# Precocious induction of oocyte maturation and ovulation in coho salmo Oncorhynchus kisutch

Bernard Jalabert, Frederick W. Goetz, Bernard Breton, Alexis Fostier, Edouard M. Donaldson

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Bernard Jalabert, Frederick W. Goetz, Bernard Breton, Alexis Fostier, Edouard M. Donaldson. Precocious induction of oocyte maturation and ovulation in coho salmo Oncorhynchus kisutch. Journal of the Fisheries Research Board of Canada, 1978, 35, pp.1423-1429. hal-01600088

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# Precocious Induction of Oocyte Maturation and Ovulation in Coho Salmon, *Oncorhynchus kisutch*

BERNARD JALABERT<fnref n="1" />, FREDERICK W. GOETZ²

Department of Fisheries and the Environment, Fisheries and Marine Service, Resource Services Branch, West Vancouver, B.C. V7V 1N6

BERNARD BRETON, ALEXIS FOSTIER

Institut National de la Recherche Agronomique, Laboratoire de Physiologie des Poissons, Campus de Beaulieu, 35042 — Rennes-Cedex — France

AND EDWARD M. DONALDSON

Department of Fisheries and the Environment, Fisheries and Marine Service, Resource Services Branch, West Vancouver, B.C. V7V 1N6

JALABERT, B., F. W. GOETZ, B. BRETON, A. FOSTIER, AND E. M. DONALDSON. 1978. Precocious induction of oocyte maturation and ovulation in coho salmon, *Oncorhynchus kisutch*. J. Fish. Res. Board Can. 35: 1423-1429.

Coho salmon (*Oncorhynchus kisutch*) were induced to ovulate 3-5 wk ahead of the normal time using partially purified salmon gonadotropin (SG-G100) alone or in combination with 17α-hydroxy 20β dihydroprogesterone (17α-20βP). Treatment with 17α-20βP alone induced final oocyte maturation but not ovulation and was associated with a negative feedback upon endogenous gonadotropin. The most successful treatments consisted of a "priming" injection of 0.1 mg/kg SG-G100 followed within 2-3 d by an injection of either 1 mg/kg SG-G100 or 3 mg/kg 17α-20βP. Ova from the ovulated fish were successfully fertilized. This is the first occasion in which the time of spawning has been controlled in adult migrant Pacific salmon.

Key words: coho salmon, *Oncorhynchus kisutch*, induced ovulation, final maturation, SG-G100, gonadotropin, 17α-20β dihydroprogesterone

JALABERT, B., F. W. GOETZ, B. BRETON, A. FOSTIER, AND E. M. DONALDSON. 1978. Precocious induction of oocyte maturation and ovulation in coho salmon, *Oncorhynchus kisutch*. J. Fish. Res. Board Can. 35: 1423-1429.

Chez le saumon coho, la date d'ovulation a pu être avancée de 3 à 5 sem grâce à l'injection de gonadotropine partiellement purifiée (SG-G100), seule ou combinée à la 17α-hydroxy 20β dihydroprogesterone (17α-20βP). Le traitement par la 17α-20βP seule n'a induit que la maturation ovocytaire sans ovulation, et s'est accompagné d'une rétroaction négative sur la secrétion gonadotrope endogène. Le traitement le plus efficace a consisté en une injection de « sensibilisation » de 0.1 mg/kg de SG-G100 suivie après 2-3 d par l'injection soit de 1 mg/kg de SG-G100, soit de 3 mg/kg de 17α-20βP. Les ovules obtenus ont été fécondés avec succès. C'est la première fois que la date de fraie a pu être contrôlée chez un saumon migrateur adulte du Pacifique.

Received March 30, 1978
Accepted August 1, 1978

Reçu le 30 mars 1978
Accepté le 1 août 1978

IN *Oncorhynchus* species, fish returning to hatcheries must often be kept from several days to several weeks or months to allow the completion of the natural maturation of the gonads. During this period heavy losses can occur before spawning possibly due to the high density of captive fish and associated diseases. These

losses could be avoided by inducing precocious ovulation.

