LHRHa and pimozide-induced spawning of Asian catfish Ciarias macrocephalus (Gunther) at different times during an annual reproductive cycle

Aquaculture 148 (1997) 323-331

LHRHa and pimozide-induced spawning of Asian catfish Clarias macrocephalus ( Gunther) at different times during an annual reproductive cycle Josefa D. Tan-Fermin Southeast Asian Fisheries

*, Rosenio R. Pagador, Rosemarie C. Chavez

Deuelqmzent

Center, Aquuculture Iloilo,

Depurtment

(SEAFDEC/

AQD),

5021 Tighoucrn,

Philippines

Accepted 24 August 1996

Abstract Captive C/arias macrocephalus were induced to spawn during the off-season (February), before (May), during (August) and end (November) of the natural breeding period to test their seasonal responsiveness to hormone treatment, and assess the resulting egg and larval quality. Intramuscular injections were given to five fish in each treatment consisting of 0.05 pg LHRHa + 1 pg PIM g-’ body weight (BW), 0.05 pg LHRHa, 1 pg PIM, or LHRHa vehicle (0.9% NaCI) and PIM vehicle (1 dimethylsulfoxide: 9 propylene glycol, v/v>. At any phase of the annual cycle, only fish injected with a combination of LHRHa and PIM spawned, although initial egg size was similar among fish within a season. However, initial egg size was largest in fish induced to spawn in May (1.59 mm), followed by fish induced in August and November (I 54 mm), and smallest in fish induced in February (1.49 mm>. All fish ovulated when induced to spawn in May and August, but ovulation rates decreased to 80% and 60% when fish were injected in November and February, respectively. Catfish induced to spawn in May, August and November had higher egg production, fertilization and larval survival rates than the fish induced in February. Hatching rates were higher in fish induced in May and August than in February, while hatching rate of fish induced in November was similar to those spawned at other times of the year. These results provide useful information regarding the broodstock management and hatchery production of C. macrocephalus. Keywords: Spawning; Hormones; Catfish; Annual; Reproduction;

Off-season

* Corresponding author Southeast Asian Fisheries Development Center, Aquaculture Department (SEAFDEC/AQD), 5021 Tigbauan, Iloilo, Philippines, Tel: 63 (33) 335-1009; fax: 63 (33) 335-1008; e-mail: TMS-SEAFDEC @ PHIL.GN. APC.ORG. Copyright SOO44-8486(96)01423-S

0044.8486/97/$17.00 PfI

0 1997 Elsevier Science B.V. All rights reserved.

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1. Introduction The Asian catfish Clurias macrocephalus is a tropical freshwater fish belonging to the order Siluriformes. Although the fish complete vitellogenesis within the first year of captivity, final maturation does not occur unless gravid females are induced to spawn by hormonal manipulation (Tan-Fermin, 1992). The effects of various hormones on oocyte maturation and ovulation, plasma gonadotropin and steroid hormone levels have been extensively investigated in the ayu Plecoglossus ultivelis (Hirose et al., 1983) catfishes Heteropneustes fossilis (Goswami et al., 1985), Heterobrunchus longifilis (Legendre, 1986) Clurius guriepinus (Richter et al., 1987a), and Clurius mucrocephulus (Tan-Fermin, 1992), carp Cyprinus curpio (Kime and Dolben, 1985; Levavi-Zermonsky and Yaron, 1986; Drori et al., 1994) goldfish Curussius uurutus (Sokolowska et al., 1985) walleye Stizostedion vitreum (Pankhurst et al., 1986), sablefish Anoplomu jimbriu (Solar et al., 1990), sea bass Dicentrurchus lubrux (Alvariiio et al., 1992), and snapper Pugrus uurutus (Pankhurst and Carragher, 1992). Only a few of the studies assess the effects of hormone-induced spawning on egg and larval quality, which is important in evaluating induced spawning protocols (Mylonas et al., 1992), and most of these were carried out within the natural breeding period (Hogendoom and Vismans, 1980; De Leeuw et al., 1985; Manickam and Joy, 1989; Mylonas et al., 1992; Tan-Fermin and Emata, 1993) or under different environmental conditions (Richter et al., 1987b; Harmin and Crim, 1992). The objective of the present study was to test the efficiency of the induced spawning protocol in C. mucrocephulus at different times of the annual reproductive cycle but using similar broodstock and hatchery management. This protocol involved the simultaneous intramuscular injection of 0.05 pg luteinizing hormone-releasing hormone analogue (LHRHa) and 1 pg pimozide (PIM) g-’ body weight (BW), followed by stripping of ovulated eggs 16-20 h thereafter. Initial egg size, ovulation rate, egg production, fertilization, hatching and survival rates of the larvae upon yolk resorption were used to assess the effectiveness of the hormone treatment as described by Tan-Fermin and Emata (1993).

