean egg diameter f SD. (mm)
-_ -
-
93.0 97.3 95.7 94.5 96.6
+ f f f f
7.6 1.1 2.0 9.1 1.5
461.0 443.4 395.0 434.8 466.0
f f + * f
46.7 58.7 24.0 40.6 40.1
466.8 + 11.9 461.4 f 33.5
474 485.1 + 15.0
5 37.5 f 24.8 89.0 f 6.6 93.0 f. 7.6
449
89
-
Mean degree days to hatching f S.D.
Survival to hatching (%)
Size, survival and degree days to hatching of eggs from salmon pituitary extract (SPE) treated coho salmon. Survival to the eyed stage and mean egg diameter are presented for the November 1977 experiment. Egg diameter, percent survival and mean degree days to the eyed stage and to hatching are presented for the September, October and November 1978 experiments. Group numbers correspond to those of Table I
TABLE II
124 100.
x
10 SPE. 25 SPE, 25 SPE.
o
10 SPE. 50 SPE
-ism
o
10 SPE. M SPE
-OCT
I
33 SPE, ,p
NW
.
66 SPE,Irn
NO”
0
33 SPEJP
70.
n
66 SPE,ip
-NO”
.
CONTROL
-S.,ine
-NO”
60.
.
CONTROL
-Soline
-XT
.
CONTROL
-Soline
.SEPT
PO.
80.
6 ; 4 ; 6
50.
5 ;
40.
25 SPE
-WEPT
NO”
?
? 30.
2O-
10
Fig. 2. September, October and November 1978 experiments. Induced ovulation of coho salmon (Oncorhynchus kisutch). The numbers of fish which ovulated in each experimental group are expressed ss a cumulative percentage of the total number of fish in each group. In the September and October 1978 experiment, groups were administered a saline control or a primer of 10 mg/kg salmon pituitary extract (SPE) followed by either a 60 mg/kg SPE or 3-25 mg/kg SPE injection. In the November 1978 experiment, groups were administered a saline control or a single injection of 33 mg/kg SPE or 66 mg/kg SPE via intraperitoneal (ip) or intramuscular (im) routes. Arrows indicate the days on which the injections were administered. The normal peak spawning period for Big Qualicum coho is 20-25 November.
The results of the September, October and November 1978 experiments are presented in Fig. 2 and Tables I and II. In each of the three experiments, groups which were administered pituitary preparations ovulated significantly earlier than their respective controls. Comparison of the mean days to ovulation, Table I, indicates that approximate gains of 7 weeks, 2 weeks and 4 days were made relative to controls as a result of pituitary treatment in the Septem ber, October and November experiments. It is notable that in the case of this latter experiment, the actual difference in time to 100% ovulation between treatment and control groups was 12 days (Fig. 2). The mean ovulation dates for each of the three control groups occurred between 8-16 November. While the cumulative ovulations of the SPE lo-50 and SPE 10-25-2525 groups in the September 1978 experiment reached 44.4% and 11.8% respectively, 100% of the fish in both groups reached GVBD. Only 60% of the fish in the control group of the September 1978 experiment reached GVBD. Germinal vesicle breakdown reached 100% in all groups in the October 1978 and November 1978 experiments. The cumulative percent ovulations for test and control groups were similar within each experiment with the exception of the September SPE 10-25-
125
25-25 group and increased with each subsequent experimental period (Table I). Three fish from the September 19’78 control group were accidentally killed on Day 40. There were no significant differences in egg diameters between the groups in the October and November 1978 experiments. The eggs from the 1978 fish were, however, smaller than those from the 1977 fish. Also, the mean weight of females from the 1978 experimental groups, while having no significant differences between themselves, were significantly smaller than the 1977 fish. The percent survival to the eyed stage of eggs from the October and November 1978 experiments ranged from 93-98s respectively (Table II). The mean egg diameters for the September 1978 saline, SPE lo-50 and SPE 10-25-25-25 groups were 7.0 mm, 5.5 mm and 6.5 + 1.4 mm. Difficulty in obtaining sufficient milt for fertilization prevented the rearing of the eggs of six females from the September 1978 lo-50 SPE group. The respective percentage survivals to the eyed stage were 94%, 5% and 43%. The small number of egg samples obtained from the September 1978 experiment precludes statistical analysis of egg size and percent survival. Degree days to the eyed stage and to hatching determined for the 1978 experiments all fell within the normal range of values for coho salmon. All the males in the November 1977 and November 1978 given a 50 mg/kg injection of SPE were found to be spermiating within 3 days of the injection. Of the four males given a lo-50 SPE injection in the September 1978 experiment only small quantities of milt could be obtained. Four of the five males given similar injections in the October 1978 experiment were found to be spermiating by Day 6 of the experiment. DISCUSSION
