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Sustained production of milt in rabbitfish, Siganus guttatus Bloch, by weekly injection of luteinizing hormone-releasing hormone analogue (LHRHa)
Aquaculture, 113 (1993) 261-267 Elsevier Science Publishers B.V., Amsterdam
261
AQUA 60028
Sustained production of milt in rabbitfish, Siganus guttatus Bloch, by weekly injection of luteinizing hormone-releasing hormone analogue (LHRHa) Luis Maria B. Garcia Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEUAQD), Tigbauan, Iloilo, Philippines (Accepted 20 November
1992)
ABSTRACT Mature male rabbitfish (Siganus guttatus Bloch) received weekly injections of 200 &g of luteinizing hormone-releasing hormone analogue ( D-Ala6, Prog-LHRH-ethylamide) per kg body weight for 5 consecutive weeks. Mean spermatocrit, or packed sperm volume (27-5 lo/o), and mean sperm density (3.2-9.6 x lo6 spermatozoa per kg body weight) decreased significantly 24 h after each injection. The amount of expressible milt (mean: 5.8-l 1.7 ml per kg) in response to weekly injections of LHRHa increased significantly relative to saline-injected fish ( 1.0-2.9 ml per kg), but only during the initial 4 weeks of regular hormone treatment. Three weekly injections of LHRHa likewise augmented mean sperm production (29.2-l 12.5 x lo9 spermatozoa per kg) in rabbit&h. However, no significant enhancement in sperm production by LHRHa-injected fish was observed over the last 2 weeks of hormone injection. These results demonstrate that weekly injection of LHRHa can sustain milt production in mature rabbitfish, although their capacity to produce spermatozoa is limited to only 3 consecutive weeks of regular hormone treatment.
INTRODUCTION
Although rabbitfish (Siganus guttatus Bloch) appear to follow a lunar-synchronized spawning rhythm (Hara et al., 1986), artificial manipulation of reproduction is still required to synchronize spawning of captive broodstock. Induction of spawning of rabbitfish typically involves pairing of a human chorionic gonadotropin (HCG)-injected female with males possessing viscous milt (Ayson, 199 1) . While this protocol is quite effective when only a single spawning bout is desired, it is constrained especially when the number of mature fish from a captive source is limited. The same group of spawners Correspondence fo: L.Ma.B. Garcia, velopment Center (SEAFDEC/AQD),
0044-8486/93/$06.00
Aquaculture Tigbauan,
Department, Southeast Iloilo, Philippines.
0 1993 Elsevier Science Publishers
Asian Fisheries
B.V. All rights reserved.
De-
262
L.Ma.B. GARCIA
could therefore be used repeatedly to maintain the demand for finfish seed in a hatchery. To circumvent this limitation, treatment of rabbitfish with pelleted luteinizing hormone-releasing hormone analogue (LHRHa) has been shown to extend the period of availability of vitellogenic oocytes for hormone-induced spawning (Harvey et al., 1985). The use of LHRHa in male rabbitfish also holds some potential as indicated in a recent report wherein the milt production response of mature fish peaked 24 h after a single injection of LHRHa before declining to pre-treatment levels 48 h post-treatment (Garcia, 199 1) . In the common carp, daily injection for 9 days or weekly stimulation by carp pituitary extract over a month sustained high levels of sperm and milt production (Courtois et al., 1986; Saad and Billard, 1987). While levels of serum gonadotropic hormone (GTH) and 11-ketotestosterone ( 11XT ) were elevated, milt volume and sperm concentration in common carp also remained high in response to daily injection for 5 days of LHRHa dissolved in saline (Takashima et al., 1984). However, HCG treatment failed to stimulate a similar response in carp (Courtois et al., 1986). This report investigated the possibility of making milt available for an extended period by repeated injections of LHRHa to the same group of rabbitfish. MATERIALS AND METHODS