The injection of gonadotropic pituitary extract, or hypophysation, which is extensively used to artificially spawn several fish species, has not been applied so far in salmonid culture except in some inconclusive laboratory experiments. Thus, the lack of information on the type and dose of the pituitary extract to be used prevents any speculation about the economic feasibility of such a treatment.

On the other hand, it is generally recognized that the effect of pituitary gonadotropins in inducing oocyte maturation (resumption of meiosis) is mediated by

<footnote n="1">¹,²Present address: Institut National de la Recherche Agronomique, Laboratoire de Physiologie des Poissons, Campus de Beaulieu, 35402 — Rennes-Cedex — France.</footnote>
steroid hormones in fish (Hirose 1976; Jalabert 1976; Sundararaj and Goswami 1977). Strong experimental evidence indicates that the specific steroid mediator for maturation in salmonids is probably a progesterone derivative, 17α-hydroxy-20β dihydroprogesterone (4 pregnen-17α, 20β diol - 3 one, abbreviation: 17α-20βP) (Jalabert 1976).

Thus, it was tempting to try using this steroid, which can be prepared easily from 17α-hydroxy progesterone (Fostier et al. 1973), to replace pituitary treatment. This was shown to be possible in trout having oocytes with the germinal vesicle (GV) in the subperipheral position, that is, approximately 2 wk before natural ovulation (Jalabert et al. 1976), and also in carp, *Cyprinus carpio*, which had received a "priming" treatment of pituitary extract to induce GV migration (Jalabert et al. 1977).

The present study was undertaken to determine whether in vivo final maturation and ovulation could be induced in the coho salmon and whether the resultant ova could be successfully fertilized. Specifically it was of interest to determine whether the synthetic hormone 17α-20βP could be used to partially or completely replace salmon gonadotropin. To better understand the mechanisms involved in induced ovulation, gonadotropin and estradiol levels were monitored during the investigation.

## Materials and Methods

Two successive experiments were carried out in October 1976 at the West Vancouver Laboratory of the Resource Services Branch on returning adult females of coho salmon, *Oncorhynchus kisutch*, from the Capilano Salmon Hatchery. They were selected according to the state of maturity of a few oocytes squeezed out by abdominal stripping after anesthesia in an aqueous solution (0.5%) of 2-phenoxyethanol (Eastman). Fish giving no oocytes by abdominal stripping, or presenting oocytes with peripheral GV, or already engaged in the process of maturation (as described in trout by Jalabert 1978) were eliminated.

Experiment A (Table 1) was started on October 8 and experiment B (Table 2) on October 28. The natural spawning period at Capilano Hatchery in 1976 was November 2–December 29 (peak November 15–December 5). The fish were kept in aerated well water at 11°C during both

experiments. Experiment A was aimed at investigating the effect of two doses of SG-G100, partially purified gonadotropin from chinook salmon (*Oncorhynchus tshawytscha*) pituitaries (Donaldson et al. 1972; Donaldson 1973) (0.25 and 2.5 mg/kg), and the effect of 17α-20βP alone or administered 2 d after SG-G100 (0.25 mg/kg). Because it appeared from experiment A that the optimum doses of SG-G100 had been overestimated and that 17α-20βP could not be administered alone in the absence of a preliminary priming by a low dose of SG-G100, experiment B was planned as indicated in Table 2.

Injections were made intraperitoneally; the doses of injected material were calculated in order to always inject the same volume of vehicle (physiological saline, 1 mL/kg).

17α-20βP was prepared from 17α-hydroxy progesterone according to Fostier et al. 1973. The preparation was used directly after crystallization in methanol-water (1:2), with nonreduced 17α-hydroxy progesterone as main contaminant (less than 20%).