The present studies have been designed to facilitate the effective use of the induced ovulation technique within a production framework. The results of these initial experiments indicate that the technique of induced ovulation with salmon pituitary extract can be used to accelerate the process of final maturation and ovulation. The technique was effective over the observed natural range of spawning time at Capilano hatchery. Furthermore, a simple single injection regime has been used in relatively mature salmon for the first time. An examination of the November 1977 and 1978 experiments indicates that SPE administered in a single injection, in two separate injections or as a second injection following a SG-GlOO primer was as effective as the 0.1-2.0 SG-GlOO injection regime. SPE has been shown (T.G. Owen, personal eommuni~ation, 1980) to contain the equivalent of 1.5-2.0 mg SG-Gloomy wet weight pituitaries when assayed using a modification of the ovarian cyclic AMP assay described by Idler et al. (1975). The effective dose of SPE is thus lower than that which would have been predicted based on its SG-GlOO content suggesting that a synergistic factor or factors may be present in the whole
126
pituitary preparation. Alternatively, the ovarian cyclic AMP assay may not have given an accurate estimation of the ovulating potency of SPE. The similarity in effect of the single SPE injection at dosage levels of 100,66 and 33 mg/kg implies that an even lower effective dosage is possible. While no difference in effect was found between the intraperitoneal or intramuscular injection routes, differences may exist at lower dosages. The successful use of a single injection regime increases the potential application of this technique in a production framework. Further studies will be required to determine the optimal injection dosage and the period of time prior to normal peak ovulation during which a single injection regime will be effective. The results of the November 1977, September 1978 and October 1978 experiments show that the potential exists for inducing ovulation up to 2 months prior to the normal peak spawning period. The level of success achieved both in terms of percentage of fish ovulated, which is comparable with the respective controls, and the survival to hatching of the eggs, appears to be inversely related to the number of days the treatment period preceeds the normal spawning period. This suggests that there is a limit to the period of time prior to the normal spawning period that a single or series of injections of salmon pituitary preparations or salmon gonadotropin can be used to induce ovulation of maturing salmon. However, it is notable that even in the earliest treatment period, 100% of the fish in the treated groups had eggs which reached GVBD. The specific parameters which determine whether the eggs of a particular fish may be induced to ovulate by the above preparations have yet to be determined. Further studies are also needed to determine whether easily measurable indicators for these parameters exist. The identification of such indicators would allow for the isolated treatment of fish capable of responding or the determination of an accurate expected percent success for any given group of fish. Further consideration must also be given to factors associated with success of fertilization. The eggs of fish induced very early in the spawning period show lower fertilization rates than eggs obtained at a later date. The relative contributions of sperm and egg quality to these low rates warrants further examination. These studies should include consideration of means of accelerating the process of vitellogenesis. At present the decision of whether or not to induce ovulation at an early stage must be weighed against the value of the eggs to be obtained at that stage and the risk factor associated with holding the adults until normal spawning. CONCLUSION
This study demonstrates that a crude salmon pituitary extract may be used to successfully induce ovulation in coho salmon over the entire normal spawning period. It has also been demonstrated that a simplified single injection of salmon pituitary extract will induce relatively mature fish. Further studies are necessary to determine how early in the normal spawning period a single injection regime will be effective. The further development of a single injec-
127
tion procedure work.