2. Materials

and methods

2.1. Fish Two year-old hatchery-bred catfish broodstock were alternately fed chopped fish by-catch and a pelleted feed containing 36% protein at a daily ration of 5% biomass. Both male and female fish were kept together in 9.5 X 1.5 X 1 m outdoor concrete tanks with a mud bottom, under natural photoperiod (12L: 12D) at water temperature of 27-30°C. A day before each experiment, 25 of these gravid females were stocked individually in 60 1 fiberglass tanks lined with mud substrate, while the males were placed together in a 1 ton fiberglass tank. Induced spawnings were conducted during the off-season (February), before (May), at the peak (August), and end of the natural breeding season (November). Although

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different females were used for each induced spawning run, the fish belonged to the same batch and age group. Gravid females spawned in February, May, August and November had average body weights of 144.2 + 5.7, 167.9 + 5.4, 198.3 k 6.7 and 175.7 + 4.2, respectively. 2.2. Hormone preparation An analogue of luteinizing hormone-releasing hormone (LHRH), D-Ala6 Pro’LHRH-N-ethylamide (LHRHa) was purchased from Lam Hua Dragon Co. Limited (Wanchai, Hong Kong). LHRHa was dissolved in 0.9% sodium chloride (NaCl). Pimozide (PIM) was a gift from Janssen Pharmaceuticals Limited (Beerse, Belgium). PIM was first dissolved in dimethylsulfoxide (DMSO, Ajax Chemicals, NSW, Australia), to which propylene glycol (PG, BDH Chemicals Ltd., Poole, UK) was added at 1:9 v/v (Omeljaniuk et al., 1987). LHRHa and PIM were prepared a few hours before injection with disposable tuberculin syringes and given simultaneously between 16.00-17.00 h. Fish received 1 BW on both sides of the dorsal musculature (Tan-Fermin, 1992). Treatments, pl g-’ with five fish in each group, consisted of either LHRHa (0.05 pg g- ’ ) or PIM (1 pg g-l> and in combination, and their vehicles as the control. 2.3. Egg size, ovulation

rate and egg production

Prior to injection, each female fish was biopsied for egg samples using a silastic medical grade tubing (O.D. = 1.96 mm, I.D. = 1.47 mm, Dow Coming Corporation, MI, USA). At least 20 eggs were fixed in 1% buffered formalin and measured within 120 h (Tan-Fermin, 199 1). Mean egg diameter was calculated from the average of the long and short axes measured to the nearest 0.01 mm. Each female was checked within 16-24 h post-injection for ovulation. Ovulation rate was calculated as the ratio of females emitting translucent brown eggs upon gentle pressure on the abdomen, to the total number of fish per treatment in each experiment. Egg production was calculated from the product of the weight of stripped eggs and 500 (approximate number of eggs in one gram), divided by the body weight of the female catfish taken prior to injection. 2.4. Artificial fertilization Procedures for artificial fertilization, and determination of egg production, fertilization and hatching rates were as described in Tan-Fermin and Emata (1993). There were three replicates for the calculation of the mean values of fertilization, hatching and survival rates. Three wire boxes were placed in each trough to serve as replicates for computing the hatching rate of each fish. Survival rate was assessed by counting the number of larvae 4 days after placing 100 newly-hatched catfish larvae in 1500 ml beakers.

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2.5. Statistics

Cumulative ovulation rate was analyzed usin, e the Z-test (Sokal and Rohlf, 1981) starting at 16 h and continuing until 22 h post-injection. Egg size, egg production, fertilization, hatching and survival rates among the four experiments were compared by analysis of variance (ANOVA), followed by Duncan’s multiple range test (DMRT) at P = 0.05. Fertilization, hatching and survival rates were arc-sine transformed prior to statistical analyses.