Five- and 6-year-old rabbitfish broodstock, weighing 0.33 + 0.0 1 kg (mean body weight t s.e.m. ) and maintained in a 3-m-diameter rubberized canvas tank were used. Fish were fed daily on a pelleted shrimp diet (42% protein; Universal Robina, Pasig, Metro Manila) at 3% of total biomass. Seawater was continuously drained through a l-m standpipe at the centre of the rearing tank. Salinity and water temperature in the tank in April ranged from 33 to 35 ppt and from 30 to 32”C, respectively. A day before the experiment began, 16 mature rabbitfish with expressible milt were randomly chosen from the stock tank. A numbered opercular tag was attached to each fish (Garcia and Gapasin, 1988) after anaesthetization in a shallow basin of seawater containing 100 ppm of 2-phenoxyethanol (Sigma Chemical Company, USA). Fish were likewise weighed to the nearest 5 g. Tagged fish were then equally assigned to two treatment groups: control and LHRHa. Luteinizing hormone-releasing hormone analogue ( D-Ala6, Pro9-LHRHethylamide) was obtained from a commercial source (Jackson Industrial Company, Hongkong). A fresh hormone solution was prepared by dissolving 200 pg of LHRHa in 0.5 ml of 0.9% NaCl (saline). With a disposable plastic tuberculin syringe, fish were intramuscularly injected 200 pg of LHRHa per kg body weight below the dorsal fin. An injection
MILT PRODUCTION IN RABBITFISH
263
volume of 0.5 ml per kg body weight was used. Saline-injected fish served as controls. Fish were injected once weekly for 5 consecutive weeks. The time of injection was fixed between 08.30 h and 09.30 h. Experimental fish were placed in a communal rearing tank after injection. Milt was stripped by gently stroking the abdomen of fish and collected at 24 h after each weekly injection. In a previous report (Garcia, 199 1)) peak milt response was observed 24 h after an injection of LHRHa to mature rabbit&h and declined significantly to pre-treatment conditions 48 h post-injection. The collection and processing of milt samples to measure spermatocrit (packed sperm volume) and milt production has been described elsewhere (Garcia, 1991). For each sample, sperm density ( x lo6 spermatozoa per ~1 milt) was estimated from spermatocrit by a regression equation modified from a line equation described by Garcia ( 199 1): spermatocrit = 15.25+3.69xsperm density (n=65; r-=0.78; P~0.01). This modification was necessary since a few milt samples had low spermatocrit levels of less than 30%, which is below the linear portion of the regression line described earlier (Garcia, 199 1). However, analysis of covariance revealed that the slope of the presently modified regression line is not significantly different from the previous line described earlier by Garcia ( 1991). Sperm production, expressed as the number of spermatozoa per kg body weight, represents the product of sperm density and expressible milt volume. Data were expressed as mean +-s.e.m. of eight fish. Treatment means were compared by Student’s t-test or by analysis of variance followed by Duncan’s multiple range test at P= 0.05 (Snedecor and Cochran, 1980). RESULTS
Compared with saline-injected fish, weekly injections of LHRHa for 5 consecutive weeks caused a significant reduction in mean spermatocrit levels 24 h after each injection (Fig. IA). Saline-injected fish had high spermatocrit levels of 67-84%, which remained relatively constant throughout the experiment. Among LHRHa-injected fish, spermatocrit significantly declined during the second (34%) and third (27%) weeks of treatment, but increased in the fourth (46%) and fifth (5 1%) weeks to levels comparable to the first week (49%) of treatment. LHRHa stimulated an increase in mean milt production levels 24 h after rabbitfish were injected weekly for 4 consecutive weeks (Fig. 1B ) . No significant stimulation by LHRHa injection was observed on the last week of hormone treatment. While milt production among control fish ( 1.O-2.9 ml per kg) remained relatively low during the experiment, the volume of expressible milt among LHRHa-injected fish peaked in the first 3 weeks (9.2-l 1.7 ml per kg) of hormone injection before declining during subsequent weeks. Relative to control fish, weekly injections of LHRHa for 5 consecutive weeks
264
L.Ma.B. GARCIA
A
0 Saline El LHRk!
Fig. 1. Spermatoctit (A), milt production (B), sperm density (C), and sperm production (D) of mature rabbitfish 24 h after weekly injections of 200 ,ug LHRHa per kg body weight for 5 consecutive weeks. Bars are mean 31s.e.m. of eight fish. A, P< 0.05; A A, P< 0.01 compared with saline-injected fish at respective sample time. For each experimental group, bars with a different letter (saline) or number ( LHRHa ) are significantly different from each other (P< 0.05 ) .
