Sigal Drori, Michal Ok, Berta Levavi-Sivan, Zvi Yaron. Department of Zoology, Tel-Aviv University, Tel-Aviv, Israel (Accepted 20 September 1993)

Aquacuhre, 119 (1994) 393-407 Elsevier Science B.V., Amsterdam 393 AQUA 60068 Spawning induction in common carp (Cyprivtus carpio) using pituitary extract or GnRH superactive analogue combined with metoclopramide: analysis of hormone profile, progress of oocyte maturation and dependence on temperature Sigal Drori, Michal Ok, Berta Levavi-Sivan, Zvi Yaron Department of Zoology, Tel-Aviv University, Tel-Aviv, Israel (Accepted 20 September 1993) ABSTRACT Spawning experiments under routine hatchery conditions on Israeli farms showed that successful ovulation in the common carp (Dor 70 x Yugoslavian lines) can be achieved by a single administration of 10 pg/kg sgnrha ( [D-Arg6,Prop-NEt I-sGnRH) combined with 20 mg/kg of the water-soluble dopamine receptor antagonist, metoclopramide (GnRH+MET). An initial rise in the maturational gonadotropin (cgth) level occurred 3 h after treatment, gradually increasing thereafter, and reaching a peak of 227f 41.8 ng/ml (mean? s.e.m., n= 10) 14 h after treatment, when full ovulation took place, as reflected by the presence of a few expelled eggs on the bottom of the tank. This rise was associated with increased levels of oestradiol-17/i (Ez; 19.5 f 3.4 ng/ml) and 17a,20/?-dihydroxy-4- pregnen-3-one ( 17,20-P; 23.9 f 19.7 ng/ml). Ovarian biopsies showed a gradual progress in oocyte maturation with germinal vesicle breakdown (GVBD) occurring during the rise in both steroids. At the time of initial egg release, GtH remained at a high level while the steroids started to decline. In a parallel group, fish were primed with a dose of carp pituitary extract (CPE), calibrated to contain 0.07 mg/kg cgth, and 11 h later with a resolving dose of CPE (0.34 mg/kg cgth). Following priming, circulating GtH and E, increased moderately, and the germinal vesicle migrated towards the oocytes periphery. A further increase in cgth and E2, and a sharp peak in 17,20-P, occurred concomitantly with GVBD, 6 h after the resolving dose was given. Data compiled from several spawning experiments showed that the interval between hormone administration and initial egg release (latency) was negatively correlated with water temperature over the range of 20-26 C. While latency was always longer in GnRH + MET than in CPE treatment, in each treatment it was nonetheless constant between 22.5 C and 25 C. GnRH + MET given at various hours of the day or evening resulted in identical spawning with the same latency. This fact, together with the predicted latency at a given temperature, may provide the aquaculturist with a protocol to accurately schedule spawning induction in the common carp. Correspondence to: Dr. Z. Yaron, Department of Zoology, Tel-Aviv University, Tel-Aviv 69978, Israel. 0044-8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved. ssdi0044-8486(93)e0240-a

394 S. DRORI ET AL. INTRODUCTION Induced spawning in cyprinid fish is currently carried out in Israel by the hypophyseal approach, utilizing a calibrated carp pituitary extract (CPE) containing a predetermined dose of gonadotropin ( GtH) (Yaron et al., 1984). Spawning carp in Israel is generally performed in hatcheries equipped with recirculating water systems; the females are introduced into the hatcheries, and after hormonal treatment, given as priming and resolving doses, the eggs are stripped, fertilized and incubated in funnels (Rothbard, 198 1). The satisfactory results attained using CPE have so far delayed the introduction of the contemporary hypothalamic approach into Israeli cyprinid aquaculture. Due to the recent increase in production of cyprinids in the country, mainly ornamental fish, and the decrease in the number of processed carp, the supply of pituitaries can no longer meet the demand for the calibrated extract. This situation led to the introduction of the Linpe method (Peter et al., 1988) in local aquaculture. This method is based on the stimulation of endogenous GtH release by a superactive analogue of piscine gonadotropin-releasing hormone (sgnrha) combined with a dopamine (DA) receptor antagonist which potentiates the response to the peptide. The success of this method in spawning of fish, mainly cyprinids, is well documented in many articles and reviews (e.g. Peter et al., 1987, 1988, 1993; Zohar, 1986). In most reports on spawning induction in cyprinids using GnRH with or without DA antagonist, the latency of the response (i.e. the duration between treatment and ovulation) and its dependence on water temperature are only reported in general terms (e.g., Rottmann and Shireman, 1985; Peter et al., 1987; Glubokov et al., 199 1). Therefore, before any recommendations could be given to fish breeders for the use of these compounds, both the dose and the precise timing in relation to water temperature had to be studied under local conditions. In goldfish maintained under a long photoperiod, ovarian recrudescense, in response to repeated injection of carp GtH, varied during the day according to the time of injection, being maximal 12 h after the onset of light. This has indicated daily variation in the ovarian response to GtH (Peter et al., 1982). In the carp, too, the oocyte maturational response to GtH both in vivo and in vitro was found to fluctuate in a daily rhythm, being maximal 9 h after the onset of the photophase. Furthermore, pituitary homogenate injected at 9 a.m. resulted in a higher ovulation response than when injected at 9 p.m. (Bieniarz et al., 1985 ). Therefore, it was essential to clarify whether the ovulatory response of carp to sgnrha combined with a DA antagonist, under local conditions, differs when treatment is given at various hours of the day. Although changes in the profile of maturational gonadotropin (GtH II) following GnRH and DA antagonists have been reported in a number of cyprinid fish, the ensuing alterations in ovarian steroids are rarely reported (see,

