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Induced spawning of maturing milkfish (Chanos chanos) using human chorionic gonadotropin and mammalian and salmon gonadotropin releasing hormone analogues
A~UUCU~~UF~, 73 (1988) 333-340 Elsevier Science Publishers B.V., Amsterdam
333
-
Printed
in The Netherlands
Induced Spawning of Maturing Milkfish (Chanos chanos) Using Human Chorionic Gonadotropin and Mammalian and Salmon Gonadotropin Releasing Hormone Analogues CLARISSA
L. MARTE, NANCY SHERWOOD’,
Aquaculture
Department,
City (The Philippines) ‘Department of Biology,
Southeast University
LAWRENCE
GRIM* and JOSEFA TAN
Asian Fisheries Development
Center, P.O. Box 256, Iloilo
of Victoria, P.O. Box 1700, Victoria, B.C. VB W 2Y2
(Canada) ‘Marine Science Research Laboratory, Al C 5S7 (Canada)
(Accepted 27 January
Memorial
University
of Newfoundland,
St. John’s,
Nfld
1988)
ABSTRACT Marte, C.L., Sherwood, N., Crim, L. and Tan, J., 1988. Induced spawning of maturing milkfish (Chanos chanos) using human chorionic gonadotropin and mammalian and salmon gonadotropin releasing hormone analogues. Aquaculture, 73: 333-340. The response of maturing female milkfish to D-Ala’-des Gly” mammalian GnRH ethylamide (mGnRH-A), D-Arg’-des Gly” salmon GnRH ethylamide (sGnRH-A) and human chorionic gonadotropin (hCG) was investigated. The GnRH analogues and hCG were equally effective when administered by intramuscular injection at doses of 10 pg/kg and 100 pg GnRH-A/fish or 1000 IU hCG/fish. All of the females injected with HCG and 87.5% (7/8) of females injected with GnRH-A spawned. Pellet implantation of the GnRH analogues, however, was less effective based on 100 pg of pellet per fish, which provided from 20 to 36 ,ug of analogue per kg fish. Fish implanted with mGnRH-A or sGnRH-A showed responses which varied from oocyte hydration to spawning. Only 3/7 implanted with mGnRH-A and l/7 implanted with sGnRH-A spawned; in the latter group,the average eggdiameter was ll-17% smaller at the time of treatment compared with the other treated groups. Except for one, all fish with egg diameters above 0.65 mm had hydrated/ ovulated oocytes or spawned. Females which spawned had egg diameters above 0.71 mm.
INTRODUCTION
Human chorionic gonadotropin (hCG) is widely used for spawning various fish species. It is also used alone or in combination with fish gonadotropins for spawning milkfish (Lam, 1984; Kuo, 1985 ). However, most fish require at least two injections of hCG and are hand-stripped of ovulated eggs. Gonadotropin-
0044-8486/88/$03.50
0 1988 Elsevier Science Publishers
B.V.
334
releasing hormone analogues (GnRH-A) are also used successfully to induce spawning in a variety of fish which include carps (Cooperative Team for Hormonal Application in Pisciculture, 1977; Billard et al., 1982, 1984a), coho salmon (Donaldson et al., 1981, 1982; Van der Kraak et al., 1983), rainbow trout (Crim et al., 1983), brown trout (Billard et al., 1984b), Atlantic salmon (Crim and Glebe, 1984), sea bass (L&es calcarier) and rabbitfish (Harvey et al., 1985 ), and Dicentrarchus Z&ax (Barnabe and Barnabe-Quet, 1985; Zanuy et al., 1986). The spontaneous spawning of captive milkfish breeders occurs following injection of GnRH-A or implantation of GnRH-A in a cholesterol-cellulose pellet or osmotic pump (Marte et al., 1987). LHRH-A injection or implantation is also found to be effective in spawning milkfish previously treated with longacting testosterone and LHRH-A or LHRH-A alone (Lee et al., 1986). Although milkfish spawn naturally in floating cages (Lacanilao and Marte, 1980; Marte and Lacanilao, 1986) or ponds (Lin, 1985) without hormone induction, controlled spawning may be advantageous in optimizing hatchery operations. D-Ala’--des Gly’O-mGnRH ethylamide (mGnRH-A) is the most commonly used GnRH analogue and is effective for spawning milkfish. In experiments to determine the gonadotropin-releasing activity of various GnRH analogues, Peter et al. (1985) reported that D-Arg’-des Gly” salmon-GnRH ethylamide was the most effective in inducing pituitary gonadotropin release in goldfish. This study compares the efficacy in spawning milkfish using analogues of mGnRH and sGnRH as well as hCG. MATERIALS AND METHODS
The experiments were conducted at SEAFDEC, Igang substation, Guimaras Island on 31 May-4 June, 28-29 June, and 12-14 July 1986 and at the National Bangus Breeding Project station of the Bureau of Fisheries and Aquatic Resources at Lucap, Alaminos, Pangasinan on 13-14 June 1986. The fish were obtained from 6- and 10-m diameter cages at Igang and from 10-m diameter cages at Alaminos. Adult female milkfish at Igang were 6-9 years old (3.606.95 kg); those at Alaminos were 5 years old and maturing for the first time (2.60-4.50 kg). The fish were netted individually and anesthetized in a l-m polyethylene bag containing about 20 1 of seawater (Igang) or a plastic tank containing 40 1 of seawater (Alaminos) and 200 ppm 2-phenoxyethanol. Maturing females were identified by aspirating oocyte samples through a polyethylene cannula (PElOO) (Marte et al., 1987); mature males expressed milt after slight pressure was applied to the abdomen. Fish were weighed, measured and marked by fin clips. Females with oocyte diameters greater than 0.50 mm were selected for the experiments. Temperature and salinity at Igang substation were 2930' C and 31-34 ppt, respectively.