also significantly reduced mean sperm density levels of rabbitfish 24 h after each injection (Fig. 1C). Sperm densities of 14.0 to 18.6 X lo6 spermatozoa per ~1 milt among control fish remained elevated throughout the test. Compared to those observed in the first, fourth, and fifth weeks, sperm densities of LHRHa-injected fish significantly declined during the second and third weeks of treatment. Relative to saline-injected fish, mean sperm production significantly increased when rabbitfish received weekly injections of LHRHa for 3 consecutive weeks (Fig. 1D ) . A peak sperm production level of 112.5 x 1O9spermatozoa per kg was attained by LHRHa-injected fish after the first week of treatment before declining ( 19.6-50.8x lo9 spermatozoa per kg) in subsequent weeks. Although the mean sperm output of control fish appeared to decline during the second and third weeks ( 14.4- 14.6 x 1O9spermatozoa per kg), these levels were not significantly different from those observed during the previous and subsequent weeks (3 1.5-43.2 x 1O9spermatozoa per kg).
MILT PRODUCTION IN RABBITFISH
265
DISCUSSION
The present study demonstrates that weekly injections of LHRHa to mature rabbitfish prolong for several weeks their milt production response to the hormone. The effect of a single injection of LHRHa on mature male rabbitfish is rather brief. Hence, milt must be collected 24 h after each injection. Beyond this time, the milt production response of LHRHa-treated fish declines to pre-injection levels (Garcia, 1991). Nonetheless, this study opens the possibility of using repeatedly the same group of spawners for production of milt in a linfish hatchery and, therefore, alleviates the need to use several batches of mature fish broodstock for induced spawning operations. The milt production response of rabbitfish measured 24 h after a single injection of LHRHa given weekly, paralleled that reported in an earlier study (Garcia, 199 1) . Mature rabbitfish which were also injected once with LHRHa had decreased spermatocrit, thinning of semen, and an increased volume of expressible milt one day after exogenous hormone treatment (Garcia, 199 1) . Exogenous GTH administration likewise caused a similar response in intact (Clemens and Grant, 1964) or in hypophysectomized goldfish (Yamazaki and Donaldson, 1968 ) . Milt and sperm production among LHRHa-injected rabbitfish showed a 2to 3-fold elevation during the first 3 weeks of treatment. Although milt production remained high during the fourth week, sperm production decreased to control levels during the last 2 weeks of treatment. Therefore, while the volume of expressible milt appeared to be extended by repeated injections of LHRHa, true augmentation of sperm production occurred only during the initial 3 weeks of treatment. Compared with an earlier study (Garcia, 199 1) , the present work utilized a relatively higher dose of LHRHa (200 pg/kg), expecting that such a dose would extend the response of fish to the hormone. Perhaps the observed decline in milt production following extended hormone treatment could be a reflection of the relatively high dose of LHRHa injected weekly to male rabbitfish. There are indications in the goldfish that prolonged administration of high levels of GTH-releasing hormone may lead to a partial refractoriness of the GTH-release response mechanism (Habibi, 199 1) , which eventually influences gonadal activity. In contrast, weekly injections of a pituitary extract to common carp for 9 months stimulated regular production of milt and sperm (Saad and Billard, 1987). Although sperm output was not measured, milt production of common carp was likewise continuously augmented by weekly injections of LHRHa for a month (Courtois et al., 1986). Therefore, the present data suggest a limited capacity of rabbitfish to respond to weekly injections of LHRHa. Although it remains to be demonstrated, increased milt production induced by LHRHa injected weekly to mature rabbitfish for 3 consecutive weeks may find practical application for fertilizing eggs for seed production in a hatchery.