would facilitate
use of the technique
in a production
frame-
ACKNOWLEDGEMENTS
The authors wish to thank Dick Harvey, Eldon Stone and the staff of the Big Qualicum and Capilano hatcheries for their assistance in obtaining the required experimental animals. We also wish to thank Phil Edge11 for his assistance during the weighing, tagging and injection procedures. The help of Morva Young, who typed the manuscript, is greatly appreciated. REFERENCES Donaldson, E.M., 1973. Reproductive endocrinology of fishes. Am. Zool., 13: 909-927. Donaldson, E.M., Yamazaki, F., Dye, H.M. and Philleo, W.W., 1972. Preparation of gonado. tropin from salmon (Onocorhynchus tshawytschn) pituitary glands. Gen. Comp. Endocrinol., 18: 469-481. Goetz, F.W., 1976. The in vitro induction of final maturation and ovulation in brook trout (Solvelinus fontinalis) and yellow perch (Perca flauescens) ova and the hormonal control of final maturation in ova of other fish species. Ph.D. Thesis, Univ. of Wyoming, Laramie, WY, 99 pp. Hunter, G.A., Donaldson, E.M., Stone, E.T. and Dye, H.M., 1978. Induced ovulation of female chinook salmon (Oncorhynchus tshawytscha) at a production hatchery. Aquaculture, 15: 99-112. Hunter, G.A., Donaldson, E.M., Dye, H.M. and Peterson, K., 1979. A preliminary study of induced ovulation in coho salmon (Oncorhynchus kisutch) at Robertson Creek Salmon Hatchery. Fish. Mar. Serv. Tech. Rep. No. 899,15 pp. Idler, D.R., Hwang, S.J. and Bazar, L.S., 1975. Fish gonadotropin(s). I. Bioassay of salmon gonadotropin(s) in vitro with immature trout gonads. Endocrinol. Res. Commun., 2: 199-213. Jalabert, B., Bry, C., Breton, B. and Campbell, C., 1976. Action de la 17e hydroxy-200 dihydroprogest&one et de la progesterone sur la maturation et l’ovulation in vivo et sur le niveau d’hormone gonadotrope plasmatique, t-Gth chez la Truite Arc-en-ciel Salmo gairdneri. C.R. Acad. Sci. Paris, Ser. D., 283: 1205-1208. Jalabert, B., Goetz, F.W., Breton, B., Fostier, A. and Donaldson, E.M., 1978. Precocious induction of oocyte maturation and ovulation in coho salmon (Oncorhynchus kisutch). J. Fish Res. Board Can., 35: 1423-1429.
Aq~ec~lfure,
INDUCED OVULATION IN COHO SALMON (O~CO~~Y~CHUS I. FURLER STUDIES ON THE USE OF SALMON PITU~A~Y TIONS
117
KISUTCH). PREPARA-
GEORGE A. HUNTER, EDWARD M. DONALDSON and HELEN M. DYE
West Vuncouuer Laboratory, Resource Services Branch, Repartment of Fisheries and Oceans, 4160 Marine Drive, West Vancouver, B.C. V7V IN6 ~Canada} (Accepted
25 February 1981)
ABSTRACT Hunter, G.A., Donaldson, E.M. and Dye, H-M., 1981. Induced oblation in coho sahnon (Oneor~ync~us kisutch). I. Further studies on the use of salmon pituitary preparations.
Aquaculture,
26: 117-127.
The work presented extends previous studies on the use of pituitary preparations to induce ovulation in coho salmon. It has been demonstrated that coho salmon may be successfully induced to ovulate up to 2 months prior to their normal peak spawning period and that the use of a single injection of salmon pituitary extract (SPE) to induce ovulation is possible. In November 1977, the experimental fish were divided into six groups. Three groups were administered injections of 0.1 mg/kg partially purified salmon gonadotropin (SGGlOO) followed 3 days later by either 2.0 mg/kg SG-GlOO, 25.0 SPE or 50.0 SPE. A fourth group was administered 10.0 mg/kg SPE followed 3 days later by 50.0 mg/kg. The fifth group received a single injection of X00.0 mg/kg SPE. A control group was injected with saline. All treated groups ovulated significantly earlier than the control. Egg survival and mean egg diameter were within the normal range for coho from Big Qua&urn. In September and October 1978, the 10.0 mg/kg SPE primer followed by a 50.0 mg/kg secondary SPE injection were compared to a saline control. In addition, in September a second test group was administered the same primer but was followed by three daily 25.0 m&kg SPE injections. All treatment groups ovulated significantly earlier than their controls. However, only the lo-50 SPE treatment groups reached cumulative ovulation levels comparable to the controls. Further, these levels increased with closer proximity to the normal spawning period from app~ximately 40.0% in September to approximately 80% in October. Egg survival and mean degree days to the eyed stage and to hatching for the October experiment fell within the normal range for the Big Qualicum stock. In November 1978, four groups of seven fish received single intraperitoneal or intramuscular injections of either 33.0 mg/kg or 66.0 mg/kg SPE. A control group was injected with saline. All groups ovulated significantly earlier than the control: No significant differences were found between test groups related to dosage level or route of injection. Egg survival and mean degree days to the eyed stage and to hatching fell within the normal range for this stock.