335
D-Ala’-des Gl”-mGnRH ethylamide (mGnRH-A) was purchased from Syndel Laboratories Ltd. (Vancouver, B.C., Canada); D-Arg’-des Gly’O-sGnRH ethylamide (sGnRH-A) was from the Institute Armand-Frappier (Laval, Quebec, Canada), and human chorionic gonadotropin was from Sigma Chemical Co. The fish were treated as follows: (1) mGnRH-A in a cholesterol-cellulose pellet (100 pg/pellet) implanted intraperitoneally (IP), N= 7; (2) sGnRH-A in a cholesterol-cellulose pellet implanted IP (100 pug/pellet), N= 7; (3 ) sGnRH-A given by intramuscular injection (IM) (100 ,ug/fish), N= 4; (4) mGnRH-A given by intramuscular injection (10 pg/kg body weight), N= 4; (5) hCG given by intramuscular injection (1000 III/kg body weight ), N=6; (6) cholesterol-cellulose pellet implanted IP, N=3; and (7) saline IM injection, N=3. The GnRH-A cholesterol-cellulose pellet was prepared as previously described (Marte et al., 1987). Injected GnRH-A and hCG were prepared in Cortland’s saline immediately before use; injections were delivered using a #22 needle introduced into the epaxial muscle. Injected volume was from 0.40 ml to 0.80 ml. The cholesterol-cellulose pellets were implanted IP by way of a 2mm trochar inserted through a 2-3-mm abdominal incision. In experiments conducted at Igang, two to three treated females were transferred to 6 m diameter x 0.7 m deep canvas-lined tanks. At Alaminos, four to five treated females were returned to 4 x 4 x 2 m cages constructed of fine mesh TABLE 1 Response of maturing milkfish to mGnRH-A, sGnRH-A and hCG Treatment
Numberper treatment
Numberof fish spawned
Initial oocyte diameter (Mean? SD)
Remarks
mGnRH-A pellet implant ( 100 pug/pellet )” sGnRH-A pellet implant ( 100 pg/pellet ) h
7
3
0.73 & 0.10
7
1
0.65? 0.07
1 fish ovulated; 3 showed no response 2 fish ovulated; 1 with hydrated oocytes; 3 showed no response
sCnRH-A injection (100 pg/fish)’ mGnRH-A injection
4
4
0.78 f 0.02
4
3
0.74 f 0.02
hCG injection (1000 IU/kg)
6
6
0.76 * 0.02
Control pellet implant Control saline injection
3 4
0 0
0.62 ? 0.10 0.64kO.14
1 fish ovulated
i 10 m/kg)
“20.6-27.4 ng/kg body weight. “20.4-35.7 ,ug/kg body weight. ‘24.4-33.3 ng/kg body weight.
