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Acclimatization and induced spawning of sardine Sardina pilchardus Walbaum in captivity
.I. Exp. Mar. Biol. Ecol., 1990, Vol. 140, pp. 61-67 Elsevier
61
JEMBE 01442
and induced spawning of sardine Sardina pilchardus Walbaum in captivity
Acclimatization
Mercedes Oltnedo, Jo& Iglesias, JosC Benito Peleteiro, Rafael Forks and Ana Miranda Centro Costerode1InstitutoEspaiiolde Oceanograflade Vigo, Vigo,Spain (Received 29 November 1989; revision received 19 February 1990; accepted 22 March 1990) Abstract: Sardines ~ard~ap~chard~ Walbaum captured in the Ria de Vigo with a mean length of 17.13 cm and a mean weight of 49.66 g were acclimatized to captivity for a year, with an overall survival of 39.5 %. They adapted to dry commercial pellets of 500 pm diameter within 3 days of capture. Hormonal induction gave positive results in recently captured mature specimens but those which had been acclimatized for 1 month did not spawn in captivity. The induction method consisted of an initial injection of 125 IU of human chorionic gonadotropin (HCG) followed 24 h later by 100 IU of pregnant mare serum (PMS) combined with 10 mg of carp pituitary for each specimen. The incubation period of the eggs lasted 4.9-1.7 days at temperatures between 11 and 20 “C, and the mean hatching rate was 56%. The larvae obtained showed a positive response to living food consisting of Isociq& galbana Parke and Brachionusplicaiilir Miiller. Key words: Induced spawning; Rearing; Sardinapilchardus INTRODUCTION
The sardine Sardinapilchardus Walbaum is a species of great ecological and economic importance on the Atlantic coast of the Iberian Peninsula and over the last 4 yr, several field methods have been developed to determine the biological and oceanographic processes that produce annual variations in its recruitment (Chesney & Alonso, 1988; Cabanas et al., 1989). To validate these field data in the laboratory, it was considered essential to adapt adult specimens to captivity, and subsequently to obtain spawning, eggs and larvae. If this were successful, it should then be possible: (1) To determine the presence of postovulatory follicles in females which have spawned in captivity, This objective is necessary to validate the rate of postovulatory follicle d~eneration in order to obtain the fraction spawned by females per day, one of the parameters in the egg production method. (2) To relate the stage of eggs obtained by induced spawning to their different incubation temperatures. (3) To verify the ages of larvae at different stages using the daily ring technique. (4) To conduct laboratory experiments on starvation in recently hatched larvae. Correspondence address: M. Olmedo, Centro Costero de1 Instituto Espagol de Oceanografia de Vigo, Apartado 1.552, 36280 Vigo, Spain. 0022-0981/90/%03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
6:
M. OLMEDOET AL
Publications on the acclimatization and spawning of clupeoid species in captivity are very scarce and limited to the works of Muzinic (1966, 1977) and Leong (1971) who induced spawning in anchovy. The present work is the first in which induced spawning in sardines in the laboratory has been achieved.