266
L.Ma.B. GARCIA
Since sperm production of hormone-treated rabbitlish was not significantly different from that of control fish during the fourth and fifth weeks of treatment, a decrease in spermatocrit and sperm density may be largely due to sperm dilution, as suggested by high milt volumes from the first until the fourth week of treatment. On the other hand, it is difficult to explain the decline in spermatocrit and sperm density levels during the last week of treatment since milt production remained low. Other factors (e.g., physical stress due to handling, high hormone dose, etc.) may have been involved, resulting in low milt production. Nonetheless, it is likely that LHRHa caused the release of pituitary GTH which, in turn, stimulated seminal thinning in rabbitfish, resulting in low spermatocrit and sperm densities of stripped milt. Administration of GTH preparations to hypophysectomized goldfish also resulted in the hypertrophy of sperm duct epithelial cells and interstitial cells (Yamazaki and Donaldson, 1968). These authors, therefore, suggested that hypertrophied cells secreted some fluid into the sperm duct causing seminal thinning and eventual release of milt due to increased intralobular pressure. Taken together, repeated injections of a relatively high dose of LHRHa have a limited period of successful stimulation of milt and sperm production in mature rabbit&h. The present procedure of hormone administration also entails frequent, handling of fish which increases the risk of handling-related stress among broodfish, and involves added labour and handling costs. Implantation of LHRHa incorporated in a cholesterol pellet matrix offers an alternative to a regular injection protocol. As demonstrated in some salmonids (Weil and Crim, 1983; Crim and Glebe, 1984; Crim et al., 1988) and suggested in sea bass (Garcia, 1989), LHRHa released from its cholesterol pellet matrix stimulates a chronic and prolonged elevation of GTH in circulation, resulting in the induction of the final stages of gonadal maturation and spawning. This aspect remains to be investigated in rabbitfish. ACKNOWLEDGEMENTS
The author is grateful to L.A.T. Espada, V. Futalan, A. Gamuza, C.M.H. Garcia, E.T. Panes, and A.M. Tan for their technical assistance; to M.N. Duray and M.N. Parazo for the fish; and to A.C. Fermin and G.H. Garcia for reviewing the manuscript.
REFERENCES Ayson, F.G., 1991. Induced spawning of rabbitfish, Siganus guttatus (Bloch), using human chorionic gonadotropin (HCG). Aquaculture, 95: 133-I 37. Clemens, H.P. and Grant, F.B., 1964. The seminal thinning response of carp (Cyprinus carpio) and goldfish (Curussius auratus) after injections of pituitary extracts. Zoologica (N.Y.), 49: 193-210.
MILT PRODUCTION
IN RABBITFISH
267
Courtois, F., Takashima, F. and Billard, R., 1986. Stimulation of spermiation following repeated injection of carp pituitary homogenates in the carp. Bull. Jpn. Sot. Sci. Fish., 52: 995997. Crim, L.W. and Glebe, B.D., 1984. Advancement and synchrony of ovulation in Atlantic salmon with pelleted LHRH analog. Aquaculture, 43: 47-56. Crim, L.W., Sherwood, N.M. and Wilson, C.E., 1988. Sustained hormone release. II. Effectiveness of LHRH analog (LHRHa) administration by either single time injection or cholesterol pellet implantation on plasma gonadotropin levels in a bioassay model fish, the juvenile rainbow trout. Aquaculture, 74: 87-95. Garcia, L.Ma.B., 1989. Dose-dependent spawning response of mature female sea bass, Lutes culcarijh (Bloch), to pelleted luteinizing hormone-releasing hormone analogue (LHRHa). Aquaculture, 77: 85-96. Garcia, L.Ma.B., 199 1. Spermiation response of mature rabbi&h, Siganus g&tutus Bloch, to luteinizing hormone-releasing hormone analogue (LHRHa) injection. Aquaculture, 97: 29 l299. Garcia, L.Ma.B. and Gapasin, RSJ., 1988. An inexpensive tag for short-term studies in milktish (Chanos chanos Forsskal) and in sea bass (Lutes calcarifir Bloch). J. Appl. Icthyol., 4: 10 l104. Habibi, H.R., 1991. Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in the goldfish pituitary: effects of native GnRH peptides and a synthetic GnRH antagonist. Biol. Reprod., 44: 275-283. Hara, S., Duray, M.N., Parazo, M. and Taki, Y., 1986. Year-round spawning and seed production of the rabbitfish, Siganus guttatus. Aquaculture, 59: 259-272. Harvey, B., Nacario, J., Crim, L.W., Juario, J.V. and Marte, C.L., 1985. Induced spawning of sea bass, Lutes calcarifer, and rabbitfish, Sigunus guttatus, after implantation of pelleted LHRH analogue. Aquaculture, 47: 53-59. Saad, A. and Billard, R., 1987. Spermatozoa production and volume of semen collected after hormonal stimulation in the carp, Cyprinus carpio. Aquaculture, 65: 67-77. Snedecor, G.W. and Cochran, W.G., 1980. Statistical Methods, 7th Edn. Iowa State University Press, Ames, IA, 507 pp. Takashima, F., Weil, C., Billard, R., Crim, L.W. and Fostier, A., 1984. Stimulation of spermiation by LHRHa analogue in carp. Bull. Jpn. Sot. Sci. Fish., 50: 1323-1329. Weil, C. and Crim, L.W., 1983. Administration of LHRH analogues in various ways: effect of the advancement of spermiation in prespawning landlocked salmon, Salmo salar. Aquaculture, 35: 103-l 15. Yamazaki, F. and Donaldson, E.M., 1968. The spermiation of goldfish (Curassius auratus) as a bioassay for salmon (Oncorhynchus tshawytscha) gonadotropin. Gen. Comp. Endocrinol., 10: 383-391.
261
AQUA 60028
Sustained production of milt in rabbitfish, Siganus guttatus Bloch, by weekly injection of luteinizing hormone-releasing hormone analogue (LHRHa) Luis Maria B. Garcia Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEUAQD), Tigbauan, Iloilo, Philippines (Accepted 20 November
1992)
ABSTRACT Mature male rabbitfish (Siganus guttatus Bloch) received weekly injections of 200 &g of luteinizing hormone-releasing hormone analogue ( D-Ala6, Prog-LHRH-ethylamide) per kg body weight for 5 consecutive weeks. Mean spermatocrit, or packed sperm volume (27-5 lo/o), and mean sperm density (3.2-9.6 x lo6 spermatozoa per kg body weight) decreased significantly 24 h after each injection. The amount of expressible milt (mean: 5.8-l 1.7 ml per kg) in response to weekly injections of LHRHa increased significantly relative to saline-injected fish ( 1.0-2.9 ml per kg), but only during the initial 4 weeks of regular hormone treatment. Three weekly injections of LHRHa likewise augmented mean sperm production (29.2-l 12.5 x lo9 spermatozoa per kg) in rabbit&h. However, no significant enhancement in sperm production by LHRHa-injected fish was observed over the last 2 weeks of hormone injection. These results demonstrate that weekly injection of LHRHa can sustain milt production in mature rabbitfish, although their capacity to produce spermatozoa is limited to only 3 consecutive weeks of regular hormone treatment.
INTRODUCTION
Although rabbitfish (Siganus guttatus Bloch) appear to follow a lunar-synchronized spawning rhythm (Hara et al., 1986), artificial manipulation of reproduction is still required to synchronize spawning of captive broodstock. Induction of spawning of rabbitfish typically involves pairing of a human chorionic gonadotropin (HCG)-injected female with males possessing viscous milt (Ayson, 199 1) . While this protocol is quite effective when only a single spawning bout is desired, it is constrained especially when the number of mature fish from a captive source is limited. The same group of spawners Correspondence fo: L.Ma.B. Garcia, velopment Center (SEAFDEC/AQD),
0044-8486/93/$06.00
Aquaculture Tigbauan,
Department, Southeast Iloilo, Philippines.
0 1993 Elsevier Science Publishers
Asian Fisheries
B.V. All rights reserved.