1 fish escaped 1 fish escaped
336
net (0. l-mm mesh). Two to three males per female were placed in each spawning tank or cage. Males were implanted with a GnRH-A pellet in implantation experiments or injected with 10 pg/kg of mGnRH-A or sGnRH-A for the injection experiments. Spawning was monitored by periodically examining seawater samples for spawned eggs. Fish were netted, anesthetized and cannulated on the day spawning was observed. Spawned eggs were collected by seining with a fine mesh nylon net (0.08-mm mesh); fertilization rate was determined from 100-ml subsamples. RESULTS
The potencies of mGnRH-A and sGnRH-A were similar. Both analogues were more effective when administered by injection compared with pellet imTABLE 2 Duration of response following a single treatment of GnRH-A and hCG; fertilization rate and number of eggs collected Treatment
mGnRH-A pellet implant
Number of fish spawned
Date and time of hormone treatment
Date and time of spawning
1
31.5.1986 15.00-18.00 h
1.6.1986 10.00-11.00 h 22.00-23.00 h 1.61986 22.00-23.00 h 14.7.1986 11.00 h
1 1 mGnRH-A injected
1
sGnRH-A pellet implant
1
sGnRH-A injected
4
hCG injected
3 3
31.5.1986 15.00-18.00 h 12.7.1986 12.00 h 29.6.1986 09.00-12.00 h 12.7.1986 09.00-12.00 h
Time interval (h)
Number of eggs collected
Fertilization rate (%o)
16-20 28-32
56 700 68 300
27.6 20.0
28-32 47
151600 72 900
24.0 79.0
29-32
648 000
88.0
30.6.1986 17.30 h 13.7.1986 08.00 h 14.7.1986 10.00 h
20-23
227 500
25.0
46-49
2 081000
80.0
13.6.1986 15.00-18.00 h
14.6.1986 10.00 h
16-19
680 000
57.7
13.6.1986 09.00-12.00 h
14.6.1986 04.00-05.00 h
16-20
825 400
68.8
13.6.1986 09.00-12.00 h 12.7.1986 09.00-12.00 h
14.6.1986 04.00-05.00 h 13.7.1986 08.00 h 14.7.1986 10.00 h
16-20
647 300
50.0
20-23
633 600
14.0
46-49
1512 000
71.0
337
plants. All four fish injected with 100 pg/fish (24.4-33.3 pg/kg) sGnRH-A spawned, while three of four injected with 10 bg/kg (34-62 pg/fish) mGnRHA spawned (Table 1). One fish injected with 10 pg/kg mGnRH-A did not spawn but ovulated; the eggs were stripped and artificially fertilized 50 h after the injection. Fish implanted with GnRH-A showed variable responses. Three of seven fish implanted with mGnRH-A spawned, one ovulated and three did not respond. Of those implanted with sGnRH-A only one spawned, two had ovulated eggs, one had hydrated eggs and three did not respond. Both sGnRHA-implanted fish that ovulated were stripped but artificial fertilization was not successful. hCG at 1000 IU/kg was as effective as GnRH-A at 100 pug/fish as all six fish injected with 1000 IU/kg hCG spawned. Cholesterol pellet-implanted or saline-injected control fish regressed. Spawnings were observed from 16 to 49 h after the hormone treatment (Table 2). Total number of eggs per spawning ranged from 56 000 to over two million. Fertilization rates varied from 14 to 88%. Two successive spawnings, 12 h apart were observed on 31 May in a fish implanted with mGnRH-A, and at a 26-h interval on 12 July in fishes injected with mGnRH-A and hCG. The number of eggs collected and rate of fertilization during each spawning on 31 May were similar but in the 12 July experiment, fewer eggs were collected and fertilization rate was lower during the first spawning. DISCUSSION
The spontaneous spawning of milkfish after a single injection or pellet-implantation of GnRH-A confirms our previous results (Marte et al., 1987). GnRH-A administered as pellet implants, however, was less effective than injections. This is in contrast to our earlier result which showed similar effectiveness of pellet implantation and injection. One reason for the poor response of sGnRH-A-implanted fish may be that the mean diameter of oocytes was 0.65 mm whereas in the other four treatment groups the mean diameter was 0.73-0.78 mm at the time of hormone administration. These results confirm earlier reports; they indicate that milkfish oocytes with diameters below 0.63 mm have not completed vitellogenesis and will be unresponsive to GnRH-A or gonadotropin induction. The varied response of milkfish having oocyte diameters between 0.63 mm and 0.69 mm also indicates that maturation of these oocytes following hormone induction may take a long time. In-vitro assays to determine the release rate of GnRH-A from the cholesterol-cellulose pellets showed that over 90% of the hormone was released within 24 h (N.M. Sherwood, unpubl., 1987 ). However, over 90% of an IP injection of GnRH-A is gone from the blood in less than 5 h (L.W. Crim and N.M. Sherwood, unpubl., 1987) showing that pellets and injections have different release profiles. A single injection of 1000 IU/kg of hCG was as effective as a 10 pug/kgor 100
338