3. Results

The initial egg size of catfish in each experiment was similar, hence egg sizes from the different treatments per run were pooled and compared among experiments. Mean initial egg size was highest in May, followed by those examined in August and November, and lowest in fish examined in February (Table 1). Ovulation was observed only in fish given a simultaneous intramuscular injection of LHRHa and PIM at all experimental runs (Table 1). In contrast, fish injected singly with LHRHa or PIM alone, or with the vehicle-control solutions did not ovulate at all. The number of females ovulated was significantly higher only at 16 h in fish induced to spawn in May and August (four out of five) than those spawned in November and February (one out of five). However, the total number of fish ovulated after 20 h and 22 h that corresponded to the final ovulation rate, was comparable at different times of the reproductive cycle. Catfish induced to spawn in May, August and November had higher egg production (Fig. 1A), fertilization (Fig. lB> and larval survival rates (Fig. lD> than in February. Hatching rates were higher in fish induced to spawn in May and August than in

Table 1 Initial egg size and ovulation response of cattish Clurius mucrocephalus (Gunther) induced to spawn with luteinizing hormone-releasing hormone analogue (LHRHa) and pimozide (PIM) during the off-season (February), before (May), at the peak (August) and the end (November) of the natural breeding period Month

Initial egg size (mm)

’

Number of ovulated fish n hours after injection * * 16

February May August November

1.49f0.01 I .59 f 0.01 1.54+0.01 1.54+0.00

a c b b

4b I’

Ovulation rate + (o/o)

20

22

2

_

60

I I 1

_ _

loo loo

2

80

* Values are mean * SEM. Mean values marked with the same letter superscript are not significantly different different (P > 0.05). ’ Number of (P > 0.05). * * Values with the same superscript are not significantly ovulated fish/number of replicates.

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80

b

b

B

-1 8

60

2

40

a

20

i Feb

May

Aw

0 Nov

Feb

100 80

100 -

C

D

b W----L_

b

b

May

Aug

Nov

b 80 -

1

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a

60-

fJ5 40-

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20 -

20

8

Nov

Month

% 7 nz 1

M?

May

Month

i

60 0 Ll Feb

May

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Nov

0 Feb

Month

Month

Fig. I. Egg production (EP = (weight of stripped eggs X 500)/BW; 1A), fertilization rate (FR; IB), hatching rate (HR; 1C) and larval survival rate (SR; ID) of catfish during the off-season (February), before (May), at the peak (August) and the end (November) of the natural breeding season. All female catfish were induced to spawn with 0.05 pc(8 LHRHa+ 1 pg PIM g -’ BW. Values are mean f SEM. ANOVA followed by Duncan’s Multiple Range Test showed that mean values of each parameter marked with the same letter are not significantly different (P > 0.05).

February, while hatching rate of fish induced in November at other times of the year (Fig. 1C).

was similar to those spawned

4. Discussion This study demonstrates the efficiency of injecting LHRHa in combination with PIM to induce spawning of gravid catfish C. macrocephalus at different times of the reproductive cycle. The failure of fish to ovulate after treatment with LHRHa or PIM, and the control-vehicles may suggest that plasma gonadotropin (GTH) levels in those fish had remained low. A surge in GTH can initiate final events of oocyte maturation and ovulation in C. macrocephalus, as in C. guriepinus (Richter et al., 1987b). In other fish, administration of LHRHa or PIM resulted in higher GTH levels than the controls, but the ability to cause oocyte maturation and ovulation depended on the dose, species (Donaldson and Hunter, 1983; Billard et al., 1984; Sokolowska et al., 1984; Lin et al., 1986; Peter et al., 1988; De Leeuw et al., 1987; Ngamvongchon et al., 1988, Thalathiah