De-
262
L.Ma.B. GARCIA
could therefore be used repeatedly to maintain the demand for finfish seed in a hatchery. To circumvent this limitation, treatment of rabbitfish with pelleted luteinizing hormone-releasing hormone analogue (LHRHa) has been shown to extend the period of availability of vitellogenic oocytes for hormone-induced spawning (Harvey et al., 1985). The use of LHRHa in male rabbitfish also holds some potential as indicated in a recent report wherein the milt production response of mature fish peaked 24 h after a single injection of LHRHa before declining to pre-treatment levels 48 h post-treatment (Garcia, 199 1) . In the common carp, daily injection for 9 days or weekly stimulation by carp pituitary extract over a month sustained high levels of sperm and milt production (Courtois et al., 1986; Saad and Billard, 1987). While levels of serum gonadotropic hormone (GTH) and 11-ketotestosterone ( 11XT ) were elevated, milt volume and sperm concentration in common carp also remained high in response to daily injection for 5 days of LHRHa dissolved in saline (Takashima et al., 1984). However, HCG treatment failed to stimulate a similar response in carp (Courtois et al., 1986). This report investigated the possibility of making milt available for an extended period by repeated injections of LHRHa to the same group of rabbitfish. MATERIALS AND METHODS
Five- and 6-year-old rabbitfish broodstock, weighing 0.33 + 0.0 1 kg (mean body weight t s.e.m. ) and maintained in a 3-m-diameter rubberized canvas tank were used. Fish were fed daily on a pelleted shrimp diet (42% protein; Universal Robina, Pasig, Metro Manila) at 3% of total biomass. Seawater was continuously drained through a l-m standpipe at the centre of the rearing tank. Salinity and water temperature in the tank in April ranged from 33 to 35 ppt and from 30 to 32”C, respectively. A day before the experiment began, 16 mature rabbitfish with expressible milt were randomly chosen from the stock tank. A numbered opercular tag was attached to each fish (Garcia and Gapasin, 1988) after anaesthetization in a shallow basin of seawater containing 100 ppm of 2-phenoxyethanol (Sigma Chemical Company, USA). Fish were likewise weighed to the nearest 5 g. Tagged fish were then equally assigned to two treatment groups: control and LHRHa. Luteinizing hormone-releasing hormone analogue ( D-Ala6, Pro9-LHRHethylamide) was obtained from a commercial source (Jackson Industrial Company, Hongkong). A fresh hormone solution was prepared by dissolving 200 pg of LHRHa in 0.5 ml of 0.9% NaCl (saline). With a disposable plastic tuberculin syringe, fish were intramuscularly injected 200 pg of LHRHa per kg body weight below the dorsal fin. An injection
MILT PRODUCTION IN RABBITFISH
263
volume of 0.5 ml per kg body weight was used. Saline-injected fish served as controls. Fish were injected once weekly for 5 consecutive weeks. The time of injection was fixed between 08.30 h and 09.30 h. Experimental fish were placed in a communal rearing tank after injection. Milt was stripped by gently stroking the abdomen of fish and collected at 24 h after each weekly injection. In a previous report (Garcia, 199 1)) peak milt response was observed 24 h after an injection of LHRHa to mature rabbit&h and declined significantly to pre-treatment conditions 48 h post-injection. The collection and processing of milt samples to measure spermatocrit (packed sperm volume) and milt production has been described elsewhere (Garcia, 1991). For each sample, sperm density ( x lo6 spermatozoa per ~1 milt) was estimated from spermatocrit by a regression equation modified from a line equation described by Garcia ( 199 1): spermatocrit = 15.25+3.69xsperm density (n=65; r-=0.78; P~0.01). This modification was necessary since a few milt samples had low spermatocrit levels of less than 30%, which is below the linear portion of the regression line described earlier (Garcia, 199 1). However, analysis of covariance revealed that the slope of the presently modified regression line is not significantly different from the previous line described earlier by Garcia ( 1991). Sperm production, expressed as the number of spermatozoa per kg body weight, represents the product of sperm density and expressible milt volume. Data were expressed as mean +-s.e.m. of eight fish. Treatment means were compared by Student’s t-test or by analysis of variance followed by Duncan’s multiple range test at P= 0.05 (Snedecor and Cochran, 1980). RESULTS