,ug/fish GnRH-A injection for spawning milkfish. All hCG-injected fish spawned spontaneously. In previous studies, milkfish were invariably stripped after one to three injections of hCG or hCG combined with various pituitary preparations (Lam, 1984; Kuo, 1985 ) , except for one fish that spawned spontaneously after one hCG injection (Lin, 1984). Spawning occurred from 16 to 49 h after the GnRH-A or hCG administration. In general, spawning occurred earlier in fish returned to a spawning cage than in those transferred to spawning tanks. The longer response latency in fish transferred to tanks may be a result of added stress arising from a change in holding conditions. The fish in the 31 May experiment spawned twice at a 12-h interval. In the 12 July experiment, two mGnRH-A-injected and three hCG-injected fish may have spawned at different times, although it is possible that the fish in both tanks spawned twice. The total number of eggs collected from the second spawning of the mGnRH-A-injected fishes was over 2 million. Fecundity from gonad samples of captive milkfish is around 300 OOO/kg(Marte and Lacanilao, 1986) or from 1 to 1.2 million in the injected fish (body weight 3.45 and 4.2 kg). Fewer eggs, mostly unfertilized, were collected during the first spawning. It is likely that the first hatch of eggs released corresponds to the phenomenon of “egg dribbling” (Lam, 1984). The low fertilization rate in the first batch of spawned eggs probably indicates that oocyte maturation had not been completed or sperm release may not have been fully stimulated at this time. Lam ( 1984) suggested that egg dribbling is a natural phenomenon which may serve to release pheromone (s) for stimulation of males. D-Arg’-des Gly’O-GnRH ethylamide was reported to induce significantly higher serum gonadotropin levels in goldfish with or without pimozide compared to other GnRH analogues including D-Ala’-des Glyl’-GnRH ethylamide (Peter et al., 1985 ). Our results show that in milkfish the two analogues were equally effective in inducing ovulation and spawning. It may, however, be possible to detect differences in activity at the minimal effective dose of each analogue for inducing ovulation and spawning in milkfish, and this needs to be investigated. GnRH-A and hCG at the respective doses used are equally effective for spawning milkfish. The choice of spawning agent will depend on availability, cost of the hormones and side reactions. hCG is a large protein from a distantly related species; it may trigger antibody formation and hence cause problems in subsequent spawning seasons if repeatedly injected. hCG is widely available and used as a spawning agent by fish breeders. It is, however, more expensive for spawning fish. The cost of spawning milkfish with hCG is about US$2.OO/kg (hCG, Sigma - US$9.95/5000 IU) or about ten times the cost of mGnRH-A at 10 pg/kg spawning dose (US$0.25/kg; mGnRH-A, SyndelUS$Z5.00/mg).
339
ACKNOWLEDGEMENTS
We would like to thank Mr. J. Toledo, Mr. R. Gapasin, Ms. E. de Jesus, Mr. R. Ortega and the Igang substation staff for their help in experiments conducted at Igang; and Mr. N. Lopez, Mr. D. Rasca and the National Bangus Breeding Project staff for providing assistance at Lucap, Alaminos, Pangasinan. This work was supported by a grant from the International Development Research Centre (IDRC ) , Canada.
REFERENCES Barnab& G. and Barnab&Quet, R., 1985. Avancement et am&lioration de la ponte induite chez le loup Dicentrarchus Zabrax(L.) ?Il’aide d’un analogue de LHRH inject.& Aquaculture, 49: 125132. Billard, R., Marcel, J., Matei, D. and Rusu, C., 1982. Induction de l’ovulation par un analogue du LHRH chez Hypophthalmichthys molitrix (Poisson, TBlCostCen) plack dans diverses conditions d’environnement. Bul. Cercet. Piscic., 4: 29-34. Billard, R., Bieniarz, K., Peter, R.E., Sokolowska, M., Weil, C. and Crim, L.W., 1984a. 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. Billard, R., Reinaud, P., Hollebecq, M.G. and Breton, B., 198413.Advancement and synchronisation of spawning in Salmo gairdneri and S. trutta following administration of LRH-A combined or not with pimozide. Aquaculture, 43: 57-66. Cooperative Team for Hormonal Application in Pisciculture, 1977. A new highly effective ovulation agent for fish reproduction. Sci. Sin., 20: 468-474. Crim, L.W. and Glebe, B.D., 1984. Advancement and synchrony of ovulation in Atlantic salmon with pelleted LHRH analogue. Aquaculture, 43: 47-56. Crim, L.W., Sutterlin, A.M., Evans, D.M. and Weil, C., 1983. Accelerated ovulation hy pelleted LHRH analogue treatment of spring-spawning rainbow trout (Salmo gairdneri) held at low temperature. Aquaculture, 35: 299-307. Donaldson, E.M., Hunter, G.A. and Dye, H.M., 1981. Induced ovulation in coho salmon (Oncorhynchus kisutch). II. Preliminary study of the use of LH-RH and two high potency LH-RH analogues. Aquaculture, 26: 129-141. Donaldson, E.M., Hunter, G.A., Van der Kraak, G. and Dye, H.M., 1982. Application of LH-RH and LH-RH analogues to the induced final maturation and ovulation of coho salmon Oncorhynchus kisutch. In: H.J.Th. Goos and J.J. Richter (Editors), Proceedings of the International Symposium on Reproductive Physiology of Fish. PUDOC, Wageningen, pp. 177-180. Harvey, B ., Nacario, J., Crim, L.W., Juari0,J.V. and Marte, C.L., 1985. Induced spawning of sea bass, Lates calcarifer, and rabbitfish, Siganus guttutus, after implantation of pelleted LHRH analogue. Aquaculture, 47: 53-59. Kuo, C.M., 1985. A review of induced breeding of milkfish. In: C.S. Lee and I.C. Liao (Editors), Reproduction and Culture of Milkfish. Oceanic Institute, Hawaii, and Tungkang Marine Laboratory, Taiwan, pp. 57-77. Lacanilao, F.J. and Marte, C.L., 1980. Sexual maturation of milkfish in floating cages. Asian Aquacult., 3: 4-6. Lam, T.J., 1984. Artificial propagation of milkfish: present status and problems. In: J.V. Juario, R.P. Ferraris and L.V. Benitez (Editors), Advances in Milkfish Biology and Culture. Island Publishing House, Metro Manila, The Philippines, pp. 21-39.