0044-8486l81/0000~000~$02.50
0 1981 Elsevier Scientific Publishing Company
1
118 INTRODUCTION
In earlier publications we have outlined the potential benefits of using induced ovulation techniques to reduce prespa~ing mortality, synchronize hatchery production and obtain fertilized ova at an earlier date (Jalabert et al., 1978; Hunter et al., 1978,1979). In the first of these studies (Jalabert et al., 1978) final maturation and ovulation of coho salmon (Oncorhynchus kisutch) ova were successfully induced 3-5 weeks ahead of the normal time. The most effective treatment used in this experiment consisted of an intrape~tone~ primer (ip) injection of 0.1 mg/kg body weight partially purified salmon gonadotropin (SG-GlOO) followed within 2-3 days by an ip injection of either 1 mg/kg SG-GlOO or 3 mg/kg 17a-200 dihydroprogesterone. Later work on chinook salmon (0. tshawytscha) (Hunter et al., 1978) and coho salmon (Hunter et al., 1979) demonstrated that a crude salmon pituitary extract (SPE) could also be used to effectively induce final maturation and ovulation. The purpose of the present investigation has been to extend these initial studies with salmon pituitary gonadotropin with the intention of simplifying the injection regime, establishing the most effective route of administration, comparing the crude salmon pituitary extract with SG-GlOO, and determining how long prior to normal spawning coho salmon can be ovulated. In addition, this ~vestigation with salmon pituitary materials was conducted to provide a basis against which we could compare the effectiveness of synthetic hormones such as gonadotropin releasing hormones and an antiestrogen. MATERIALS AND METHODS
The four experiments contained in this study were conducted at the West Vancouver Laboratory of the Resource Services Branch in the fall of 1977 and 1978. Origin and transportation of fish All experiments were conducted with adult 3-year-old female coho salmon (0. ~is~tc~) which o~~nated from the Big Qualicum hatchery, V~couver Island. The normal spawning period for coho salmon from Big Qua&urn is 20 October-5 January with a peak spawning period 20-25 November. The fish used in the 1977 experiment were transported by truck directly from the Big Qualicum hatchery. Water in the holding tank was cooled with ice (6-7” C) to tranquilize the fish during transport. In 1978, the test fish were transported by truck from the Capilano hatchery, North Vancouver, B.C. These fish were Big Qu~icum stock which had been reared, fin clipped for identification and released from the Capilano facility.
119
Holding facilities Fish were held under natural photoperiod in five 3-m diameter fiberglass tanks supplied with aerated well water (60 l/min) to a depth of 0.8 m. The water temperature range was lO.O-10.5”C for all experiments. Handling and injection procedure The procedures used for the anaesthetization, weighing and tagging of fish have been described by Hunter et al. (1978). Intraperitoneal injections (ip) were directed anteriorly from a point just posterior of the pelvic fins (Hunter et al., 1978). The intramuscular injections (im) were administered at a point just ventral of the posterior limit of the dorsal fin and on the side opposing the identification tag. Following treatment fish were examined at intervals of not less than 2 days. Test solutions Physiological saline (0.65%) was used as the vehicle for all pituitary preparations tested. The partially purified salmon gonadotropin SG-GlOO used was prepared according to procedures described by Donaldson et al. (1972) and Donaldson (1973) from chinook salmon (0. tshawytscha) pituitaries obtained from mature fish at the Spring Creek National Fish Hatchery, Washington, U.S.A. The SG-GlOO powder was dissolved in saline at the required concentration. Chinook salmon pituitaries collected in 1973 at the Spring Creek hatchery were used in the preparation of the salmon pituitary extract SPE according to the method described by Hunter et al. (1978). Determination
of egg maturity
and ovulatory
responses
Egg samples were taken on each sampling day and cleared formaldehyde, acetic acid and Cortland’s salt solution (5 : 4 1976). The stage of egg maturation was determined according of Jalabert et al. (1976). The days to maturation determined vesicle breakdown (GVBD), and ovulation were recorded for Spawning
of ripe females and determination
in a solution
of
: 100) (Goetz,
to the criteria by germinal each fish.
of egg size and survival
Fish showing a strong ovulatory response were normally spawned within 24 h. Where possible the eggs from each female were fertilized with milt from at least four males. Only one male was available for the fertilization of the September 1978 SPE lo-50 fish. Excess ovarian fluid was removed prior to fertilization. Eggs and milt were allowed to stand together for 5-15 min. before contact with water. The eggs were incubated in Heath trays (Heath
7 7
October 1 1978 (Day 0 =Oct .13) 2
1 2 3 4 5
aReceived injections on Days 3, 6 and 9. bReceived injections on Days 3,4 and 5. ‘Received injections intermuscularly. dSignificant at 01< 0.05.