MATERIALSAND METHODS CAPTUREOF ADULTS The attempt to capture adults was planned in two steps. The first was designed to acquire adult specimens for acclimatization and subsequent spawning, and the second was aimed at the capture of already mature individuals for the immediate induction of spawning. In November 1987, the first capture was made off Sada (La Con&la) in a large commercial purse-seine. The samples were placed in 500-l circular deck tanks with open water circulation. The fish were transported to the laboratory in an aerated and refrigerated tank with closed circulation. The stocking density during transport was 20 kg * m- 3. On arrival, 173 individuals were placed in tanks and immediately disinfected for 1 h in a bath of Sanisal, a product which contains 95% copper sulphate. During 1988, several samples were taken in February, March and November in the outer part of Ria de Vigo using a smaller purse-seine. Less manipulation of the fish and a lower stocking density during transport (12 kg * mp3) were utilized in this second attempt. By increasing the frequency of capture, fewer fish were caught on each occasion, but they were in better condition. Since spawning could not be induced in fish adapted to captivity, mature specimens (Stages III and IV) were captured in February 1989 to induce spawning immediately. The mean weight of these specimens was 49.7 g and the mean length was 17.1 cm. ACCLIMATrZATION The fish were maintained in three 9-m3 rectangular tanks each measuring 4.5 x 2 x 1 m with rounded corners, and with a continuous water flow of 3.5 m3 . h ~ ’ and a diffuser in each corner. At first a natural photoperiod was used with a m~imum surface light intensity of %0 lux. After seeing how the fish behaved, the initial conditions were changed. Aeration was stopped, the water intake was positioned at the bottom of the tanks, and the light intensity was reduced to 70 lux. With these new conditions a considerable reduction was observed in the stress suffered by the fish, and with a less turbulent regime in the water the fish were distributed more evenly in the tank. Feeding in the laboratory was first based on published data on the food of clupeoids in the natural environment (Lasker, 1978; Varela et al., in press). As it is difficult to provide living planktonic food in large qu~tities, and as the sardine is a filter-feeder
ACCLIMATIZATION
AND INDUCED
SPAWNING
OF SARDINA
PILCHARDUS
63
(Varela et al., in press), an attempt was made to begin feeding with commercial products kept in suspension. At first, inert food for aquarium fish was used, which is buoyant and which contains attractants. Later a sea bass dry pellet adapted for sardines was used (500 pm diameter) supplied by ATP SA. The diet was supplemented periodically with fish eggs from the hatchery whenever they were available. The amount of food given daily was 2% of the wet weight of the fish stocked and four rations were given daily. Preventative disinfection was carried out monthly, maintaining the lish for 1 h in a bath of Sanisal (ATP, SA) at a concentration of 1.5 g * m- 3. INDUCTION
OF SPAWNING
The method used was similar to that described by Leong (1971) for anchovy Engruuh mordax and later modified by the same author for Surdinops caerufea (Leong, pers. comm.). The fish selected were separated into 0.5-m3 cylindrical polyester tanks with open circulation, and with an egg collector of 500 pm mesh size attached to the outflow. The stocking density in these tanks was 30 fish * m - 3. To avoid problems related to handling, the fish were anaesthetized with MS-222 at a dosage of 55 mg* 1-l (after successive tests with lower doses) before being handled. Three experiments were done with distinct groups of fish. Intramuscular injections were made in the dorsal region of all specimens selected without regard to their sex. (1) In March 1988, a group of 50 sardines which had been in captivity for 1 month were kept for 24 h without food, and then given a first injection of 250 IU of human chorionic gonadotropin (HCG) and 24 h later, a second injection of 200 IU of pregnant mare serum (PMS). After the first dose, some fish produced sperm when pressed. On this basis, the second injection was only given to those thought to be females. (2) In April 1988, 57 fish, which had also been acclimatized in the laboratory for 1 month, were injected. The technique used was as in the previous group, but the dose was reduced by half due to the lower weight of sardine compared with Sardinops. In addition, 10 mg of carp pituitary was added to the second injection of each fish. (3) In February 1989, after it had been shown that samples from the natural environment were females with gonads in Stage III of sexual development, a third sample of 64 fish was injected 24 h after their capture. The doses were the same as in the previous experiment. INCUBATION
Once a relatively small number of fertilized eggs had been obtained, a system was designed comprising 10 incubation flasks of 11 each, and z 200 eggs were put into each flask. The range of temperatures used was from 11 to 20 “C. The system is described in detail by Miranda et al. (1990). The bottoms of the flasks were siphoned daily to remove dead eggs, and two thirds of the water was replaced. Antibiotics were not used during embryonic development.
M. OLMEDOETAL.
64 LARVAL
CULTURE
Recently hatched larvae were immediately transferred to a larval cultivation system composed of five 2-l flasks immersed in a water bath with open circulation and controlled temperature, gentle aeration and a natural photoperiod. The temperature and salinity were the same as during the incubation period. The initial density was 60 larvae-l-‘. The literature indicates that the natural food of larval clupeoids consist of diatoms and zooplankton (Hunter, 1976). In this work, the first food used was the phytoplanktonic alga Zsochrysisgdbana and the rotifer Brachionusplicatilis (50:/, < 175 pm and previously fed for 24 h on the same microalga) at an initial concentration of 20 rotifers * ml- ‘. I. gulbana is an alga used often in marine fish cultivation on account of its high content of poly-unsaturated fatty acids, series o 3, which are of fundamental importance to salt water fish (Owen et al., 1975; Watanabe, 1982). The bottoms of the tanks were cleaned daily by siphoning and the volume removed replaced with I. gulbuna culture. Daily measurements were made of the numbers of rotifers, temperature and salinity.