Before each injection and every 2 or 3 d, the fish were anesthetized, sampled for ova and blood, then immersed in an aqueous solution of malachite green (10 mg/L) for 2–3 min to prevent *Saprolegnia* attacks. Blood samples of 0.7 mL were taken from a caudal blood vessel using a 1-mL syringe previously rinsed with heparin solution (700 IU/mL). Plasma obtained after centrifugation was kept frozen until subsequent determination of salmon gonadotropin s-GtH by double antibody radioimmunoassay (RIA). A highly purified salmon gonadotropin s-GtH (Breton et al. 1978) prepared using the trout oocyte maturation bioassay (Jalabert et al. 1974) was used as a standard and labeled according to Breton et al. (1971); the antibody, prepared against trout gonadotropin s-GtH (Breton et al. 1976), was used at a final dilution of 0.5 × 10⁻⁶. Estradiol 17β (E2) was measured only in the plasma of control fish by double antibody RIA according to Fostier et al. 1978.

Two categories of response were considered: maturation characterized by germinal vesicle breakdown (GVBD); ovulation, release of mature oocytes from the ovary into the abdominal cavity.

Usually, when ovulation occurred, it followed maturation by 2–4 d. As soon as ovulated fish were detected in the different groups, approximately 200 eggs were taken by abdominal stripping and fertilized with diluted coho sperm (1/100) according to Billard et al. 1974; fertilization success was estimated from the proportion of eggs with apparently normal embryos after more than 10 d of development.

<table><caption>TABLE 1. Protocol for Experiment A.</caption><thead><tr><th rowspan="2">Lot</th><th rowspan="2">No. of fish</th><th colspan="3">Treatment</th></tr><tr><th>Oct. 8, 1976</th><th>Oct. 10, 1976</th><th>Oct. 18, 1976</th></tr></thead><tbody><tr><td>1<br>(control)</td><td>16</td><td>Physiological saline</td><td>Physiological saline</td><td></td></tr><tr><td>2</td><td>13</td><td>SG-G100 0.25 mg/kg</td><td>Physiological saline</td><td></td></tr><tr><td>3</td><td>13</td><td>SG-G100 0.25 mg/kg</td><td>17α-20βP 2 mg/kg</td><td></td></tr><tr><td>4</td><td>13</td><td>SG-G100 2.5 mg/kg</td><td>Physiological saline</td><td></td></tr><tr><td>5</td><td>17</td><td>17α-20βP 2 mg/kg</td><td>Physiological saline</td><td>17α-20βP 4 mg/kg</td></tr></tbody></table>
<table><caption>TABLE 2. Protocol for Experiment B.</caption><thead><tr><th rowspan="2">Lot</th><th rowspan="2">No. of fish</th><th colspan="2">Treatment</th></tr><tr><th>Oct. 28, 1976</th><th>Oct. 31, 1976</th></tr></thead><tbody><tr><td>1<br>(control)</td><td>12</td><td>Physiological saline</td><td>Physiological saline</td></tr><tr><td>2</td><td>12</td><td>SG-G100 0.1 mg/kg</td><td>Physiological saline</td></tr><tr><td>3</td><td>12</td><td>SG-G100 0.1 mg/kg</td><td>17α-20βP 3 mg/kg</td></tr><tr><td>4</td><td>12</td><td>SG-G100 0.1 mg/kg</td><td>SG-G100 1 mg/kg</td></tr></tbody></table>

## Results

### EXPERIMENT A

Maturation and ovulation responses of fish in experiment A are shown in Fig. 1. In the control group (lot 1) some fish began to mature and ovulate 8-10 d after day 0, and the percentage of ovulated fish rose to 36% by day 23. In lots 2, 3, and 4, oocyte maturation (GVBD) was observed on day 4 in 30-80% of the fish, and in 100% of them on day 12. But the number of fish that subsequently underwent normal ovulation reached a maximum of around 60%, except in lot 4 where some further ovulations occurred after 12 d. However, these latter fish were only partially ovulated (30-70%).

In lot 5, 85% of the fish exhibited mature oocytes on day 6, and only one fish ovulated on day 10. Surprisingly, the percentage of fish with mature oocytes appeared to decrease after day 6; in fact, autopsy of fish

at the end of experiment revealed that 17α-20βP injected intraperitoneally could induce heterogeneous maturation in the ovaries of some fishes, with parts exhibiting fully mature oocytes (after GVBD) and other parts where oocytes remained at the initial stage. As some follicles containing mature oocytes are more fragile than the others, these are emitted first by abdominal stripping and can be followed by emission of oocytes in less advanced stages. The injection of a further dose of 17α-20βP on day 10 increased the percentage of fish emitting mature oocytes, but no more ovulations occurred.