November 1978 (Day O=Nov.l)
15 18 17
September 1 1978 (Day O=Sept. 13) i
i 4 5 6
12 13 13 13 11 9
1
November 1977 (Day O=Nov. 4)
Number of females per group
Group number
Experiment
saline 33 SPE 33 SPEC 66 SPE 66 SPEC
saline 10.0 SPE
saline 10.0 SPE 10.0 SPE
0.1 SG-GlOO 0.1 SGGlOO 10.0 SPE 100.0 SPE
0.1 SG-GlOO
Day 0
-
salineb -
saIineb 50.0 SPE
salinea 50.0 SPE 25.0 SPEa
saline 2.0 SGGlOO 25.0 SPE 50.0 SPE 50.0 SPE
Day 3
Injection protocol dosage mg/kg body weight
f * + t + +
0.71 1.14 0.72 0.55 0.66 1.15
2.77 2.26 2.41 2.30 2.46
f f f f *
0.72 0.32 0.52 0.38 0.72
2.30 f 0.79 2.27 f 0.64
2.50 + 0.69 2.51 * 0.64 2.56 f 0.50
3.70 3.73 3.50 3.02 3.16 3.55
Mean weight of females + S.D. (kg)
100.0 100.0 100.0 100.0 100.0
100.0 100.0
60.0 100.0 100.0
75.0 100.0 100.0 100.0 100.0 100.0
Cumulative percent GVBD
7 7 6 7
9
6 5
6 8 2
7 11 13 13 11 9
Number of females reaching ovulation
100.0 100.0 100.0 85.7 100.0
85.7 71.4
40.0 44.4 11.8
58.3 84.6 100.0 100.0 100.0 100.0
Cumulative percent ovulation
+ + + f + ?
O.Od 0.9d 3.1d
o.od
6.8 l.5d
11.4 7.0 7.3 7.7 7.6
+ 4.8 * O.Od + 1.8d f 1.6d f O.gd
26.0 + 9.9 12.8 + 3.0d
64.3 + 13.2 17.1 + 12.1d 14.0 + o.od
16.9 11.1 10.0 10.0 10.3 11.6
Mean days to ovulation + S.D.
Ovulatory response of coho salmon to treatment with saimon pituitary extract (SPE). Injection protocol, number and mean weight of females, cumulative percent germinal vesicle breakdown (GVBD) and ovulation, and mean days to ovulation are presented for each experimental group in the November 1977 and the September, October and November 1978 experiments
TABLE I
121
Techna Corp.) supplied with aerated well water 15 l/min at 10°C. Approximately one half of the eggs from the 1978 experiment were incubated in city water at 6°C. Degree days to the eyed stage and to hatching were determined’ for the 1978 egg samples. Mean egg diameter based on the measurement of five samples of ten eggs, and percent survival were determined at the eyed stage. The percent survivals for the 1977 experiment were determined by incubation of all of the eggs from four females from each group. In 1978, a 200 ml (approx. 1100 eggs) sample of eggs from each of the fish in the experimental groups were used to determine egg survival. Plexiglas dividers were used to separate the Heath trays into quadrants, each of which held one 200 ml sample. Experimental protocol The experimental 1978, October 1978 I. Dates of initiation 1977,13 September
protocols for the November 1977 and the September and November 1978 experiments are presented in Table (Day 0) for these four experiments were 4 November 1978,13 October 1978 and 4 November 1978.
Induction of spermiation To permit the fertilization of ova from induced females in the November 1977 experiment, 11 males which had not yet spermiated were given a 50 mg/kg body weight ip injection of SPE on Day 10. Four males in September 1978 and five males in October 1978 were given two injections of 10 and 50 mg/kg SPE on Days 0 and 3. In the November 1978 experiment, 14 males were given a 50 mg/kg injection of SPE on Day 0. Statistical analysis One way analysis of variance (MIDAS Statistical Research Laboratory, University of Michigan) was conducted on the logarithmic transformations of the days to ovulation data. Days to ovulation data were randomized within sample intervals to permit the use of analysis of variance. In addition, measure ments of egg diameter and weight of females were subjected to analysis of variance. RESULTS
The results of the November 1977 study are presented in Fig. 1 and Tables I and II. In the SPE 25 and SPE 50 groups, all fish were found ovulated in the sampling interval preceding Day 10. Statistical analysis indicated that the five groups treated with pituitary preparations ovulated significantly earlier than the control group. No significant differences were found between treated
122
90-
30-
20-
IO-
DAY NW
0 4
3 i
6 10
9 13
12 16
v
0.1
SG-GlOO.