RESULTS
AND DISCUSSION
The first sardines captured in Sada (La Corufia) showed a high mortality rate. During transportation (z 150 km over 3 h), mortality was x 50%. After the remaining 173 fish had been established in the laboratory for 48 h, the mortality was 73 p/, and after 6 days
100
;; > .n 2 2
~~~.
50
.
l
.
40 M
l
L
.
1 2
30 20 1.‘
t 3 .
IO z0-
,,...~,~,.~,.,.,.U_L-L_YL.,,,,,,..,.,,,..,.,,.,,,,,,,.,., 0
1
2
3
4
5
6
7
8
9
10
I;
12
Months
Fig. 1. Mean percentage of adult sardines survived after 1 year in captivity. Points represent observed values, and regression line theoretical path calculated from first 7 months, without taking handling mortality into account. 1, survival rate after 1 month in captivity; 2, mortality caused by handling; 3, final petcent survival.
ACCLIMATIZATION
AND INDUCED SPAWNING OF SARI)Ih% P~L~H~~US
65
all were dead. The causes of this fast experience were excessive handling of the tish on board, the very long journey and the high stocking density during transport. The captures made off the Ria de Vigo in 1988 under more favourable conditions of proximity, handling and stocking during transport gave better results. The mean value of total mortality in the three tanks after 6 days was 13%, and after 1 month in the laboratory was only 17.6 % of the number initially stocked (Fig. 1). From this time until a year after the beginning of the experiment, survival was high in all cases (58,2%), with the exception of one tank which in the 8th month suffered 47.2% mortality due to a handling problem (Fig. 1). Mean survival in the three tanks was 39.5 %, a moderate value which allows sardines to be kept in captivity for experimental purposes. With respect to feeding, the sardine showed a gradual adaptation to the inert food provided. At first, the fish ate the inert aquarium food from the 3rd day after stocking, and by the end of a week had adapted perfectly to the dry 500~,umpellets. Later, as a routine, the captured fish were adapted directly to this last meal 3 days after collection. The fish already acclimatized to captivity in 1988 neither spawned spontaneously, nor in response to the hormonal induction method used after 1 month in captivity. Furthermore, the samples taken to study mat~ation (Perez et al., 1985) showed a progressive regression of the gonads (Table I).
TABLE I
Maturity of sardines analysed during fust 9 months of acclimatization to captivity. Month
Total no. of ~di~du~s
Maturity stage I 9:
24 9 6 8 6 1 1 36 25
6 3 3 4 3 1 21 8
III
II d -I 1 2 2 3 1
IV
9
rp
9
d
2
1
2 1
3 1
4 1 1
1
2 1
1
6
15 17
This gonadal regression was also evident in the negative results of the fast hormonal induction experiment in 1988, in which none of the fish which had been in captivity for 1 month spawned. In 1989, mature specimens captured in the Ria de Vigo for immediate hormonal induction gave positive results. Kuo et al. (1974) and Zohar & Gordin (1979), using mullet M@l cephalur and sea bream Spurus auratu, respectively, have already
M. OLMEDO
66
ET AL
shown that the best results with hormonal induction are obtained using females in an advanced stage of maturity with eggs whose diameter is > 600 pm. Following the scheme described above (Leong, 197 1) with two consecutive injections within 48 h, 64 fish were injected at random without distinguishing the sexes. Viable spawning was only obtained in a tank containing two females and four males. Although viable spawning was only achieved in two female sardines, it is the first time that such a result has been obtained in this species in captivity. The incubation period for embryonic development lasted 4.9-1.7 days at temperatures in the range 1 l-20 “C (Miranda et al., 1990) (Table II). These data agree with those of Blaxter (1969) who incubated sardine eggs from the natural environment, which hatched in 2-5 days at temperatures from 17 to 10 “C.