In any case, when ovulation did not follow oocyte maturation, autopsy revealed that mature oocytes were completely free within their follicle, so that they could be expelled easily after tearing the follicular envelopes with small forceps.

Plasma gonadotropin levels are presented in Fig. 2. In control fish (lot 1) s-GtH levels fluctuated around 0.5-5 ng/mL but a considerable variation between fish was seen, as shown by the large standard deviation. In lots 2 and 3, injection of SG-G100 (0.25 mg/kg) brought about a surge in plasma s-GtH up to 40-50 ng/mL, which is significantly higher than in the controls, followed by a gradual decrease; the difference with controls remained highly significant until day 8 and significant on day 10. In lot 4 injected with SG-G100 (2.5 mg/kg), the surge of s-GtH was much higher exceeding 300 ng/mL on day 2, and the difference with the controls remained highly significant, even on day 14. In lot 5, injection of 17α-20βP (2 mg/kg) induced a significant decrease in plasma s-GtH on days 2 and 4 in

<figure><img src="image_2.png" /><figcaption>FIG. 1. Percentages of fish exhibiting oocyte maturation (oocytes after GVBD) (---●---) or ovulation (—▲—) in the five experimental lots of experiment A.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>FIG. 2. Mean plasma gonadotropin levels (s-GtH) in the five experimental lots of experiment A. Vertical bars show SE; number of determinations is indicated for each mean. Statistical significance of difference between means is indicated as follows: ***very highly significant (probability level 0.001); **highly significant (probability level ≤0.01); *significant (probability level ≤0.05).</figcaption></figure>

comparison to the control; s-GtH rose again to the control level on days 8 and 10, but the new injection of 17α-20βP on day 10 was again followed by a significant decrease on days 12 and 14.

Taking into account that the variability of the hormonal level in plasma of control fish might be due partially to their asynchronism regarding maturation time, gonadotropin and estradiol levels have been presented in Fig. 3 as a function of time before ovulation. The mean gonadotropin level, around 2 ng/mL, does not exhibit any significant variation and shows only a slight tendency to rise (3.5 ng/mL) at the time of ovulation. On the contrary, estradiol-17β does exhibit a significant decrease, from 15 ng/mL (21 days before ovulation) to 1 ng/mL at the time of ovulation; the decrease is particularly important between the 12th and 3rd d before ovulation.

<figure><img src="image_4.png" /><figcaption>FIG. 3. Mean levels of s-GtH and estradiol 17β as a function of time before ovulation in control fish (lot 1) of experiment A. Significant differences indicated as in Fig. 2.</figcaption></figure>

Finally, the success of embryonic development in eggs from ovulated fish is shown in Table 3: the mean values for control lot 1, lot 4, and untreated control fish (not submitted to the same severe handling) are normal and do not differ from one another. On the other hand, the percentages of embryonic development in eggs from fish in lots 2, 3, and 5, although still high, are significantly lower than controls.

## EXPERIMENT B

Maturation and ovulation responses in the different lots are shown in Fig. 4. Maturation occurred faster than in experiment A; moreover, these fish, closer to natural ovulation, were much more delicate than those in experiment A, and the experiment was stopped after 14 d

<table><caption>TABLE 3. Percentage of eggs from fish in experiment A that showed embryonic development after 10-d incubation (approximately 200 eggs per fish). N.S. — No significance; *Significant at 5% level.</caption><thead><tr><th>Lot</th><th>Untreated<br>control fish<br>(no handling)</th><th>1<br>Control</th><th>2<br>SG-G100<br>0.25 mg/kg</th><th>3<br>SG-G100<br>0.25 mg/kg<br>+<br>17α-20βP<br>2 mg/kg</th><th>4<br>SG-G100<br>2.5 mg/kg</th><th>5<br>17α-20βP<br>2 mg/kg</th></tr></thead><tbody><tr><td>Number of fish</td><td>6</td><td>7</td><td>7</td><td>7</td><td>7</td><td>2</td></tr><tr><td>Mean percentage,<br>calculated through<br>transformation</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Arc sin √proportion</td><td>95.1</td><td>98.2</td><td>90.3</td><td>90.4</td><td>98.2</td><td>85.3</td></tr><tr><td>Significance of the<br>difference with control 1</td><td>N.S.</td><td></td><td>*</td><td>*</td><td>N.S.</td><td>*</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>FIG. 4. Percentages of fish exhibiting oocyte maturation (oocytes after GVBD) (---■---) or ovulation (—●—) in the 4 experimental lots of experiment B.</figcaption></figure>

because of heavy prespawning mortality in the control group (lot 1).