25
SPE
a
0.1
SG-GlOO.
50
SPE
q
10 SPE,
o
100
.
0.1
l
CONTROL
15 ,P
50
SPE
SPE SG-GlOO.
2.0
SG-GlW
-Saline
18 ??
21 25
24 28
1
Fig. 1. November 1977 experiment. Induced ovulation of coho salmon (Oncorhynchus kisutch). The numbers of fish which ovulated in each experimental group are expressed as a cumulative percentage of the total number of fii in each group. Experimental groups were administered injections of either a saline control 0.1 mg/kg SG-GlOO followed by a 25 or 50 mg/kg salmon pituitary extract (SPE), 10 mg/kg SPE followed by 50 mg/kg SPE, 0.1 mg/kg SG-GlOO followed by 2.0 mg/kg SG-GlOO or a single injection of 100.0 mg/kg SPE. Arrows indicate the days on which injections were administered. The normal peak spawning period for Big Qualicum coho is 20-25 November.
groups. All of the fish in the five treated groups and 75% of the fish in the saline control group reached final maturation as determined by GVBD. With the exception of the SG-GlOO 0.1-2.0 groups (84.6%), the treated groups reached 100% cumulative ovulations. Only 58.3% of the fish in the control group ovulated. The percent survival to the eyed stage of eggs from the six groups ranged from 78 to 99%. Of the five test groups, only salmon from the SG-GlOO 0.1-2.0 group had eggs which were significantly smaller than the control. The mean egg diameters from all six groups fell well within the normal range of egg diameters for coho from Big Qualicum. There were no significant differences between the initial body weights of females in the experimental groups.
Group number
1 2 3 4 5 6
1 2 3
1 2
1 2 3 4 5
Experiment
November 1977
September 1978
October 1978
November 1978
6 7 7 6 6
3 4
1 1 2
4 4 4 4 4 4
Egg samples incubated per group
97.3 98.4 97.6 97.7 97.4
f f * f *
92.7 f 98.0 + 2.3 1.1 1.8 3.8 1.1
5.0 2.5 213.0 221.6 210.0 219.5 239.9
+ f f f f
236.2 f 233.9 +
249 279 279.0 f
-
94 5 42.5 f 24.8
-
79 84 8S 99 78
19.6 23.0 17.2 21.5 24.1
1.1 8.8
0.0
Mean degree days to eyed stage f S.D.
93
Survival to eyed stage (%) + + f f f *
0.12 0.08 0.11 0.14 0.12 0.08
7.1 6.6 7.0 6.6 6.8
f * f f +
0.48 0.43 0.21 0.24 0.30
6.6 * 0.70 6.7 f 0.31
7.0 5.5 6.5 + 1.4
7.5 7.1 7.4 7.3 7.4 7.3
Mean egg diameter f SD. (mm)
-_ -
-
93.0 97.3 95.7 94.5 96.6
+ f f f f
7.6 1.1 2.0 9.1 1.5
461.0 443.4 395.0 434.8 466.0
f f + * f
46.7 58.7 24.0 40.6 40.1
466.8 + 11.9 461.4 f 33.5
474 485.1 + 15.0
5 37.5 f 24.8 89.0 f 6.6 93.0 f. 7.6
449
89
-
Mean degree days to hatching f S.D.
Survival to hatching (%)
Size, survival and degree days to hatching of eggs from salmon pituitary extract (SPE) treated coho salmon. Survival to the eyed stage and mean egg diameter are presented for the November 1977 experiment. Egg diameter, percent survival and mean degree days to the eyed stage and to hatching are presented for the September, October and November 1978 experiments. Group numbers correspond to those of Table I
TABLE II
124 100.
x
10 SPE. 25 SPE, 25 SPE.
o
10 SPE. 50 SPE
-ism
o
10 SPE. M SPE
-OCT
I
33 SPE, ,p
NW
.
66 SPE,Irn
NO”
0
33 SPEJP
70.
n
66 SPE,ip
-NO”
.
CONTROL
-S.,ine
-NO”
60.
.
CONTROL
-Soline
-XT
.
CONTROL
-Soline
.SEPT
PO.
80.
6 ; 4 ; 6
50.
5 ;
40.
25 SPE
-WEPT
NO”
?
? 30.