Compared with saline-injected fish, weekly injections of LHRHa for 5 consecutive weeks caused a significant reduction in mean spermatocrit levels 24 h after each injection (Fig. IA). Saline-injected fish had high spermatocrit levels of 67-84%, which remained relatively constant throughout the experiment. Among LHRHa-injected fish, spermatocrit significantly declined during the second (34%) and third (27%) weeks of treatment, but increased in the fourth (46%) and fifth (5 1%) weeks to levels comparable to the first week (49%) of treatment. LHRHa stimulated an increase in mean milt production levels 24 h after rabbitfish were injected weekly for 4 consecutive weeks (Fig. 1B ) . No significant stimulation by LHRHa injection was observed on the last week of hormone treatment. While milt production among control fish ( 1.O-2.9 ml per kg) remained relatively low during the experiment, the volume of expressible milt among LHRHa-injected fish peaked in the first 3 weeks (9.2-l 1.7 ml per kg) of hormone injection before declining during subsequent weeks. Relative to control fish, weekly injections of LHRHa for 5 consecutive weeks
264
L.Ma.B. GARCIA
A
0 Saline El LHRk!
Fig. 1. Spermatoctit (A), milt production (B), sperm density (C), and sperm production (D) of mature rabbitfish 24 h after weekly injections of 200 ,ug LHRHa per kg body weight for 5 consecutive weeks. Bars are mean 31s.e.m. of eight fish. A, P< 0.05; A A, P< 0.01 compared with saline-injected fish at respective sample time. For each experimental group, bars with a different letter (saline) or number ( LHRHa ) are significantly different from each other (P< 0.05 ) .
also significantly reduced mean sperm density levels of rabbitfish 24 h after each injection (Fig. 1C). Sperm densities of 14.0 to 18.6 X lo6 spermatozoa per ~1 milt among control fish remained elevated throughout the test. Compared to those observed in the first, fourth, and fifth weeks, sperm densities of LHRHa-injected fish significantly declined during the second and third weeks of treatment. Relative to saline-injected fish, mean sperm production significantly increased when rabbitfish received weekly injections of LHRHa for 3 consecutive weeks (Fig. 1D ) . A peak sperm production level of 112.5 x 1O9spermatozoa per kg was attained by LHRHa-injected fish after the first week of treatment before declining ( 19.6-50.8x lo9 spermatozoa per kg) in subsequent weeks. Although the mean sperm output of control fish appeared to decline during the second and third weeks ( 14.4- 14.6 x 1O9spermatozoa per kg), these levels were not significantly different from those observed during the previous and subsequent weeks (3 1.5-43.2 x 1O9spermatozoa per kg).
MILT PRODUCTION IN RABBITFISH
265
DISCUSSION
The present study demonstrates that weekly injections of LHRHa to mature rabbitfish prolong for several weeks their milt production response to the hormone. The effect of a single injection of LHRHa on mature male rabbitfish is rather brief. Hence, milt must be collected 24 h after each injection. Beyond this time, the milt production response of LHRHa-treated fish declines to pre-injection levels (Garcia, 1991). Nonetheless, this study opens the possibility of using repeatedly the same group of spawners for production of milt in a linfish hatchery and, therefore, alleviates the need to use several batches of mature fish broodstock for induced spawning operations. The milt production response of rabbitfish measured 24 h after a single injection of LHRHa given weekly, paralleled that reported in an earlier study (Garcia, 199 1) . Mature rabbitfish which were also injected once with LHRHa had decreased spermatocrit, thinning of semen, and an increased volume of expressible milt one day after exogenous hormone treatment (Garcia, 199 1) . Exogenous GTH administration likewise caused a similar response in intact (Clemens and Grant, 1964) or in hypophysectomized goldfish (Yamazaki and Donaldson, 1968 ) . Milt and sperm production among LHRHa-injected rabbitfish showed a 2to 3-fold elevation during the first 3 weeks of treatment. Although milt production remained high during the fourth week, sperm production decreased to control