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et al., 1988) and the maturational stage of the oocytes (Yaron, 1995). The present results confirm the current view that PIM antagonizes the action of DA on GTH cells and thus facilitates the stimulation of GTH release by LHRHa (Billard et al., 1983; Chang and Peter, 1983; Sokolowska et al., 1984; De Leeuw et al., 1985) and Asian catfish (Manickam and Joy, 1989). The observed differences in reproductive and larval performance were primarily due to seasonal responsiveness of the fish rather than age and size, as shown by previous data using gravid females of different size and age. Since all experimental fish belong to the same batch and age group, and experiments were conducted four times during the annual cycle, the fish induced to spawn in February is 3 months younger than those tested in May, etc. If variations were due to age, the fish induced in November being 9 months older would have been expected to have the biggest egg size, best reproductive and larval performance. However, none of these effects were observed in the present study. Previous data showed that a gravid, 39 g female catfish can be successfully induced to spawn and its initial egg size, reproductive and larval performance were comparable with those fish with body weights over 100 g, indicating that the postvitellogenic eggs in both size groups are equipotential (Tan-Fermin and Emata, 1993). Furthermore, a monthly study of the ovarian histology, GSI, oocyte size frequency and egg size on 3-year-old C. macrocephalus during an annual cycle similarly showed smaller egg sizes of fish in January-April than those taken at other times of the year (Tan-Fermin et al., 1997). The 2-year-old catfish used as experimental fish in the present investigation not only showed a similar trend as the 3-year-old fish used in that study, but that the egg sizes in both age groups are within the same size range. Reproductive and larval performance of catfish in February were consistently lower than those spawned in May, August and November even though the captive females similarly contain post-vitellogenic eggs before hormone injection, and were induced to spawn with the same protocol and dose of LHRHa + PIM. The largest egg size observed during pre-spawning (May) reflects a recruitment of the largest oocytes for release during the peak season, which coincides with the onset of the rainy season. Rainfall provides the stimulus to trigger spawning in most catfishes (Owiti and Dadzie, 1989). The smallest egg size and lower egg production obtained during the off-season month of February may be a result of atresia of some oocytes, resorption of larger oocytes or incomplete vitellogenesis. A significant decrease in oocyte diameter during the dry season was also reported in other siluroids, Heterobrunchus longifilis (Legendre, 1986) and Clarias mossambicus (Owiti and Dadzie, 1989). In the present study, water temperature did not seem important in causing differences in reproductive and larval performance since temperature fluctuations (27-30°C) in the broodstock tanks during the experiment was almost insignificant. Variability in spawning response and larval quality in other teleosts have been attributed to the developmental stage of the oocytes, sperm quality or GnRH dose used (Mylonas et al., 1992), higher sea water temperatures (Carrillo et al., 1989), or time of LHRHa administration (Zohar, 1988). Seasonal responsiveness was indicated not in the total number of ovulating females but in the shorter latency period. Four out of five fish (80%) that were induced to spawn before and during the natural breeding period had ovulated eggs 16 h after injection. In contrast, only one out of five fish (20%) ovulated after such time lag when induced to

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spawn towards the end of the breeding period and off-season. The probable reason for the shorter latency is that prematurational processes such as germinal vesicle migration (GVM) in oocytes of catfish spawned before and during the natural breeding period had already initiated (Tan-Fermin and Emata, 1993). GnRH-treated winter flounder Pseudopleuronectes americanus similarly held at 5°C had a latency period of 13 days in spring and 25 days during winter (Harmin and Crim, 1992). This study did not determine the causes of the annual differences in reproductive and larval performance of captive catfish. However, studies investigating the seasonal effects on GTH secretion showed changes in the gonadotrophs in the pituitary, presence or absence and the quantity of gonadotropin hormone-releasing hormone (GnRH) receptors (Grim et al., 1983; Sokolowska et al., 1985; Peute et al., 1986; Habibi et al., 1989; Manna and Bhattacharya, 1993). Morphological and physiological studies on the hypothalamo-pituitary-gonadal axis of captive C. macrocephalus at various times of the annual cycle may also explain the differences in reproductive and larva1 performance. With the recommended dose combination of LHRHa + PIM (Tan-Fermin and Emata, 19931, the best time to induce C. macrocephalus to spawn is before (May), at the peak (August), and even towards the end of the natural breeding season (November). This is the first study that shows the effectiveness of the LHRHa + PIM-induced spawning protocol on egg and larva1 quality at different times of the annual reproductive cycle in this genus. The present data provide useful information in broodstock management and hatchery production of catfish fry. Monthly studies of the ovarian histology, gonadosomatic index (GSI), oocyte size frequency and egg size will provide a better understanding on the dynamics of the ovary in relation to spawning response at different times of the year. A similar study on the males would also determine whether the decrease in fertilization, hatching and survival rates of catfish eggs during the off-season was partly due to a decrease in sperm quality.