TABLE
Incubation T. (“C)
11 13 15 18 20
period
and percentage
of sardine
II
eggs hatched
Initial no. of eggs
Incubation period (h)
400 400 200 200
117 91 68 44 39
~
i
at different
experimental
No. of larvae hatched
255 229 90 115
temperatures. % hatched
~~~~~~~___~~ 63.75 51.25 45.00 57.50
Mortality was low during incubation, reaching a final mean value at hatching of 55.9%. Table II shows the percentages of eggs that hatched during incubation. A batch of only 300 larvae (3.4 mm mean length) was available for feeding tests and so this study only tested the efficacy of the classical method of using live food for the experimental cultivation of sardine larvae. These first experiments on the feeding of sardine larvae permit the following conclusions. Most of the sardine larvae, after absorbing their yolk sacs, were found to contain 1. galbana. The fact that clupeoid larvae feed first on phytoplankton is already known from the literature (Hunter, 1976; Varela et al., in press). After 5 days, the larvae ate rotifer eggs and small rotifers which had previously been enriched for 24 h with I. galbana. Hunter (1976) used the same system for anchovy, and enriched the rotifers with Nunnochloris sp. Due to the scarcity of larvae available, it was not possible to test the next step of feeding using Arfemiu nauplii. In conclusion, despite the fact that the results of this experiment must be considered preliminary, it can be concluded that sardine larvae obtained in captivity show a positive response to a feeding regime based on I. galbana and enriched B. plicatilis.
ACCLIMATIZATION
AND INDUCED SPAWNING OF SARBINA PILCHARDUS
67
We particularly thank Nelida Perez of the Spanish Institute of Oceanography, Vigo, for her cooperation in the development of this work. We would also like to thank the collaboration of Alexandra Almeida e Silva of Instituto National de Investigaqao das Pescas, Lisboa, Portugal.
REFERENCES Blaxter, J. H. S., 1969. Experimental rearing ofpilchard larvae, Sard~napjlchard~. J. Mar. Biol. Assoc. U.K., Vol. 49, pp. 551-575. Cabanas, J.M., C. Porteiro & M. Varela, 1989. A possible relation between sardine fisheries and oceanographic conditions in NW Spanish coastal waters. ICES CM 1989/H, p. 18. Chesney, E. J. & M. Alonso-Naval, 1988. Coastal upwelling and the early life history of sardines (Surdina pilchardus) along the Galician coast of Spain. In, The early I@ history of&h, edited by J. H. S. Blaxter et al., Rapp. Proc. Verb. R&m. Cons, Perm. fnt. Explor. bier, Vol. 191, pp. 63-69. Hunter, J.R., 1976. Culture and growth of northern anchovy, Engrtxdis mordux, larvae. Fish. Bull. NOAA, Vol. 74,
pp. 81-88.
Kuo, C. M., C. E. Nash L Z. Shehadeh, 1974. A procedural guide to induce spawuing in grey mullet (Mugil cephalus L. ). Aquaculture, Vol. 5, pp. 19-29. Lasker, R., 1978. Relation between oceanographic conditions and larval anchovy food in the California current: identification of factors contributing to recruitment failure. Rapp. V. R&n. Int. ExpZor. Mer, Vol. 173, pp. 212-230. Leong, R., 1971. Induced spawning of the northern anchovy Engruulir mordax Giaud. Fish. Bull. NOAA, Vol. 69, pp. 357-360. Miranda, A., R. M. Cal & J. Iglesias, 1990. Effect of temperature on the development of eggs and larvae of sardine Sardina pil~ard~ Walbaum in captivity. J. Exp. h&r. Bioi. EcoL, Vof. 140, pp. 69-77. Muzinic, R., 1966. Mortal&C initiale de la sardine dans les ~n~tions ex~~rnent~es et mortalite due au marquage. Gen. Fish. Count. Meditetr. Stud. Rev., Vol. 31, pp. l-20. Muzinic, R., 1977.On the shoaling behaviour of sardines (Sardinapilchurdus)in aquaria.J. Cons. Int. Explar. Mer, Vol. 37, pp. 147-155. Owen, J. M., J. W. Adron, C. Middleton & C. B. Cowey, 1975. Elongation and desaturation of dietary fatty acids in turbot Scophthalmw maximus L. and rainbow trout, Salmo gairdneri Rich. Lipids, Vol. 10, pp. 528-531. Perez, N., C. Porteiro & F. Alvarez, 1985. Contribucidn al conocimiento de la biologia de la sardina de Galicia. Bol. Inst. Esp. Oceanogr., Vol. 2, pp. 27-37. Varela, M., A. Larranaga, E. Costas & B. Rodriguez, in press. Contenido estomacal de la sardina (Sardina pikhardus Walbaum) durante la campafla Saracus 1987 en las plataformas cantabrica y de Galicia en Febrero de 1987. Bol. inst. Esp. Oceanogr. Watanabe, T., 1982. Lipid nutrition in fish. Comp. Biochem. Physiol., Vol. 738, pp. 3-15. Zohar, Y. & H. Gordin, 1979. Spawning kinetics in the gilthead sea-bream (Sparus ouratu L.) after low doses of human chorionic gonadotropin. J. Fish. Biol., Vol. 15, pp. 665-670.