340 Lee, C.S., Tamaru, C.S., Kelley, CD. and Banno, J.E., 1986. Induced spawning of milkfiih, Chums chanos, by a single application of LHRH-analogue. Aquaculture, 58: 87-98. Lin, L.T., 1984. Studies on the induced breeding of milkfish (Chanos chanos Forsskal) reared in ponds. China Fish., 318: 3-29. Lin, L.T., 1985. My experience in artificial propagation of milkfish - studies on natural spawning of pond-reared broodstock. In: C.S. Lee and I.C. Liao (Editors), Reproduction and Culture of Milkfish. Oceanic Institute, Hawaii, and Tungkang Marine Laboratory, Taiwan, pp. 185-203. Marte, C.L. and Lacanilao, F.J., 1986. Spontaneous maturation and spawning of milkfish in floating net cages. Aquaculture, 53: 115-132. Marte, C.L., Sherwood, N.M., Crim, L.W. and Harvey, B., 1987. Induced spawning of maturing milkfish (Charzos charms Forsskal) with gonadotropin-releasing hormone (GnRH) analogues administered in various ways. Aquaculture, 60: 303-310. Peter, R.E., Nahorniak, C.S., Sokolowska, M., Chang, J.P., Rivier, J.E., Vale, W.W., King, J.A. and Millar, R.P., 1985. Structure-activity relationships of mammalian, chicken and salmon gonadotropin releasing hormones in vivo in goldfish. Gen. Comp. Endocrinol., 58: 231-242. Van der Kraak, G., Lin, H.R., Donaldson, E.M., Dye, H.M. and Hunter, G.A., 1983. Effects of LH-RH and des-Gly” (D-Ala’)LH-RH-ethylamide on plasma gonadotropin levels and oocyte maturation in adult female coho salmon (Oncorhynchus kisutch). Gen. Comp. Endocrinol., 49: 470-476. Zanuy, S., Carillo, M. and Ruiz, F., 1986. Delayed gametogenesis and spawning of sea bass (Dicentrarchus Zabrax L.) kept under different photoperiod and temperature regimes. Fish Physiol. Biochem., 2: 53-63.
333
-
Printed
in The Netherlands
Induced Spawning of Maturing Milkfish (Chanos chanos) Using Human Chorionic Gonadotropin and Mammalian and Salmon Gonadotropin Releasing Hormone Analogues CLARISSA
L. MARTE, NANCY SHERWOOD’,
Aquaculture
Department,
City (The Philippines) ‘Department of Biology,
Southeast University
LAWRENCE
GRIM* and JOSEFA TAN
Asian Fisheries Development
Center, P.O. Box 256, Iloilo
of Victoria, P.O. Box 1700, Victoria, B.C. VB W 2Y2
(Canada) ‘Marine Science Research Laboratory, Al C 5S7 (Canada)
(Accepted 27 January
Memorial
University
of Newfoundland,
St. John’s,
Nfld
1988)
ABSTRACT Marte, C.L., Sherwood, N., Crim, L. and Tan, J., 1988. Induced spawning of maturing milkfish (Chanos chanos) using human chorionic gonadotropin and mammalian and salmon gonadotropin releasing hormone analogues. Aquaculture, 73: 333-340. The response of maturing female milkfish to D-Ala’-des Gly” mammalian GnRH ethylamide (mGnRH-A), D-Arg’-des Gly” salmon GnRH ethylamide (sGnRH-A) and human chorionic gonadotropin (hCG) was investigated. The GnRH analogues and hCG were equally effective when administered by intramuscular injection at doses of 10 pg/kg and 100 pg GnRH-A/fish or 1000 IU hCG/fish. All of the females injected with HCG and 87.5% (7/8) of females injected with GnRH-A spawned. Pellet implantation of the GnRH analogues, however, was less effective based on 100 pg of pellet per fish, which provided from 20 to 36 ,ug of analogue per kg fish. Fish implanted with mGnRH-A or sGnRH-A showed responses which varied from oocyte hydration to spawning. Only 3/7 implanted with mGnRH-A and l/7 implanted with sGnRH-A spawned; in the latter group,the average eggdiameter was ll-17% smaller at the time of treatment compared with the other treated groups. Except for one, all fish with egg diameters above 0.65 mm had hydrated/ ovulated oocytes or spawned. Females which spawned had egg diameters above 0.71 mm.