TEMPERATURE AND SPAWNING INDUCTION IN CARP 395 however, Peter et al., 1984). Changes in GtH level, in parallel with progress of oocyte maturation, was reported in carp treated with LHRH-A, but without dopamine antagonists, resulting in ovulation of only 50% (three out of six females) (Billard et al., 1984). The objectives of the present work were: (a) to calibrate the dose of a piscine GnRH superactive analogue in combination with a dopamine antagonist, given in a single injection, that would be suitable for spawning induction in common carp ( Dor 70 line) under local conditions; (b) to determine the precise latency period between treatment and ovulation as a function of temperature; (c) to clarify whether the ovulatory response varies when fish are treated at various hours of the day; (d) to record the progress in oocyte maturational stages with the circulating levels of GtH II, and two ovarian hormones: oestradiol, representing the main steroid involved in vitellogenesis, and 17cr,20/3-dihydroxy-4-pregnen-3-one ( 17,20-P), considered to be the maturation-inducing hormone in cyprinids; (e) to compare these parameters in fish induced to spawn by sgnrha and a dopamine antagonist with those treated with CPE. MATERIALS AND METHODS Fish andjish maintenance The experiments were conducted on female common carp (Cyprinus carpio) of the Dor 70 line (Wolfahrt et al., 1980) or on F1 of a cross between Dor 70 and Yugoslavian lines. Females ( 1.0-l.5 kg BW) stocked without males in earthen ponds were first selected for ripeness by the softness of their abdomens, and transferred for acclimation into 650-litre tanks with circulating water at 20-26 C and ample aeration. Two to three days later, ovarian biopsies were taken, clarified and examined as described in Levavi-Zermonsky and Yaron ( 1986). The stage of oocyte maturation was determined as follows: Stage I, central germinal vesicle (GV); Stage II, migrating (eccentric) GV; Stage III, peripheral GV, Stage IV, GV breakdown (GVBD ) ; Stage V, ovulated eggs in ovarian lumen. Only fish in which more than 60% of the oocytes were at Stage II were selected for the spawning experiments. Hormones and drugs were injected into the dorsal musculature at a volume smaller than 0.5 ml/kg. Following injection, the bottom of each tank was examined every 30 min for the presence of released eggs (initial egg release) which indicates that ovulation has occurred. Each female, emitting some eggs, was anaesthetised with ethyl aminobenzoate (0.15 g/l), blotted, and the eggs then stripped by abdominal pressure. Fertilization and incubation were carried out according to Rothbard ( 198 1).