2O-
10
Fig. 2. September, October and November 1978 experiments. Induced ovulation of coho salmon (Oncorhynchus kisutch). The numbers of fish which ovulated in each experimental group are expressed ss a cumulative percentage of the total number of fish in each group. In the September and October 1978 experiment, groups were administered a saline control or a primer of 10 mg/kg salmon pituitary extract (SPE) followed by either a 60 mg/kg SPE or 3-25 mg/kg SPE injection. In the November 1978 experiment, groups were administered a saline control or a single injection of 33 mg/kg SPE or 66 mg/kg SPE via intraperitoneal (ip) or intramuscular (im) routes. Arrows indicate the days on which the injections were administered. The normal peak spawning period for Big Qualicum coho is 20-25 November.
The results of the September, October and November 1978 experiments are presented in Fig. 2 and Tables I and II. In each of the three experiments, groups which were administered pituitary preparations ovulated significantly earlier than their respective controls. Comparison of the mean days to ovulation, Table I, indicates that approximate gains of 7 weeks, 2 weeks and 4 days were made relative to controls as a result of pituitary treatment in the Septem ber, October and November experiments. It is notable that in the case of this latter experiment, the actual difference in time to 100% ovulation between treatment and control groups was 12 days (Fig. 2). The mean ovulation dates for each of the three control groups occurred between 8-16 November. While the cumulative ovulations of the SPE lo-50 and SPE 10-25-2525 groups in the September 1978 experiment reached 44.4% and 11.8% respectively, 100% of the fish in both groups reached GVBD. Only 60% of the fish in the control group of the September 1978 experiment reached GVBD. Germinal vesicle breakdown reached 100% in all groups in the October 1978 and November 1978 experiments. The cumulative percent ovulations for test and control groups were similar within each experiment with the exception of the September SPE 10-25-
125
25-25 group and increased with each subsequent experimental period (Table I). Three fish from the September 19’78 control group were accidentally killed on Day 40. There were no significant differences in egg diameters between the groups in the October and November 1978 experiments. The eggs from the 1978 fish were, however, smaller than those from the 1977 fish. Also, the mean weight of females from the 1978 experimental groups, while having no significant differences between themselves, were significantly smaller than the 1977 fish. The percent survival to the eyed stage of eggs from the October and November 1978 experiments ranged from 93-98s respectively (Table II). The mean egg diameters for the September 1978 saline, SPE lo-50 and SPE 10-25-25-25 groups were 7.0 mm, 5.5 mm and 6.5 + 1.4 mm. Difficulty in obtaining sufficient milt for fertilization prevented the rearing of the eggs of six females from the September 1978 lo-50 SPE group. The respective percentage survivals to the eyed stage were 94%, 5% and 43%. The small number of egg samples obtained from the September 1978 experiment precludes statistical analysis of egg size and percent survival. Degree days to the eyed stage and to hatching determined for the 1978 experiments all fell within the normal range of values for coho salmon. All the males in the November 1977 and November 1978 given a 50 mg/kg injection of SPE were found to be spermiating within 3 days of the injection. Of the four males given a lo-50 SPE injection in the September 1978 experiment only small quantities of milt could be obtained. Four of the five males given similar injections in the October 1978 experiment were found to be spermiating by Day 6 of the experiment. DISCUSSION
The present studies have been designed to facilitate the effective use of the induced ovulation technique within a production framework. The results of these initial experiments indicate that the technique of induced ovulation with salmon pituitary extract can be used to accelerate the process of final maturation and ovulation. The technique was effective over the observed natural range of spawning time at Capilano hatchery. Furthermore, a simple single injection regime has been used in relatively mature salmon for the first time. An examination of the November 1977 and 1978 experiments indicates that SPE administered in a single injection, in two separate injections or as a second injection following a SG-GlOO primer was as effective as the 0.1-2.0 SG-GlOO injection regime. SPE has been shown (T.G. Owen, personal eommuni~ation, 1980) to contain the equivalent of 1.5-2.0 mg SG-Gloomy wet weight pituitaries when assayed using a modification of the ovarian cyclic AMP assay described by Idler et al. (1975). The effective dose of SPE is thus lower than that which would have been predicted based on its SG-GlOO content suggesting that a synergistic factor or factors may be present in the whole
126