61
JEMBE 01442
and induced spawning of sardine Sardina pilchardus Walbaum in captivity
Acclimatization
Mercedes Oltnedo, Jo& Iglesias, JosC Benito Peleteiro, Rafael Forks and Ana Miranda Centro Costerode1InstitutoEspaiiolde Oceanograflade Vigo, Vigo,Spain (Received 29 November 1989; revision received 19 February 1990; accepted 22 March 1990) Abstract: Sardines ~ard~ap~chard~ Walbaum captured in the Ria de Vigo with a mean length of 17.13 cm and a mean weight of 49.66 g were acclimatized to captivity for a year, with an overall survival of 39.5 %. They adapted to dry commercial pellets of 500 pm diameter within 3 days of capture. Hormonal induction gave positive results in recently captured mature specimens but those which had been acclimatized for 1 month did not spawn in captivity. The induction method consisted of an initial injection of 125 IU of human chorionic gonadotropin (HCG) followed 24 h later by 100 IU of pregnant mare serum (PMS) combined with 10 mg of carp pituitary for each specimen. The incubation period of the eggs lasted 4.9-1.7 days at temperatures between 11 and 20 “C, and the mean hatching rate was 56%. The larvae obtained showed a positive response to living food consisting of Isociq& galbana Parke and Brachionusplicaiilir Miiller. Key words: Induced spawning; Rearing; Sardinapilchardus INTRODUCTION
The sardine Sardinapilchardus Walbaum is a species of great ecological and economic importance on the Atlantic coast of the Iberian Peninsula and over the last 4 yr, several field methods have been developed to determine the biological and oceanographic processes that produce annual variations in its recruitment (Chesney & Alonso, 1988; Cabanas et al., 1989). To validate these field data in the laboratory, it was considered essential to adapt adult specimens to captivity, and subsequently to obtain spawning, eggs and larvae. If this were successful, it should then be possible: (1) To determine the presence of postovulatory follicles in females which have spawned in captivity, This objective is necessary to validate the rate of postovulatory follicle d~eneration in order to obtain the fraction spawned by females per day, one of the parameters in the egg production method. (2) To relate the stage of eggs obtained by induced spawning to their different incubation temperatures. (3) To verify the ages of larvae at different stages using the daily ring technique. (4) To conduct laboratory experiments on starvation in recently hatched larvae. Correspondence address: M. Olmedo, Centro Costero de1 Instituto Espagol de Oceanografia de Vigo, Apartado 1.552, 36280 Vigo, Spain. 0022-0981/90/%03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
6:
M. OLMEDOET AL
Publications on the acclimatization and spawning of clupeoid species in captivity are very scarce and limited to the works of Muzinic (1966, 1977) and Leong (1971) who induced spawning in anchovy. The present work is the first in which induced spawning in sardines in the laboratory has been achieved.