INTRODUCTION
Human chorionic gonadotropin (hCG) is widely used for spawning various fish species. It is also used alone or in combination with fish gonadotropins for spawning milkfish (Lam, 1984; Kuo, 1985 ). However, most fish require at least two injections of hCG and are hand-stripped of ovulated eggs. Gonadotropin-
0044-8486/88/$03.50
0 1988 Elsevier Science Publishers
B.V.
334
releasing hormone analogues (GnRH-A) are also used successfully to induce spawning in a variety of fish which include carps (Cooperative Team for Hormonal Application in Pisciculture, 1977; Billard et al., 1982, 1984a), coho salmon (Donaldson et al., 1981, 1982; Van der Kraak et al., 1983), rainbow trout (Crim et al., 1983), brown trout (Billard et al., 1984b), Atlantic salmon (Crim and Glebe, 1984), sea bass (L&es calcarier) and rabbitfish (Harvey et al., 1985 ), and Dicentrarchus Z&ax (Barnabe and Barnabe-Quet, 1985; Zanuy et al., 1986). The spontaneous spawning of captive milkfish breeders occurs following injection of GnRH-A or implantation of GnRH-A in a cholesterol-cellulose pellet or osmotic pump (Marte et al., 1987). LHRH-A injection or implantation is also found to be effective in spawning milkfish previously treated with longacting testosterone and LHRH-A or LHRH-A alone (Lee et al., 1986). Although milkfish spawn naturally in floating cages (Lacanilao and Marte, 1980; Marte and Lacanilao, 1986) or ponds (Lin, 1985) without hormone induction, controlled spawning may be advantageous in optimizing hatchery operations. D-Ala’--des Gly’O-mGnRH ethylamide (mGnRH-A) is the most commonly used GnRH analogue and is effective for spawning milkfish. In experiments to determine the gonadotropin-releasing activity of various GnRH analogues, Peter et al. (1985) reported that D-Arg’-des Gly” salmon-GnRH ethylamide was the most effective in inducing pituitary gonadotropin release in goldfish. This study compares the efficacy in spawning milkfish using analogues of mGnRH and sGnRH as well as hCG. MATERIALS AND METHODS
The experiments were conducted at SEAFDEC, Igang substation, Guimaras Island on 31 May-4 June, 28-29 June, and 12-14 July 1986 and at the National Bangus Breeding Project station of the Bureau of Fisheries and Aquatic Resources at Lucap, Alaminos, Pangasinan on 13-14 June 1986. The fish were obtained from 6- and 10-m diameter cages at Igang and from 10-m diameter cages at Alaminos. Adult female milkfish at Igang were 6-9 years old (3.606.95 kg); those at Alaminos were 5 years old and maturing for the first time (2.60-4.50 kg). The fish were netted individually and anesthetized in a l-m polyethylene bag containing about 20 1 of seawater (Igang) or a plastic tank containing 40 1 of seawater (Alaminos) and 200 ppm 2-phenoxyethanol. Maturing females were identified by aspirating oocyte samples through a polyethylene cannula (PElOO) (Marte et al., 1987); mature males expressed milt after slight pressure was applied to the abdomen. Fish were weighed, measured and marked by fin clips. Females with oocyte diameters greater than 0.50 mm were selected for the experiments. Temperature and salinity at Igang substation were 2930' C and 31-34 ppt, respectively.