pituitary preparation. Alternatively, the ovarian cyclic AMP assay may not have given an accurate estimation of the ovulating potency of SPE. The similarity in effect of the single SPE injection at dosage levels of 100,66 and 33 mg/kg implies that an even lower effective dosage is possible. While no difference in effect was found between the intraperitoneal or intramuscular injection routes, differences may exist at lower dosages. The successful use of a single injection regime increases the potential application of this technique in a production framework. Further studies will be required to determine the optimal injection dosage and the period of time prior to normal peak ovulation during which a single injection regime will be effective. The results of the November 1977, September 1978 and October 1978 experiments show that the potential exists for inducing ovulation up to 2 months prior to the normal peak spawning period. The level of success achieved both in terms of percentage of fish ovulated, which is comparable with the respective controls, and the survival to hatching of the eggs, appears to be inversely related to the number of days the treatment period preceeds the normal spawning period. This suggests that there is a limit to the period of time prior to the normal spawning period that a single or series of injections of salmon pituitary preparations or salmon gonadotropin can be used to induce ovulation of maturing salmon. However, it is notable that even in the earliest treatment period, 100% of the fish in the treated groups had eggs which reached GVBD. The specific parameters which determine whether the eggs of a particular fish may be induced to ovulate by the above preparations have yet to be determined. Further studies are also needed to determine whether easily measurable indicators for these parameters exist. The identification of such indicators would allow for the isolated treatment of fish capable of responding or the determination of an accurate expected percent success for any given group of fish. Further consideration must also be given to factors associated with success of fertilization. The eggs of fish induced very early in the spawning period show lower fertilization rates than eggs obtained at a later date. The relative contributions of sperm and egg quality to these low rates warrants further examination. These studies should include consideration of means of accelerating the process of vitellogenesis. At present the decision of whether or not to induce ovulation at an early stage must be weighed against the value of the eggs to be obtained at that stage and the risk factor associated with holding the adults until normal spawning. CONCLUSION
This study demonstrates that a crude salmon pituitary extract may be used to successfully induce ovulation in coho salmon over the entire normal spawning period. It has also been demonstrated that a simplified single injection of salmon pituitary extract will induce relatively mature fish. Further studies are necessary to determine how early in the normal spawning period a single injection regime will be effective. The further development of a single injec-
127
tion procedure work.
would facilitate
use of the technique
in a production
frame-
ACKNOWLEDGEMENTS
The authors wish to thank Dick Harvey, Eldon Stone and the staff of the Big Qualicum and Capilano hatcheries for their assistance in obtaining the required experimental animals. We also wish to thank Phil Edge11 for his assistance during the weighing, tagging and injection procedures. The help of Morva Young, who typed the manuscript, is greatly appreciated. REFERENCES Donaldson, E.M., 1973. Reproductive endocrinology of fishes. Am. Zool., 13: 909-927. Donaldson, E.M., Yamazaki, F., Dye, H.M. and Philleo, W.W., 1972. Preparation of gonado. tropin from salmon (Onocorhynchus tshawytschn) pituitary glands. Gen. Comp. Endocrinol., 18: 469-481. Goetz, F.W., 1976. The in vitro induction of final maturation and ovulation in brook trout (Solvelinus fontinalis) and yellow perch (Perca flauescens) ova and the hormonal control of final maturation in ova of other fish species. Ph.D. Thesis, Univ. of Wyoming, Laramie, WY, 99 pp. Hunter, G.A., Donaldson, E.M., Stone, E.T. and Dye, H.M., 1978. Induced ovulation of female chinook salmon (Oncorhynchus tshawytscha) at a production hatchery. Aquaculture, 15: 99-112. Hunter, G.A., Donaldson, E.M., Dye, H.M. and Peterson, K., 1979. A preliminary study of induced ovulation in coho salmon (Oncorhynchus kisutch) at Robertson Creek Salmon Hatchery. Fish. Mar. Serv. Tech. Rep. No. 899,15 pp. Idler, D.R., Hwang, S.J. and Bazar, L.S., 1975. Fish gonadotropin(s). I. Bioassay of salmon gonadotropin(s) in vitro with immature trout gonads. Endocrinol. Res. Commun., 2: 199-213. Jalabert, B., Bry, C., Breton, B. and Campbell, C., 1976. Action de la 17e hydroxy-200 dihydroprogest&one et de la progesterone sur la maturation et l’ovulation in vivo et sur le niveau d’hormone gonadotrope plasmatique, t-Gth chez la Truite Arc-en-ciel Salmo gairdneri. C.R. Acad. Sci. Paris, Ser. D., 283: 1205-1208. Jalabert, B., Goetz, F.W., Breton, B., Fostier, A. and Donaldson, E.M., 1978. Precocious induction of oocyte maturation and ovulation in coho salmon (Oncorhynchus kisutch). J. Fish Res. Board Can., 35: 1423-1429.