Acknowledgements We thank E. Panes, B. Eullaran, and J. Nocillado for technical assistance, V. Balinas for the statistical analyses, E.T. Quinitio, L.Ma.B. Garcia and three unknown reviewers for their comments and suggestions on different versions of the manuscript. This work was presented during the Biology and Aquaculture of Siluriformes (BASIL) Workshop held in Montpellier, France in 24-27 May 1994 through a travel grant given to JDTF by the French Ministry of Foreign Affairs. The travel grant was made possible through the efforts of the Scientific Attache, French Embassy in Manila.

References Alvariiio, J.M.R., Zanuy, S., Prat, F., Canillo, M. and Maiianos, E., 1992. Stimulation of ovulation and steroid secretion by LHRHa injection in the sea bass (Dicentrurchus Idwax): effect of time of day. Aquaculture, 102: 177-186. Billard, R., Alagarswami, K., Peter, R.E. and Brcton, B., 1983. Potentialisation par le pimozide des effets du LHRHa sur la secretion gonadotrope hypophysaire, I’ovulation et la spermiation chez la carpc commune (Cyprinus carpio). C.R. Acad. Sci. Paris, Ser. t, 296: 181- 184.

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et ul./Aquaculture

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Billard, R., Reinaud, P., Hollebecq, M.G. and Breton, B., 1984. Advancement and synchronization of spawning in Sulmo ,guirdneri and S. truttu following administration of LRH-A combined or not with pimozide. Aquaculture, 43: 57-66. Carrillo, M., Bromage, N., Zanuy, S., Serrano, R. and Prat, F., 1989. The effect of modifications in photoperiod on spawning time, ovarian development and egg quality in the sea bass (Dicentrarchus kubrux L.). Aquaculture, 81: 351-365. Chang, J.P. and Peter, R.E., 1983. Effects of pimozide and des-GlylO [D-Ala6]-luteinizing hormone-releasing hormone ethylamide on serum gonadotropin concentrations, germinal vesicle migration and ovulation in female goldfish Curassius aurutus. Gen. Comp. Endocrinol., 52: 30-37. Grim, L.W., Evans, D.M. and Vickery, B.H., 1983. Manipulation of the seasonal reproductive cycle of the landlocked Atlantic salmon (Salmo s&r) by LHRH analogues administered at various stages of gonadal development. Can. J. Fish. Aquat. Sci., 40: 61-67. De Leeuw, R., Goos, H.J.Th. and Van Oordt, P.G.W.J., 1987. The regulation of gonadotropin release by neurohotmones and gonadal steroids in the African catfish, Clurius guriepinus. Aquaculture, 63: 43-58. De Leeuw, R., Goos, H.J.Th., Richter, C.J.J. and Eding, E.H., 1985. Pimozide-LHRHa-induced breeding of the African catfish, Clurius guriepinus (Burchell). Aquaculture, 44: 295-302. Donaldson, E.M. and Hunter, G.A., 1983. Induced final maturation, ovulation and spermiation in cultured fish. In: W.S. Hoar and D.J. Randall (Editors). Fish Physiology, Vol.IXB. Academic Press, New York, NY, pp. 