MATERIALSAND METHODS CAPTUREOF ADULTS The attempt to capture adults was planned in two steps. The first was designed to acquire adult specimens for acclimatization and subsequent spawning, and the second was aimed at the capture of already mature individuals for the immediate induction of spawning. In November 1987, the first capture was made off Sada (La Con&la) in a large commercial purse-seine. The samples were placed in 500-l circular deck tanks with open water circulation. The fish were transported to the laboratory in an aerated and refrigerated tank with closed circulation. The stocking density during transport was 20 kg * m- 3. On arrival, 173 individuals were placed in tanks and immediately disinfected for 1 h in a bath of Sanisal, a product which contains 95% copper sulphate. During 1988, several samples were taken in February, March and November in the outer part of Ria de Vigo using a smaller purse-seine. Less manipulation of the fish and a lower stocking density during transport (12 kg * mp3) were utilized in this second attempt. By increasing the frequency of capture, fewer fish were caught on each occasion, but they were in better condition. Since spawning could not be induced in fish adapted to captivity, mature specimens (Stages III and IV) were captured in February 1989 to induce spawning immediately. The mean weight of these specimens was 49.7 g and the mean length was 17.1 cm. ACCLIMATrZATION The fish were maintained in three 9-m3 rectangular tanks each measuring 4.5 x 2 x 1 m with rounded corners, and with a continuous water flow of 3.5 m3 . h ~ ’ and a diffuser in each corner. At first a natural photoperiod was used with a m~imum surface light intensity of %0 lux. After seeing how the fish behaved, the initial conditions were changed. Aeration was stopped, the water intake was positioned at the bottom of the tanks, and the light intensity was reduced to 70 lux. With these new conditions a considerable reduction was observed in the stress suffered by the fish, and with a less turbulent regime in the water the fish were distributed more evenly in the tank. Feeding in the laboratory was first based on published data on the food of clupeoids in the natural environment (Lasker, 1978; Varela et al., in press). As it is difficult to provide living planktonic food in large qu~tities, and as the sardine is a filter-feeder
ACCLIMATIZATION
AND INDUCED
SPAWNING
OF SARDINA
PILCHARDUS
63
(Varela et al., in press), an attempt was made to begin feeding with commercial products kept in suspension. At first, inert food for aquarium fish was used, which is buoyant and which contains attractants. Later a sea bass dry pellet adapted for sardines was used (500 pm diameter) supplied by ATP SA. The diet was supplemented periodically with fish eggs from the hatchery whenever they were available. The amount of food given daily was 2% of the wet weight of the fish stocked and four rations were given daily. Preventative disinfection was carried out monthly, maintaining the lish for 1 h in a bath of Sanisal (ATP, SA) at a concentration of 1.5 g * m- 3. INDUCTION
OF SPAWNING
The method used was similar to that described by Leong (1971) for anchovy Engruuh mordax and later modified by the same author for Surdinops caerufea (Leong, pers. comm.). The fish selected were separated into 0.5-m3 cylindrical polyester tanks with open circulation, and with an egg collector of 500 pm mesh size attached to the outflow. The stocking density in these tanks was 30 fish * m - 3. To avoid problems related to handling, the fish were anaesthetized with MS-222 at a dosage of 55 mg* 1-l (after successive tests with lower doses) before being handled. Three experiments were done with distinct groups of fish. Intramuscular injections were made in the dorsal region of all specimens selected without regard to their sex. (1) In March 1988, a group of 50 sardines which had been in captivity for 1 month were kept for 24 h without food, and then given a first injection of 250 IU of human chorionic gonadotropin (HCG) and 24 h later, a second injection of 200 IU of pregnant mare serum (PMS). After the first dose, some fish produced sperm when pressed. On this basis, the second injection was only given to those thought to be females. (2) In April 1988, 57 fish, which had also been acclimatized in the laboratory for 1 month, were injected. The technique used was as in the previous group, but the dose was reduced by half due to the lower weight of sardine compared with Sardinops. In addition, 10 mg of carp pituitary was added to the second injection of each fish. (3) In February 1989, after it had been shown that samples from the natural environment were females with gonads in Stage III of sexual development, a third sample of 64 fish was injected 24 h after their capture. The doses were the same as in the previous experiment. INCUBATION
Once a relatively small number of fertilized eggs had been obtained, a system was designed comprising 10 incubation flasks of 11 each, and z 200 eggs were put into each flask. The range of temperatures used was from 11 to 20 “C. The system is described in detail by Miranda et al. (1990). The bottoms of the flasks were siphoned daily to remove dead eggs, and two thirds of the water was replaced. Antibiotics were not used during embryonic development.
M. OLMEDOETAL.
64 LARVAL
CULTURE
Recently hatched larvae were immediately transferred to a larval cultivation system composed of five 2-l flasks immersed in a water bath with open circulation and controlled temperature, gentle aeration and a natural photoperiod. The temperature and salinity were the same as during the incubation period. The initial density was 60 larvae-l-‘. The literature indicates that the natural food of larval clupeoids consist of diatoms and zooplankton (Hunter, 1976). In this work, the first food used was the phytoplanktonic alga Zsochrysisgdbana and the rotifer Brachionusplicatilis (50:/, < 175 pm and previously fed for 24 h on the same microalga) at an initial concentration of 20 rotifers * ml- ‘. I. gulbana is an alga used often in marine fish cultivation on account of its high content of poly-unsaturated fatty acids, series o 3, which are of fundamental importance to salt water fish (Owen et al., 1975; Watanabe, 1982). The bottoms of the tanks were cleaned daily by siphoning and the volume removed replaced with I. gulbuna culture. Daily measurements were made of the numbers of rotifers, temperature and salinity.