396 S. DRORI ET AL. Hormones and drugs sgnrha ( [ D-Arg6,Pro9-NEt ]-sgnrh) was purchased from Bachem Inc. (Torrance, CA), domperidone (DOM) and metoclopramide (MET) from Sigma (St. Louis, MO). The sgnrha and MET were dissolved in 0.7% saline while DOM was given as a suspension in acidified saline containing 0.1% sodium metabisulphite (Chang et al., 1984). Hormone determinations Blood was sampled from the caudal vessels into a heparinized syringe. Plasma was separated and stored at - 20 o C till assayed. Maturational carp GtH II (cgth) was determined by a double-antibody radioimmunoassay (RIA) according to Levavi-Zermonsky and Yaron ( 1986 ) and Yaron and Levavi-Zermonsky ( 1986) except that the second antibody employed was donkey anti-rabbit immunogammaglobulin bound to a magnetizable compound ( Amerlex, The Radiochemical Centre, Amersham). The bound and unbound cgth fractions were separated on magnetic plates. Sensitivity of the assay was 0.8 ng/tube; the intra- and inter-assay coefficients of variation were 4.1% (n = 20) and 20% (n = 11)) respectively. Oestradiol was determined by a double-antibody RIA using standard oestradiol- 178 ( Ez; Ikapharm, Israel), 251-E2(Rotem, Israel) and anti-e2 as specified previously (Levavi-Zermonsky and Yaron, 1986 ), The bound and unbound fractions were separated as above. The sensitivity of the assay was 2.9 pg/ml. The intra- and interassay coefficients of variation were 8.3% (n= 10) and 16.9% (n=6), respectively. The progestagen, 17q20fi-dihydroxy-4-pregnen-3-one ( 17,20-P), was determined according to Scott et al. ( 1982) as modified by Levavi-Zermonsky and Yaron ( 1986 ), except that the antiserum used was developed in Rennes (Fostier et al., 198 1). The cross-reactivity of the antiserum with other steroids was 2% with 20j?-dihydroprogesterone and ~2% with progesterone, pregnenolone, 17cu-hydroxyprogesterone, 20o+dihydroprogesterone, cortisone, 20/3-dihydrocortisone, and deoxycorticosterone. No cross-reaction was found with cortisol, corticosterone, testosterone, 11 -ketotestosterone, oestradiol and oestrone (Fostier et al., 198 1). Statistical analysis When appropriate, the significance between treatment groups in the spawning experiments was analysed non-parametrically by Fisher s Exact Test. The difference among group means of hormone values was tested by AN- OVA, followed by the Least Significant Difference test for multiple range experiments, and by Student s t-test for pairs of means.

TEMPERATURE AND SPAWNING INDUCTION IN CARP 397 RESULTS The optimal dose of sgnrha The following experiment was designed to determine the lowest effective dose of sgnrha ( [ D-Arg6,Pro9-NEt ]-sgnrh). Sixty female carp were divided into six groups and administered with increasing doses of sgnrha combined with a constant dose of DOM ( 5 mg/kg). Fish injected with saline (0.7%) served as a negative control to examine the possibility of spontaneous spawning under the hatchery conditions, while CPE-injected fish served as a positive control (Table 1). None of the tish injected with saline or the lowest dose of the sgnrha spawned. The spawning rate was similar in CPE-treated fish and in fish injected with either 10 or 50 pg/kg sgnrha+ 5 mg/kg DOM (Table 1). The optimal dose of metoclopramide Ten fish injected with sgnrha ( 10 pg/kg) combined with 20 mg/kg MET spawned successfully while lower doses of MET only led to progress in oocyte maturation but not to ovulation (Table 2). Potentiation of sgnrha effect by metoclopramide The GtH level in female carp injected with either saline or MET (20 mg/ kg) alone did not increase 12 h after injection, while injection of sgnrha ( 10 pg/kg) alone was followed by a slight increase in the hormone (Fig. 1). A lo-fold-increase in circulating GtH was observed in fish injected with the TABLE 1 Spawning induction in common carp ( DOR 70 line; l-l.5 kg BW) using graded doses of sgnrha ( [ D-Arg6,Prog-NEt ]-sgnrh) alone or combined with a constant dose of domperidone (DOM), or with a calibrated carp pituitary extract (CPE) containing 0.07 mg/kg cgth for priming and 0.34 mg/ kg cgth as a resolving dose Treatment Vehicle sgnrha 50,ng/kg sgnrha 5 egg/kg + DOM 5 mg/kg sgnrha 10,ug/kg +DOM 5 mg/kg sgnrha 50 pg/kg + DOM 5 mg/kg CPE Spawning ratio Latency (h) o/10-5110 30 : o/10 lo/lo 28 lo/lo 9110 28 C 9 bc Significance a C Latency = hours elapsed from the single injection of sgnrha or the resolving injection of CPE and initial egg release. Significance relates to the spawning ratio only; groups designated by the same letter do not differ signiticantly from each other (Fisher exact test; P> 0.05). Water temperature: 24 C.