351-403. Drori, S., Ofir M., Levavi-Sivan, B. and Yaron, Z., 1994. Spawning induction in common carp (Cyprinus curpio) using pituitary extract or GnRH superactive analogue combined with metoclopramide: analysis of hormone profile, progress of oocyte maturation and dependence on temperature. Aquaculture, 119: 393-407. Goswami, S.V., Lamba, V.J. and Sundararaj, B.J., 1985. Gonadotropin-induced oocyte maturation in the cattish, Heteropneuste.sfousi[is (Bloch), requires steroidogenesis in both interrenal and ovary. Gen. Comp. Endocrinol., 57: 53-63. Habibi, H.R., De Leeuw, R., Nahomiak, C.S., Goos, H.J.Th. and Peter, R.E., 1989. Pituitary gonadotropin-releasing hormone (GnRH) receptor activity in goldfish and catfish: seasonal and gonadal effects. Fish Physiol. B&hem., 7: 109- 118. Harmin, S.A. and Grim, L.W., 1992. Gonadotropin hormone-releasing hormone analogue (GnRHa) induced ovulation and spawning in female winter flounder, Pseudopleuronectes umericunus (Walbaum). Aquaculture, 104: 375-390. Hirose, K., Nagahama, Y., Adachi, S. and Wakabayashi,K., 1983. Changes in serum concentrations of gonadotropin, I7 alpha-hydroxyprogesterone and 17 alpha, 20 beta-dihydroxy-4pregnen-3-one during synthetic LHRH-induced final oocyte maturation and ovulation in the ayu, Plecoglossus ultiuelis. Nippon Suisan Galdcaishi, 49: 1165- I 169. Hogendoom, H. and Vismans, M.M., 1980. Controlled propagation of the African catfish, Clurius luzeru (C. and V.). II. Artificial reproduction. Aquaculture, 21: 39-53. Kime, D.E. and Dolben, I.P., 1985. Hormonal changes during induced ovulation of the carp, Cyprinus curpio. Gen. Comp. Endocrinol., 58: 137- 149. Legendre, M., 1986. Seasonal changes in sexual maturity and fecundity, and HCG-induced breeding of the cattish, Heterobrunchus bngijlis Val. (Clariidae), reared in Ebrie lagoon (Ivory Coast). Aquaculture, 55: 201-213. Levavi-Zeononsky, B. and Yaron, Z., 1986. Changes in gonadotropin and ovarian steroids associated with oocyte maturation during spawnin g induction in the carp. Gen. Comp. Endocrinol., 62: 89-98. Lin, H.-R., van der Kraak, G.V.D., Liang, J.-Y.., Peng, C., Li, G.-Y., Lu, L.-Z., Zhou, X.-J., Chang, M.-L. and Peter, R.E., 1986. The effects of LHRH analogue and drugs which block the effects of dopamine on gonadotropin secretion and ovulation in fish cultured in China. In: R. Billard and J. Marcel (Editors). Aquaculture of Cyprinids. INRA, Paris, pp. 140- 150. Manickam, P. and Joy, K.P., 1989. Induction of maturation and ovulation by pimozide-LHRH analogue treatment and resulting high quality egg production in the Asian catfish, CIurias butrachus CL.). Aquaculture, 83: 193-199. Manna, P.R. and Bhattacharya, S., 1993. [1251] gonadotropin binding to the ovary of an Indian major carp, Cutlu cutlu, at different stages of reproductive cycle. J. Biosci., 18: 361-372.