335
D-Ala’-des Gl”-mGnRH ethylamide (mGnRH-A) was purchased from Syndel Laboratories Ltd. (Vancouver, B.C., Canada); D-Arg’-des Gly’O-sGnRH ethylamide (sGnRH-A) was from the Institute Armand-Frappier (Laval, Quebec, Canada), and human chorionic gonadotropin was from Sigma Chemical Co. The fish were treated as follows: (1) mGnRH-A in a cholesterol-cellulose pellet (100 pg/pellet) implanted intraperitoneally (IP), N= 7; (2) sGnRH-A in a cholesterol-cellulose pellet implanted IP (100 pug/pellet), N= 7; (3 ) sGnRH-A given by intramuscular injection (IM) (100 ,ug/fish), N= 4; (4) mGnRH-A given by intramuscular injection (10 pg/kg body weight), N= 4; (5) hCG given by intramuscular injection (1000 III/kg body weight ), N=6; (6) cholesterol-cellulose pellet implanted IP, N=3; and (7) saline IM injection, N=3. The GnRH-A cholesterol-cellulose pellet was prepared as previously described (Marte et al., 1987). Injected GnRH-A and hCG were prepared in Cortland’s saline immediately before use; injections were delivered using a #22 needle introduced into the epaxial muscle. Injected volume was from 0.40 ml to 0.80 ml. The cholesterol-cellulose pellets were implanted IP by way of a 2mm trochar inserted through a 2-3-mm abdominal incision. In experiments conducted at Igang, two to three treated females were transferred to 6 m diameter x 0.7 m deep canvas-lined tanks. At Alaminos, four to five treated females were returned to 4 x 4 x 2 m cages constructed of fine mesh TABLE 1 Response of maturing milkfish to mGnRH-A, sGnRH-A and hCG Treatment
Numberper treatment
Numberof fish spawned
Initial oocyte diameter (Mean? SD)
Remarks
mGnRH-A pellet implant ( 100 pug/pellet )” sGnRH-A pellet implant ( 100 pg/pellet ) h
7
3
0.73 & 0.10
7
1
0.65? 0.07
1 fish ovulated; 3 showed no response 2 fish ovulated; 1 with hydrated oocytes; 3 showed no response
sCnRH-A injection (100 pg/fish)’ mGnRH-A injection
4
4
0.78 f 0.02
4
3
0.74 f 0.02
hCG injection (1000 IU/kg)
6
6
0.76 * 0.02
Control pellet implant Control saline injection
3 4
0 0
0.62 ? 0.10 0.64kO.14
1 fish ovulated
i 10 m/kg)
“20.6-27.4 ng/kg body weight. “20.4-35.7 ,ug/kg body weight. ‘24.4-33.3 ng/kg body weight.
1 fish escaped 1 fish escaped
336
net (0. l-mm mesh). Two to three males per female were placed in each spawning tank or cage. Males were implanted with a GnRH-A pellet in implantation experiments or injected with 10 pg/kg of mGnRH-A or sGnRH-A for the injection experiments. Spawning was monitored by periodically examining seawater samples for spawned eggs. Fish were netted, anesthetized and cannulated on the day spawning was observed. Spawned eggs were collected by seining with a fine mesh nylon net (0.08-mm mesh); fertilization rate was determined from 100-ml subsamples. RESULTS
The potencies of mGnRH-A and sGnRH-A were similar. Both analogues were more effective when administered by injection compared with pellet imTABLE 2 Duration of response following a single treatment of GnRH-A and hCG; fertilization rate and number of eggs collected Treatment
mGnRH-A pellet implant
Number of fish spawned
Date and time of hormone treatment
Date and time of spawning
1
31.5.1986 15.00-18.00 h
1.6.1986 10.00-11.00 h 22.00-23.00 h 1.61986 22.00-23.00 h 14.7.1986 11.00 h
1 1 mGnRH-A injected
1
sGnRH-A pellet implant
1
sGnRH-A injected
4
hCG injected
3 3
31.5.1986 15.00-18.00 h 12.7.1986 12.00 h 29.6.1986 09.00-12.00 h 12.7.1986 09.00-12.00 h
Time interval (h)
Number of eggs collected
Fertilization rate (%o)
16-20 28-32
56 700 68 300
27.6 20.0
28-32 47
151600 72 900
24.0 79.0
29-32
648 000
88.0
30.6.1986 17.30 h 13.7.1986 08.00 h 14.7.1986 10.00 h
20-23
227 500
25.0
46-49
2 081000
80.0
13.6.1986 15.00-18.00 h
14.6.1986 10.00 h
16-19
680 000
57.7
13.6.1986 09.00-12.00 h
14.6.1986 04.00-05.00 h
16-20
825 400
68.8
13.6.1986 09.00-12.00 h 12.7.1986 09.00-12.00 h
14.6.1986 04.00-05.00 h 13.7.1986 08.00 h 14.7.1986 10.00 h
16-20
647 300
50.0
20-23
633 600
14.0
46-49
1512 000
71.0
337
plants. All four fish injected with 100 pg/fish (24.4-33.3 pg/kg) sGnRH-A spawned, while three of four injected with 10 bg/kg (34-62 pg/fish) mGnRHA spawned (Table 1). One fish injected with 10 pg/kg mGnRH-A did not spawn but ovulated; the eggs were stripped and artificially fertilized 50 h after the injection. Fish implanted with GnRH-A showed variable responses. Three of seven fish implanted with mGnRH-A spawned, one ovulated and three did not respond. Of those implanted with sGnRH-A only one spawned, two had ovulated eggs, one had hydrated eggs and three did not respond. Both sGnRHA-implanted fish that ovulated were stripped but artificial fertilization was not successful. hCG at 1000 IU/kg was as effective as GnRH-A at 100 pug/fish as all six fish injected with 1000 IU/kg hCG spawned. Cholesterol pellet-implanted or saline-injected control fish regressed. Spawnings were observed from 16 to 49 h after the hormone treatment (Table 2). Total number of eggs per spawning ranged from 56 000 to over two million. Fertilization rates varied from 14 to 88%. Two successive spawnings, 12 h apart were observed on 31 May in a fish implanted with mGnRH-A, and at a 26-h interval on 12 July in fishes injected with mGnRH-A and hCG. The number of eggs collected and rate of fertilization during each spawning on 31 May were similar but in the 12 July experiment, fewer eggs were collected and fertilization rate was lower during the first spawning. DISCUSSION