398 S. DRORI ET AL. TABLE 2 Spawning induction in common carp using a constant dose ( 10 &kg) of sgnrha ( [ D-Arg6,Prog- NW ]-sgnrh) combined with graded doses of metoclopramide (MET) -- Treatment Spawning ratio or oocyte stage Saline sgnrha 10 pgg/kg + MET 5 mg/kg sgnrha 10 pgg/kg + MET 10 mg/kg sgnrha 10 fig/kg + MET 20 mg/kg 6%-Stage III 94%-Stage IV 1 00%-Stage IV 9/10 Ten fish in each group. Water temperature: 20.5 C. The percentage of oocyte maturation derives from the total count of oocytes in ovarian biopsies. Oocyte Stage III = peripheral germinal vesicle; Stage IV, GVBD=geiminal vesicle breakdown.,e 125 P 100 z W 75 50 25 EzzzBmBmm i C M G GM CPE Fig. 1. Increase in circulating cgth level 12 h after injection of C (saline, 0.7%), M (metoclopramide, 20 mg/kg), G (sgnrha, 10 pguglkg), G+ M (sgnrha, 10,ug/kg combined with metoclopramide, 20 mg/kg) or CPE (calibrated carp pituitary extract containing 0.07 + 0.34 mg/kg cgth in priming and resolving doses, respectively). I = initial level. Water temperature: 23 C. Mean + s.e.m.; n = 10. Group means designated by the same letter do not differ significantly from each other (P> 0.05, Least Significant Difference test). combination of sgnrha ( 10 pg/kg) and MET (20 mg/kg). Therefore, the combination of 10 pg/kg sgnrha and 20 mg/kg MET given concomitantly in a single injection (henceforth GnRH + MET ) was chosen as the standard treatment for spawning induction in all further experiments. Timing of the treatment Initial egg release in fish kept at 20.5 o C and injected with GnRH + MET at 07.00, 12.00, 16.00 or 20.00 occurred after a latency of 19-2 1 h, irrespective

TEMPERATURE AND SPAWNING INDUCTION IN CARP 399 of the hour of injection. The spawning rate was similar in all groups (Table 3). cgth levels in the circulation of 20 fish kept at 23 C and injected with GnRH+MET at 9 a.m. did not differ from those of 20 fish injected at 8 p.m. (Fig. 2 ). Initial egg release occurred after a 14-h interval in all 40 fish. Hormone projile and progress in oocyte maturation For the following experiment, only female fish in which 65% of the oocytes possessed migrating germinal vesicles (Stage II) were selected. In saline-injected fish there was no progress in oocyte maturation; ovarian biopsies showed that 70-90% of the oocytes remained at Stage II throughout the experiment (Fig. 3A). Circulating cgth remained constant at about 6 ng/ml, except for a small but significant peak of 9.2-1-0.2 ng/ml which occurred at 10 p.m. (Fig. 3B). A similar pattern was noted in the level of cir- TABLE 3 Spawning induction in common carp by sgnrha ( 10 fig/kg) combined with metoclopramide (20 mg/kg) given at various hours during the day Hour of treatment Hour of initial egg release Spawning ratio Latency (h) Significance 07.00 12.00 16.00 20.00 02.00 07.00 11.00 15.00 lo/lo 618 5/g 10/12 19-21 19-21 19-21 19-21 Water temperature: 20.5 o C. Other details as in Table 1. -14-11 -7-3 0 HOURS PRIOR TO EGG RELEASE mu. 09.00 m NJ. 20.00 Fig. 2. Increase in circulating cgth in response to sgnrha ( 10 pg/kg) combined with metoclopramide (20 mg/kg) injected at 09.00 or at 20.00 ( 14 h before egg release). Blood was sampled just before treatment ( - 14 h), 11, 7, 3 h before, and during egg release (0). Water temperature: 23 C. Mean f s.e.m.; n = 10. The comparison (Student s t-test) was done separately for each time interval.