levels during the last 2 weeks of treatment. Therefore, while the volume of expressible milt appeared to be extended by repeated injections of LHRHa, true augmentation of sperm production occurred only during the initial 3 weeks of treatment. Compared with an earlier study (Garcia, 199 1) , the present work utilized a relatively higher dose of LHRHa (200 pg/kg), expecting that such a dose would extend the response of fish to the hormone. Perhaps the observed decline in milt production following extended hormone treatment could be a reflection of the relatively high dose of LHRHa injected weekly to male rabbitfish. There are indications in the goldfish that prolonged administration of high levels of GTH-releasing hormone may lead to a partial refractoriness of the GTH-release response mechanism (Habibi, 199 1) , which eventually influences gonadal activity. In contrast, weekly injections of a pituitary extract to common carp for 9 months stimulated regular production of milt and sperm (Saad and Billard, 1987). Although sperm output was not measured, milt production of common carp was likewise continuously augmented by weekly injections of LHRHa for a month (Courtois et al., 1986). Therefore, the present data suggest a limited capacity of rabbitfish to respond to weekly injections of LHRHa. Although it remains to be demonstrated, increased milt production induced by LHRHa injected weekly to mature rabbitfish for 3 consecutive weeks may find practical application for fertilizing eggs for seed production in a hatchery.
266
L.Ma.B. GARCIA
Since sperm production of hormone-treated rabbitlish was not significantly different from that of control fish during the fourth and fifth weeks of treatment, a decrease in spermatocrit and sperm density may be largely due to sperm dilution, as suggested by high milt volumes from the first until the fourth week of treatment. On the other hand, it is difficult to explain the decline in spermatocrit and sperm density levels during the last week of treatment since milt production remained low. Other factors (e.g., physical stress due to handling, high hormone dose, etc.) may have been involved, resulting in low milt production. Nonetheless, it is likely that LHRHa caused the release of pituitary GTH which, in turn, stimulated seminal thinning in rabbitfish, resulting in low spermatocrit and sperm densities of stripped milt. Administration of GTH preparations to hypophysectomized goldfish also resulted in the hypertrophy of sperm duct epithelial cells and interstitial cells (Yamazaki and Donaldson, 1968). These authors, therefore, suggested that hypertrophied cells secreted some fluid into the sperm duct causing seminal thinning and eventual release of milt due to increased intralobular pressure. Taken together, repeated injections of a relatively high dose of LHRHa have a limited period of successful stimulation of milt and sperm production in mature rabbit&h. The present procedure of hormone administration also entails frequent, handling of fish which increases the risk of handling-related stress among broodfish, and involves added labour and handling costs. Implantation of LHRHa incorporated in a cholesterol pellet matrix offers an alternative to a regular injection protocol. As demonstrated in some salmonids (Weil and Crim, 1983; Crim and Glebe, 1984; Crim et al., 1988) and suggested in sea bass (Garcia, 1989), LHRHa released from its cholesterol pellet matrix stimulates a chronic and prolonged elevation of GTH in circulation, resulting in the induction of the final stages of gonadal maturation and spawning. This aspect remains to be investigated in rabbitfish. ACKNOWLEDGEMENTS
The author is grateful to L.A.T. Espada, V. Futalan, A. Gamuza, C.M.H. Garcia, E.T. Panes, and A.M. Tan for their technical assistance; to M.N. Duray and M.N. Parazo for the fish; and to A.C. Fermin and G.H. Garcia for reviewing the manuscript.
REFERENCES Ayson, F.G., 1991. Induced spawning of rabbitfish, Siganus guttatus (Bloch), using human chorionic gonadotropin (HCG). Aquaculture, 95: 133-I 37. Clemens, H.P. and Grant, F.B., 1964. The seminal thinning response of carp (Cyprinus carpio) and goldfish (Curussius auratus) after injections of pituitary extracts. Zoologica (N.Y.), 49: 193-210.
MILT PRODUCTION
IN RABBITFISH
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