J.D. Tan-Fermin et ul./Ayuuculture

148 (1997) 323-331

Mylonas, C., Hinshaw, J.M. and Sullivan, C.V., 1992. GnRHa-induced and its effects on egg quality. Aquaculture, 106: 379-392.

ovulation

of brown trout (Mmo

331 truttu)

Ngamvongchon, S., Pawaputanon, O., Leelapatra, W. and Johnson, W.E., 1988. Effectiveness of an LHRH analogue for the induced spawning of carp and catfish in Northeast Thailand. Aquaculture, 74: 35-40. Omeljaniuk, R.J., Shih, S.H. and Peter, R.E., 1987. In vivo evaluation of dopamine receptor-mediated inhibition of gonadotropin secretion from the pituitary gland of the goldfish, Curussius uwutus. J. Endocrinol., 114: 449-458. Owiti, D.O. and Dadzie, S., 1989. Maturity, fecundity and the effect of reduced rainfall on the spawning rhythm of a siluroid catfish, Cfurius mossumhicus (Peters). Aquaculture and Fish. Mgt., 20: 355-368. Pankhurst, N.W., Van Der Kraak, G. and Peter, R.E.. 1986. Effects of human chorionic gonadotropin, Des-glyl0 (D-ala61 LHRH-ethylamide and pimozide on oocyte final maturation, ovulation and levels of plasma sex steroids in the walleye (Stizostedion oitreum). Fish Physiol. B&hem., I: 45-54. Pankhurst, N.W. and Carragher, J.F., 1992. Oocyte maturation and changes in plasma steroid levels in snapper Pugrus ( = Chrysophrys) uurutus (Sparidae) following treatment with human chorionic gonadotropin. Aquaculture, 101: 337-347. Peter, R.E., Lin, H.-R. and Van Der Kraak, G., 1988. Induced ovulation and spawning of cultured freshwater fish in China: advances in application of GnRH analogues and dopamine antagonists. Aquaculture, 74: I-IO. Peute, J., Zandbergen, M.A., Goes, H.J.Th., De Leeuw, R., Pinkas, R., Viveen, W.J.A.R. and Van Oordt, P.G.W.J., 1986. Pituitary gonadotropin contents and ultrastructure of the gonadotrops in the African catfish Ck~ius guriepinus during the annual cycle in a natural habitat. Can. J. Zool., 64: 17 18- 1726. Richter, C.J.J., Eding, E.H., Goes, H.J.Th., De Leeuw, R., Scott, A.P. and Van Oordt, P.G.W.J., 1987a. The effect of pimozide/LHRHa and 17 alpha-hydroxyprogesterone on plasma steroid levels and ovulation in the African catfish, Clurirrs guriepinus. Aquaculture, 63: 157- 168. Richter, C.J.J., Viveen, W.J.A.R., Eding, E.H., Sukkel, M., Rothuis, A.J., Van Hoof, M.F.P.M., Van Berg, F.G.J. and Van Oordt, P.G.W.J., 1987b. The signiticance of photoperiodicity, water temperature and an inherent endogenous rhythm for the production of viable eggs by the African catfish, Cluriuu guriepinus, kept in subtropical ponds in Israel and under Israeli and Dutch hatchery conditions. Aquaculture, 63: 169-185. Sokal, R.R. and Rohlf F.J., 1981. Biometry, 2nd edn. W.H. Freeman, San Francisco, pp. 98-126. Sokolowska, M., Peter, R.E., Nahomiak, C.S. and Chang, J.P., 1985. Seasonal effects of pimozide and des GlylO[D-Ala61 LH-RH ethylamide on gonadotropin secretion in goldfish. Gen. Comp. Endocrinol., 57: 472-479. Sokolowska, M., Peter, R.E., Nahomiak, C.S., Pan, C.H., Chang, J.P., Crim, L.W. and Weil, C., 1984. Induction of ovulation in goldfish, Curnssius uurutus, by pimozide and analogues of LHRH. Aquaculture, 36: 71-83. Solar, 1.1.. McLean, E., Baker, I.J., Sherwood, N.M. and Donaldson, E.M., 1990. Induced ovulation of sablefish ( Anoplopomu fimhrid following oral administration of des Gly lo-(D-Ala61 LH-RH ethylamide. Fish Physiol. B&hem., 8: 497-499. Tan-Fermin, J.D., 199 I Suitability of different formalin-containin g fixatives for the eggs of Clurius mucrocephufus (Gunther). Isr. J. Aquaculture-Bamidgeh, 43: 57-61. Tan-Fermin, J.D., 1992. Induction of oocyte maturation and ovulation in the freshwater Asian catfish, Clurius mucrocephalus by LHRHa and pimozide. J. Appl. Ichthyol., 8: 90-98. Tan-Fermin, J.D. and Emata, A.C., 1993. Induced spawning by LHRHa and pimozide in the Asian cattish Clurius mucrocephulus (Gunther). J. Appl. Ichthyol., 9: 89-96. Tan-Fermin, J.D., Ijiri, S., Heda, H., Adachi, S. and Yamauchi, K., 1997. Seasonal change in reproductive parameters and serum steroid hormones in hatchery-bred female catfish Clarius macrocephalus (Gunther), submitted. Thalathiah, S., Ahmad, A.O. and Zaini, M.S., 1988. Induced spawning techniques practised at Batu Berendam, MeIaka, Malaysia. Aquaculture, 74: 23-33. Yaron, Z., 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture, 129: 49-73. Zohar, Y., 1988. Gonadotropin releasing hormone in spawning induction in teleosts: basic and applied considerations. In: Y. Zohar and B. Breton (Editors). Reproduction in Fish: Basic and Applied Aspects in Endocrinology and Genetics. INRA, Paris. Colloq. INRA, 44: 47-62.