RESULTS
AND DISCUSSION
The first sardines captured in Sada (La Corufia) showed a high mortality rate. During transportation (z 150 km over 3 h), mortality was x 50%. After the remaining 173 fish had been established in the laboratory for 48 h, the mortality was 73 p/, and after 6 days
100
;; > .n 2 2
~~~.
50
.
l
.
40 M
l
L
.
1 2
30 20 1.‘
t 3 .
IO z0-
,,...~,~,.~,.,.,.U_L-L_YL.,,,,,,..,.,,,..,.,,.,,,,,,,.,., 0
1
2
3
4
5
6
7
8
9
10
I;
12
Months
Fig. 1. Mean percentage of adult sardines survived after 1 year in captivity. Points represent observed values, and regression line theoretical path calculated from first 7 months, without taking handling mortality into account. 1, survival rate after 1 month in captivity; 2, mortality caused by handling; 3, final petcent survival.
ACCLIMATIZATION
AND INDUCED SPAWNING OF SARI)Ih% P~L~H~~US
65
all were dead. The causes of this fast experience were excessive handling of the tish on board, the very long journey and the high stocking density during transport. The captures made off the Ria de Vigo in 1988 under more favourable conditions of proximity, handling and stocking during transport gave better results. The mean value of total mortality in the three tanks after 6 days was 13%, and after 1 month in the laboratory was only 17.6 % of the number initially stocked (Fig. 1). From this time until a year after the beginning of the experiment, survival was high in all cases (58,2%), with the exception of one tank which in the 8th month suffered 47.2% mortality due to a handling problem (Fig. 1). Mean survival in the three tanks was 39.5 %, a moderate value which allows sardines to be kept in captivity for experimental purposes. With respect to feeding, the sardine showed a gradual adaptation to the inert food provided. At first, the fish ate the inert aquarium food from the 3rd day after stocking, and by the end of a week had adapted perfectly to the dry 500~,umpellets. Later, as a routine, the captured fish were adapted directly to this last meal 3 days after collection. The fish already acclimatized to captivity in 1988 neither spawned spontaneously, nor in response to the hormonal induction method used after 1 month in captivity. Furthermore, the samples taken to study mat~ation (Perez et al., 1985) showed a progressive regression of the gonads (Table I).
TABLE I
Maturity of sardines analysed during fust 9 months of acclimatization to captivity. Month
Total no. of ~di~du~s
Maturity stage I 9:
24 9 6 8 6 1 1 36 25
6 3 3 4 3 1 21 8
III
II d -I 1 2 2 3 1
IV
9
rp
9
d
2
1
2 1
3 1
4 1 1
1
2 1
1
6
15 17
This gonadal regression was also evident in the negative results of the fast hormonal induction experiment in 1988, in which none of the fish which had been in captivity for 1 month spawned. In 1989, mature specimens captured in the Ria de Vigo for immediate hormonal induction gave positive results. Kuo et al. (1974) and Zohar & Gordin (1979), using mullet M@l cephalur and sea bream Spurus auratu, respectively, have already
M. OLMEDO
66
ET AL
shown that the best results with hormonal induction are obtained using females in an advanced stage of maturity with eggs whose diameter is > 600 pm. Following the scheme described above (Leong, 197 1) with two consecutive injections within 48 h, 64 fish were injected at random without distinguishing the sexes. Viable spawning was only obtained in a tank containing two females and four males. Although viable spawning was only achieved in two female sardines, it is the first time that such a result has been obtained in this species in captivity. The incubation period for embryonic development lasted 4.9-1.7 days at temperatures in the range 1 l-20 “C (Miranda et al., 1990) (Table II). These data agree with those of Blaxter (1969) who incubated sardine eggs from the natural environment, which hatched in 2-5 days at temperatures from 17 to 10 “C.