% OOCYTE STAGE GtH (ng/ml) % OOCYTE STAGE I c!l ~ em m 0 -L~~*mrn-4 0000000 100000000 GtH (ng/ml) GtH (ng/ml)

TEMPERATURE AND SPAWNING INDUCTION IN CARP 401 culating oestradiol; the level never exceeded 3 ng/ml. The level of 17,20-P was low or undetectable throughout the experiment (Fig. 3B). Oocyte maturation, culminating in ovulation, was noted in ovarian biopsies of fish treated with GnRH + MET (Fig. 3C). Circulating cgth increased progressively following treatment and reached a peak of 227 + 4 1.8 ng/ml at initial egg release, 3 h after ovulation (Fig. 3C, D ). Oestradiol level increased in parallel to that of cgth but started to decline following ovulation. The initial increase in 17,20-P occurred 7 h after treatment, when germinal vesicle breakdown (GVBD) occurred in 70% or more of the oocytes. However, the peak in the 17,20-P level was noted 4 h later (23.9 L 19.7 ng/ml; mean + s.e.m.; n = 10). There was considerable variation in the level of this steroid among individual fish at the time of ovulation ( - 3 h) and at the point of egg release (0 h). At - 3 h, 3 fish had levels lower than 1 ng/ml, while 7 fish had levels ranging from 17 to 199 ng/ml. Similarly, at the point of egg release, the level of 17,20-P in 5 fish was lower than 1 ng/ml, while in the other fish the level ranged from 1 to 40 ng/ml. All hormones examined had values close to their initial level, 26 h after treatment (Fig. 3D). In CPE-treated fish, progress in oocyte maturation occurred shortly after the priming injection; the percentage of oocytes with peripheral GV (Stage III) increased from 4 to 60%. However, GVBD occurred only after the resolving dose was given and was followed by complete ovulation (Fig. 3E). An initial rise in both cgth and oestradiol occurred following priming, while 17,20-P remained unchanged (Fig. 3F). Upon administration of the resolving dose, a peak of circulating cgth (545 +- 70 ng/ml) occurred, and was followed by a prominent surge of 17,20-P (28.5 2 9.8 ng/ml). The oestradiol level increased moderately and was still high during initial egg release. All hormones examined receded after spawning, and returned to their initial level 37 h after treatment (Fig. 3F). Dependence of latency on temperature Data on the period of latency between injection of hormone preparation and initial egg release were compiled from 16 spawning experiments carried Fig. 3. Progress of oocyte final maturation in relation to hormone profile during spawning induction in carp. Carp ( DOR 70 line; l-l.5 kg BW) were injected either with saline (A, B), sgnrha (10 pg/kg) combined with metoclopramide (20 mg/kg) (C, D) or with calibrated carp pituitary extract containing 0.07 mg+0.34 mg/kg cgth as priming and resolving doses (E, F). Blood samples and ovarian biopsies were taken at intervals expressed as hours prior to or after initial egg release (S). Stages of oocyte maturation (A, C, E) as determined in clarified ovarian samples were: I, central germinal vesicle (GV); II, migrating GV, III, peripheral GV, IV, GV breakdown; V, ovulated eggs. Circulating level of cgth (GtH), oestradiol-17/? (E,) and 17a,20&lihydroxy-4-pregnen-3-one (PRG) of the 3 groups are presented in the right panel (B, D, F). Water temperature: 23 C. Mean + s.e.m.; n = 10. The single injection of saline or GnRH + MET was given at time marked as - 14 h.

402 S. DRORI ET AL 24, 20 21 22 23 24 25 26 27 WATER TEMPERATURE (C) - GnRH+MET - CPE Fig. 4. The time interval at various temperatures, between injection of GnRH + MET (sgnrha, 10 pg/kg+metoclopramide, 20 mg/kg) or the resolving dose of CPE (pituitary extract containing 0.34 mg/kg cgth) and initial egg release (minimal). Bars represent the time range for each temperature. Numbers of fish spawned in brackets. out at various temperatures. The latency was negatively correlated with temperature over the range examined (20.5-26 C) irrespective of whether the fish were treated with GnRH+ MET or CPE. However, the latency in the CPE-treated fish was always shorter. Nonetheless, the latency in both treatments was more or less constant in the range of 22.5-25 C (Fig. 4). DISCUSSION Treatment with 10 pg/kg sgnrha ( [ D-Arg6,Pro9-NEt ] -sgnrh) was found to be the minimal dose for spawning induction in carp when given together with a dopamine antagonist. Although this superactive analogue is not more potent than the mammalian superactive analogue ( [ D-Ala6,Pro9-NEt ] - LHRH) in the rainbow trout (Crim et al., 1988; Weil et al., 1992)) the winter flounder (Crim et al., 1988) and the gilthead seabream (Zohar et al., 1989), it was reported to be superior in elevating circulating GtH and induction of ovulation in cyprinids such as the carp (Lin et al., 1988), the loach (Lin et al., 199 1) and the goldfish (Peter et al., 1987). Because of its solubility in water, metoclopramide (MET) was utilized in the present work as a dopamine antagonist although its potency, as tested in the goldfish, is inferior to that of pimozide or domperidone (Omeljaniuk et