The percentage of ovulated fish as a function of time until day 11 was not very different in lot 2 (SG-G100 alone, 0.1 mg/kg) from that of the controls (lot 1), although SG-G100 priming increased the percentage of fish undergoing maturation. Either 17α-20βP (3 mg/kg) or SG-G100 (1 mg/kg) administered 3 d after priming with SG-G100 induced nearly 100% ovulation.

Plasma gonadotropin levels are shown in Fig. 5. In the control fish (lot 1) s-GtH levels fluctuated between 1 and 5 ng/mL as in experiment A. In lots 2 and 3, injection of SG-G100 (0.1 mg/kg) caused a surge in plasma s-GtH up to 10 ng/mL which was very highly significant in comparison to control levels on day 3, highly significant on day 6, and still significant on day 9. In lot 4, the very highly significant increase on day 3 was followed by a further rise, up to 200 ng/mL following treatment by SG-G100 1 mg/kg.

As shown in Table 4, the success of embryonic development was equally high in all of the 4 experimental lots, without any statistically significant difference.

<figure><img src="image_6.png" /><figcaption>FIG. 5. Mean plasma gonadotropin levels (s-GtH) in the 4 experimental lots of experiment B. Vertical bars show SE; number of determinations is indicated for each mean. Statistical significance of the difference between means is indicated as follows: ***very highly significant (probability level 0.001); **highly significant (probability level 0.01); *significant (probability level 0.05).</figcaption></figure>

Discussion

The present work is the first consistent demonstration that precocious ovulation can be induced by hormonal treatment in adults of a migrating salmonid species, at least 5 wk in advance to the time of natural ovulation in 60% of the fish, and that the ova produced can be successfully fertilized. Moreover, it shows that all individuals could respond positively to a combined treatment consisting of an injection of SG-G100 followed within 2-3 d by an injection of either SG-G100 or 17α-20βP as shown in experiment B. However, the fish in experiment A were "greener" than in experiment B

<table><caption>TABLE 4. Percentage of eggs from fish in experiment B that showed embryonic development after 10 d (approximately 200 eggs per fish).</caption><thead><tr><th rowspan="2">Lot</th><th>1</th><th>2</th><th colspan="2">3</th><th colspan="2">4</th></tr><tr><th>Control</th><th>SG-G100</th><th>SG-G100 0.1 mg/kg</th><th>SG-G100 0.1 mg/kg<br>+<br>17α-20βP</th><th>SG-G100 0.1 mg/kg<br>+<br>3 mg/kg</th><th>SG-G100 0.1 mg/kg<br>+<br>1 mg/kg</th></tr></thead><tbody><tr><td>Number of fish</td><td>5</td><td>6</td><td>11</td><td></td><td>10</td><td></td></tr><tr><td>Mean percentage, calculated through<br>transformation</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Arc sin √proportion</td><td>97.4</td><td>95.1</td><td>97.2</td><td></td><td>97.1</td><td></td></tr><tr><td>Significance of the difference with<br>control 1</td><td></td><td>N.S.</td><td>N.S.</td><td></td><td>N.S.</td><td></td></tr></tbody></table>
and the possibility that successful treatments found in the second experiment can be as efficient in "greener" fish requires further investigation.