TABLE
Incubation T. (“C)
11 13 15 18 20
period
and percentage
of sardine
II
eggs hatched
Initial no. of eggs
Incubation period (h)
400 400 200 200
117 91 68 44 39
~
i
at different
experimental
No. of larvae hatched
255 229 90 115
temperatures. % hatched
~~~~~~~___~~ 63.75 51.25 45.00 57.50
Mortality was low during incubation, reaching a final mean value at hatching of 55.9%. Table II shows the percentages of eggs that hatched during incubation. A batch of only 300 larvae (3.4 mm mean length) was available for feeding tests and so this study only tested the efficacy of the classical method of using live food for the experimental cultivation of sardine larvae. These first experiments on the feeding of sardine larvae permit the following conclusions. Most of the sardine larvae, after absorbing their yolk sacs, were found to contain 1. galbana. The fact that clupeoid larvae feed first on phytoplankton is already known from the literature (Hunter, 1976; Varela et al., in press). After 5 days, the larvae ate rotifer eggs and small rotifers which had previously been enriched for 24 h with I. galbana. Hunter (1976) used the same system for anchovy, and enriched the rotifers with Nunnochloris sp. Due to the scarcity of larvae available, it was not possible to test the next step of feeding using Arfemiu nauplii. In conclusion, despite the fact that the results of this experiment must be considered preliminary, it can be concluded that sardine larvae obtained in captivity show a positive response to a feeding regime based on I. galbana and enriched B. plicatilis.
ACCLIMATIZATION
AND INDUCED SPAWNING OF SARBINA PILCHARDUS
67
We particularly thank Nelida Perez of the Spanish Institute of Oceanography, Vigo, for her cooperation in the development of this work. We would also like to thank the collaboration of Alexandra Almeida e Silva of Instituto National de Investigaqao das Pescas, Lisboa, Portugal.
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Kuo, C. M., C. E. Nash L Z. Shehadeh, 1974. A procedural guide to induce spawuing in grey mullet (Mugil cephalus L. ). Aquaculture, Vol. 5, pp. 19-29. Lasker, R., 1978. Relation between oceanographic conditions and larval anchovy food in the California current: identification of factors contributing to recruitment failure. Rapp. V. R&n. Int. ExpZor. Mer, Vol. 173, pp. 212-230. Leong, R., 1971. Induced spawning of the northern anchovy Engruulir mordax Giaud. Fish. Bull. NOAA, Vol. 69, pp. 357-360. Miranda, A., R. M. Cal & J. Iglesias, 1990. Effect of temperature on the development of eggs and larvae of sardine Sardina pil~ard~ Walbaum in captivity. J. Exp. h&r. Bioi. EcoL, Vof. 140, pp. 69-77. Muzinic, R., 1966. Mortal&C initiale de la sardine dans les ~n~tions ex~~rnent~es et mortalite due au marquage. Gen. Fish. Count. Meditetr. Stud. Rev., Vol. 31, pp. l-20. Muzinic, R., 1977.On the shoaling behaviour of sardines (Sardinapilchurdus)in aquaria.J. Cons. Int. Explar. Mer, Vol. 37, pp. 147-155. Owen, J. M., J. W. Adron, C. Middleton & C. B. Cowey, 1975. Elongation and desaturation of dietary fatty acids in turbot Scophthalmw maximus L. and rainbow trout, Salmo gairdneri Rich. Lipids, Vol. 10, pp. 528-531. Perez, N., C. Porteiro & F. Alvarez, 1985. Contribucidn al conocimiento de la biologia de la sardina de Galicia. Bol. Inst. Esp. Oceanogr., Vol. 2, pp. 27-37. Varela, M., A. Larranaga, E. Costas & B. Rodriguez, in press. Contenido estomacal de la sardina (Sardina pikhardus Walbaum) durante la campafla Saracus 1987 en las plataformas cantabrica y de Galicia en Febrero de 1987. Bol. inst. Esp. Oceanogr. Watanabe, T., 1982. Lipid nutrition in fish. Comp. Biochem. Physiol., Vol. 738, pp. 3-15. Zohar, Y. & H. Gordin, 1979. Spawning kinetics in the gilthead sea-bream (Sparus ouratu L.) after low doses of human chorionic gonadotropin. J. Fish. Biol., Vol. 15, pp. 665-670.