TEMPERATURE AND SPAWNING INDUCTION IN CARP 403 al., 1987). In tilapia, however, MET, pimozide and domperidone were found to be equipotent in augmenting GtH release in response to GnRHa (Gissis et al., 199 1). The MET dose of 20 mg/kg combined with sgnrha had to be chosen because lower doses only advanced oocyte maturation but did not lead to ovulation. The same dose of MET, when given together with sgnrha ( 10 pg/kg), was followed by a significant elevation in circulating cgth, while each of these compounds given separately had only a slight effect on its level. The latency between injection and initial egg release in fish treated with sgnrha+dom was 28-30 h at 24 C, while in fish administered sgnrha + MET, the latency was shorter ( 19-2 1 h) in spite of the lower temperature (20.5 C). It is possible that the solubility of metoclopramide, and hence its rapid absorption from the injection site, accounts for the faster spawning response. In an unpublished experiment, an exaggerated dose of 100 mg/kg given to carp did not result in any deleterious effects, except for a temporary darkening of the skin around the injection site, which disappeared a few hours later. Our results demonstrate that treating female common carpat any time during the day or evening may result in successful ovulation after the same latency period. The situation is apparently different in Sparus aurata, in which GnRHa treatment given at 10 p.m. was found to be more effective in inducing spawning than at any other time of the day (Zohar, 1986). Conversely, GnRHa treatment at 10 a.m. resulted in a shorter latency in Dicentrarchus Zabrax than when given at 10 p.m. (Alvarino et al., 1992). The present results, whilst demonstrating similar latency and ovulatory response, irrespective of the hour of treatment, cannot corroborate the findings on daily variations in oocyte sensitivity to gonadotropic stimulation as previously reported (Bieniarz et al., 1985). Furthermore, the increase in cgth level following sgnrha+met in the present work was found to be similar in carp injected in the morning and at night. Pimozide-injected male carp implanted with GnRH at 6 p.m. had a higher GtH level 24 and 48 h later and released more milt than those treated at 10 p.m. (Billard et al., 1987). It should be emphasised, however, that the long intervals (24 and 48 h) between treatment and blood samplings prevent precise recording of the rise in GtH. Frequent samplings in the present work, although performed at a higher temperature (22"C), demonstrate that the gonadotropin peak occurs about 15-16 h after treatment and declines thereafter. The cgth level in saline-treated fish showed a slight but significant peak at 10 p.m., which probably indicates the existence of a diurnal cycle in the level of the hormone in postvitellogenic carp. A similar peak was observed at 11 p.m. in saline-injected carp in a previous study (Levavi-Sivan and Yaron, unpublished data). A detailed study on 130 female carp in Poland also demonstrated distinct circadian changes in gonadotropin level with a peak 10 h after onset of light (Bieniarz et al., 1985 ).