The low dose of SG-G100 used with success in experiment B may still be more than is needed and can be expected to be lowered for practical application. However, this dose is already much lower than the amount of SG-G100 used to induce ovulation in the goldfish, *Carassius auratus* (Yamazaki and Donaldson 1968), the Indian catfish, *Heteropneustes fossilis* (Sundararaj et al. 1972), the striped mullet, *Mugil cephalus* (Shehadeh et al. 1973), the ayu, *Plecoglossus altivelis* (Hirose et al. 1973), or in the Japanese flounder, *Limanda yokohamae* (Hirose et al. 1976). The higher efficiency of the salmonid gonadotropin when administered to salmonid species is in accordance with the notion of the species specificity of gonadotropin among fishes (Breton et al. 1973; Fontaine 1976).

Experiment A provides the first observation that 17α-20βP administered alone too early in the process can induce oocyte maturation without subsequent ovulation and that this effect is accompanied by a negative feedback upon endogenous gonadotropin secretion. Contamination of the preparation of 17α-20βP used here with some 17α-hydroxy progesterone is probably not responsible for blocking the ovulation process, as this is also a natural steroid normally found in salmon plasma at the time of spawning (Schmidt and Idler 1962). An alternative explanation is that maturation and ovulation, although both ultimately under gonadotropic control, are controlled in fact through different mechanisms by distinct mediators, as evidenced by previous work in vitro in trout. While 17α-20βP appears to control both oocyte maturation and follicular detachment from the oocyte (Jalabert 1978), ovulation control is likely to involve the action of prostaglandins and (or) catecholamines and stimulation of α-andrenergic receptors (Jalabert 1976). Thus it is not surprising that injection of 17α-20βP alone results only in oocyte maturation and follicular detachment without ovulation, particularly when exerting a negative feedback upon endogenous gonadotropin which would be expected to favor ovulation. In contrast, 17α-20βP did induce maturation followed by successful ovulation when injected into rainbow trout at a stage closer to natural ovulation (GV subperipheral) (Jalabert et al. 1976). At that later stage plasma gonadotropin levels were higher, and injected 17α-20βP did not exhibit any negative feedback upon endogenous gonadotropin.

Thus both theoretical considerations and endocrinological data give physiological support to the favorable effect of a moderate preliminary gonadotropic priming observed in experiment B before either 17α-20βP treatment or even SG-G100 treatment. In fact, experiment A showed that 30-40% of the fish injected only once with a high dose of SG-G100 were not able to ovulate after maturation, as occurred after injection of 17α-20βP alone, which probably means that a gonadotropin overdose given without priming can upset the normal

sequence of physiological events. Such priming has already been used empirically in the current practise of hypophysation (for example, in phytophageous carp, see Vinogradov 1966); in carp, priming was found necessary for further successful action of 17α-20βP upon maturation and ovulation and was shown to induce GV migration (Jalabert et al. 1977).

The profiles of gonadotropin values found in experimental fish after injection with SG-G100 and their relationship to the injected doses appear to be in the same range as those found by Crim and Evans (1976) in immature rainbow trout, which is of interest as differences could have been expected as a result of differences between species and maturity stages.

The values of plasma gonadotropin in control coho during a preovulatory period of 21 d appear very low, with only a slight rise just before ovulation, whereas in trout a small but significant increase was seen during GV migration. These low gonadotropin values during the preovulatory period in salmonids held in artificial conditions differ from the high levels observed in sockeye salmon on natural spawning grounds, in possible relationship with spawning behavior (Crim et al. 1975). On the other hand, the profile of estradiol 17β in control coho is very similar to the profile observed in rainbow trout during the same period (Fostier et al. 1978). Estradiol has been shown to decrease the efficiency of hormones on intrafollicular maturation in vitro (Jalabert 1975). It would be interesting to investigate further the relationship between estradiol level and the ability of 17α-20βP to induce maturation without ovulation block.

## Acknowledgments

This work was made possible by an exchange and co-operation program between the National Research Council of Canada and the French Ministry of Foreign Policy (Direction générale des relations culturelles scientifiques et techniques) and by the award of a postdoctoral fellowship to F. W. G. by the National Research Council of Canada. We thank Mr E. Stone and the staff of the Capilano Salmon Hatchery for their assistance in providing the coho salmon used in this study and H. M. Dye, P. Reinaud, M. Heydorff, and S. Marlot for excellent technical assistance.

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