The spontaneous spawning of milkfish after a single injection or pellet-implantation of GnRH-A confirms our previous results (Marte et al., 1987). GnRH-A administered as pellet implants, however, was less effective than injections. This is in contrast to our earlier result which showed similar effectiveness of pellet implantation and injection. One reason for the poor response of sGnRH-A-implanted fish may be that the mean diameter of oocytes was 0.65 mm whereas in the other four treatment groups the mean diameter was 0.73-0.78 mm at the time of hormone administration. These results confirm earlier reports; they indicate that milkfish oocytes with diameters below 0.63 mm have not completed vitellogenesis and will be unresponsive to GnRH-A or gonadotropin induction. The varied response of milkfish having oocyte diameters between 0.63 mm and 0.69 mm also indicates that maturation of these oocytes following hormone induction may take a long time. In-vitro assays to determine the release rate of GnRH-A from the cholesterol-cellulose pellets showed that over 90% of the hormone was released within 24 h (N.M. Sherwood, unpubl., 1987 ). However, over 90% of an IP injection of GnRH-A is gone from the blood in less than 5 h (L.W. Crim and N.M. Sherwood, unpubl., 1987) showing that pellets and injections have different release profiles. A single injection of 1000 IU/kg of hCG was as effective as a 10 pug/kgor 100
338
,ug/fish GnRH-A injection for spawning milkfish. All hCG-injected fish spawned spontaneously. In previous studies, milkfish were invariably stripped after one to three injections of hCG or hCG combined with various pituitary preparations (Lam, 1984; Kuo, 1985 ) , except for one fish that spawned spontaneously after one hCG injection (Lin, 1984). Spawning occurred from 16 to 49 h after the GnRH-A or hCG administration. In general, spawning occurred earlier in fish returned to a spawning cage than in those transferred to spawning tanks. The longer response latency in fish transferred to tanks may be a result of added stress arising from a change in holding conditions. The fish in the 31 May experiment spawned twice at a 12-h interval. In the 12 July experiment, two mGnRH-A-injected and three hCG-injected fish may have spawned at different times, although it is possible that the fish in both tanks spawned twice. The total number of eggs collected from the second spawning of the mGnRH-A-injected fishes was over 2 million. Fecundity from gonad samples of captive milkfish is around 300 OOO/kg(Marte and Lacanilao, 1986) or from 1 to 1.2 million in the injected fish (body weight 3.45 and 4.2 kg). Fewer eggs, mostly unfertilized, were collected during the first spawning. It is likely that the first hatch of eggs released corresponds to the phenomenon of “egg dribbling” (Lam, 1984). The low fertilization rate in the first batch of spawned eggs probably indicates that oocyte maturation had not been completed or sperm release may not have been fully stimulated at this time. Lam ( 1984) suggested that egg dribbling is a natural phenomenon which may serve to release pheromone (s) for stimulation of males. D-Arg’-des Gly’O-GnRH ethylamide was reported to induce significantly higher serum gonadotropin levels in goldfish with or without pimozide compared to other GnRH analogues including D-Ala’-des Glyl’-GnRH ethylamide (Peter et al., 1985 ). Our results show that in milkfish the two analogues were equally effective in inducing ovulation and spawning. It may, however, be possible to detect differences in activity at the minimal effective dose of each analogue for inducing ovulation and spawning in milkfish, and this needs to be investigated. GnRH-A and hCG at the respective doses used are equally effective for spawning milkfish. The choice of spawning agent will depend on availability, cost of the hormones and side reactions. hCG is a large protein from a distantly related species; it may trigger antibody formation and hence cause problems in subsequent spawning seasons if repeatedly injected. hCG is widely available and used as a spawning agent by fish breeders. It is, however, more expensive for spawning fish. The cost of spawning milkfish with hCG is about US$2.OO/kg (hCG, Sigma - US$9.95/5000 IU) or about ten times the cost of mGnRH-A at 10 pg/kg spawning dose (US$0.25/kg; mGnRH-A, SyndelUS$Z5.00/mg).
339
ACKNOWLEDGEMENTS
We would like to thank Mr. J. Toledo, Mr. R. Gapasin, Ms. E. de Jesus, Mr. R. Ortega and the Igang substation staff for their help in experiments conducted at Igang; and Mr. N. Lopez, Mr. D. Rasca and the National Bangus Breeding Project staff for providing assistance at Lucap, Alaminos, Pangasinan. This work was supported by a grant from the International Development Research Centre (IDRC ) , Canada.
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