404 S. DRORI ET AL. The rise in circulating GtH following G&H+ MET was gradual and reached its peak only when some released eggs were noted in the tank 14 h after treatment. However, injection of the CPE resolving dose was followed by a rapid surge of GtH 4 h later and started to decline when eggs were initially released. The GtH peak level attained in fish treated with GnRH + MET (227 2 41 ng/ml) is similar to that observed in carp induced to spawn by elevating the temperature (range 170-260 ng/ml) (Santos et al., 1986) or by CPE treatment (Levavi-Zermonsky and Yaron, 1986). In the present work CPE treatment resulted in considerably higher levels. The elevated GtH produced in response to the administration of either CPE or GnRH+MET was followed by an increase in oestradiol levels. Such an increase was also observed, although to a lesser extent, in carp induced to spawn by elevating water temperature (Santos et al., 1986 ). CPE-treated carp in which the gonadotropin levels increased above 200 ng/ml also showed an increase in circulating oestradiol up to 10 ng/ml, while fish injected with mammalian native GnRH, in which GtH merely increased to 20 ng/ml, showed only a marginal increase in oestradiol (Weil et al., 1980). The oestradiol surge in ovulating carp has been attributed to the vitellogenic oocytes present in the ovary rather than to the maturing follicles (Weil et al., 1986). The cgth surge in fish treated with either GnRH + MET or CPE was followed by a short, but very prominent peak of the maturation inducing steroid, 17,20-P (23.9 t 19.7 and 28.5 + 9.8 ng/ml, respectively). It should be noted, however, that only the resolving dose of CPE led to the surge of this steroid while the priming dose was ineffective. Such a short peak in 17,20-P was previously reported in the local carp induced to spawn by CPE (Levavi-Zermonsky and Yaron, 1986), but the level attained was much higher ( 1115 14.6 ng/ml). It should be noted that the level of the steroid in the present work varied considerably between fish with individual values of more than 100 ng/ ml not being uncommon. Since all fish of these groups ovulated successfully, it is possible that in some fish the peak was reached earlier, and at the sampling point the steroid was already declining. A much lower peak in the level of 17,20-P ( < 7 ng/ml) was reported in carp induced to spawn in Europe by partially purified carp GtH (Weil et al., 1986). However, spawning induction in carp by raising water temperature was associated with levels conforming to those determined in the present study (Santos et al., 1986). In both treatments, GVBD occurred prior to the peak of 17,20-P, indicating that even the initial rise in the steroid was sufficient to advance oocyte maturation to this stage. In goldfish, too, treatment with LHRH superactive analogue combined with a DA antagonist, the surge of 17,20-P occurred when oocytes had already reached GVBD (Peter et al., 1984). It was not surprising to note that the latency was negatively correlated with water temperature in both GnRH + MET- and CPE-injected tish. Such a phenomenon is to be expected in poikilothermic organisms in which the rate of

TEMPERATURE AND SPAWNING INDUCTION IN CARP 405 metabolic processes is dependent on the ambient temperature. The longer latency in GnRH + MET-treated fish, as compared with CPE-treated fish, may be explained by the two sequential processes required in the former (GtH release from the pituitary and the ovarian response to the released GtH) and the single process (ovarian response to the exogenous GtH) in the latter. Latency as a function of temperature in common carp, silver carp, bighead carp and grass carp, induced to spawn by CPE, was found to be linear in the range of 19-26 C (Horvath, 1978, 1985; Zonneveld, 1984). A linear negative correlation between latency and temperature in the range of 25-3 1 C was reported in grass carp induced to spawn by LHRH (Rottmann and Shireman, 1985). In the present work, however, the 69 carp induced to spawn by CPE showed a constant minimal latency of 9 h between 22.5 C and 24 C. Furthermore, the 160 fish injected with sgnrha+met showed a constant minimal latency of 14 h in the temperature range of 22.5-25 C. This range corresponds to the optimal temperature range for spawning in the common carp (Horvath, 1978). The possibility of predicting the duration of latency according to the water temperature, together with the fact that injection of GnRH + MET can be given at any time during the day with the same results, provides the fish breeder with valuable tools. Using the combined treatment of sgnrha ( 10 lug/kg) and metoclopramide (20 mg/kg ), together with the latency prediction, local fish farmers can now plan with great certainty the stripping of common carp eggs at their convenience and in accordance with other activities in the hatchery. ACKNOWLEDGEMENTS We wish to thank the people in the hatcheries of Gan Shmuel, HaMaapil and Maagan Michael and The Fish and Aquaculture Research Station, Dor, for providing us with the fish and the facilities, and for their kind help in carrying out the spawning experiments. REFERENCES Alvarino, J.M.R., Zanuy, S., Prat, F., Carrillo, M. and Mananos, E., 1992. Stimulation of ovulation and steroid secretion by LHRHa injection in the sea bass (Dicentrarchus labrax): effect of time of day. Aquaculture, 102: 177-l 86. Bieniarz, B., Popek, W., Breton, B. and Epler, P., 1985. Daily changes in the gonadotropin levels and response of carp oocytes to hypophysial homogenate. Chronobiol. Int., 2: 93-l 0 1. Billard, R., Bieniarz, K., Peter, R.E., Sokolowska, M. and Weil, C., 1984. Effects of LHRH and LHRH-A on plasma GtH levels and maturation/ovulation in the common carp, Cyprinus carpio, kept under various environmental conditions. Aquaculture, 41: 245-254.

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