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An automated system for egg collection, hatching, and transfer of larvae in a freshwater finfish hatchery
Aquaculture 182 Ž2000. 137–148 www.elsevier.nlrlocateraqua-online
An automated system for egg collection, hatching, and transfer of larvae in a freshwater finfish hatchery Fu-Guang Liu a,b,) , Tain-Sheng Lin c , Der-Uei Huang c , Meei-Ling Perng c , I Chiu Liao b a
c
Institute of Zoology, National Taiwan UniÕersity, Taipei 107, Taiwan b Taiwan Fisheries Research Institute, Keelung 202, Taiwan Chupei Branch, Taiwan Fisheries Research Institute, Chupei 302, Taiwan Accepted 7 July 1999
Abstract An automated system for egg collection, hatching, and transfer of larvae of several cultured freshwater finfish has been developed. The system consists of a spawning tank, a filter, an incubation tank, hatching nets, and larviculture ponds. Using this system, grass carp Ž Ctenopharyngodon idellus ., bighead carp Ž Aristichtys nobilis ., silver perch Ž Bidyanus bidyanus ., palmetto bass Ž Morone saxatilis= M. chrysops . and black carp Ž Mylopharyngodon piceus . were successfully induced to spawn with a single hormone injection ŽLHRH-a at 15 mgrkg for grass carp and bighead carp; HCG at 200, 400 and 600 IUrkg for silver perch, palmetto bass and black carp, respectively.. Using this system, fertilized eggs flowed freely into nets for normal hatching, and the hatched larvae were conveyed automatically into larviculture ponds. The new automated system reduces labor. It achieved relatively high rates of spawning, fertilization and hatching. The filtration and reuse of culture water conserves water. This system offers an improvement to the existing finfish hatchery technology. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Spawning; Eggs; Incubation; Hatchery; Automated
1. Introduction Three approaches are used for finfish seed production. They are natural, induced and strip spawning. In natural spawning, fish mature and spontaneously spawn in their )
Corresponding author. Taiwan Fisheries Research Institute, 199 Hou-Ih Road, Keelung 202, Taiwan. Tel.: q886-2-24622101; fax: q886-2-24629388 0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 5 5 - 0
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natural habitat or in captivity. In induced spawning, either wild or captive broodstocks are induced to spawn through the use of a hormone or without hormone treatment but with environmental manipulation. The strip method is the manual stripping of eggs and sperm, and artificial fertilization, after hormonal treatment. Natural or induced spawning dominates the fish hatchery industry, as it is more effective than the strip method ŽJuario et al., 1984; Watson, 1987; Liao, 1993a.. In Taiwan, reproduction of the major freshwater cultured finfish, i.e., Chinese carps, such as grass carp Ž Ctenopharyngodon idellu., bighead carp Ž Aristichthys nobilis . and black carp Ž Mylopharyngodon piceus ., has for a long time followed the traditional strip method. It is time-consuming and relatively ineffective, and mortality of spawners often occurs, particularly with the large black carp ŽLiu et al., unpubl. data.. Although the induced spawning method has been used in most hatcheries in Taiwan and other countries, transporting fertilized eggs and hatched larvae by manual labor remains a bottleneck in larval fish production ŽLin et al., 1986; Leu, 1994.. We, therefore, conducted a study to improve the efficiency in hatcheries through the use of automation in egg collection and incubation, and transfer of larvae. In 1993, a prototype design for automation in egg collection and hatching was constructed and the first successful induced tank spawning of grass carp and bighead carp was obtained in Taiwan ŽLiao, 1996.. In 1994, the same facility was used successfully for induced tank spawning, egg collection, hatching and larval rearing of silver perch Ž Bidyanus bidyanus .. In March 1995, the first successful induced spawning of palmetto bass Ž Morone saxatilis= M. chrysops . in Taiwan was also achieved using the system ŽLiu et al., 1998.. Thereafter, the system was further improved in May 1995. In April 1998, the first case of induced spawning and automatic egg collecting and hatching of black carp was also recorded in Taiwan through the use of this facility. This paper describes this automated system for egg collection and hatching and for transfer of larvae.
2. Materials and methods Components of the automated system included broodstock, spawning tank, incubation tank, hatching nets, and biological filter. 2.1. Broodstock Broodstock were cultured in the freshwater ponds at Chupei Branch, Taiwan Fisheries Research Institute. They consisted of the following: 1. Grass carp: age 3–5 years, weight 3–4 kg. 2. Bighead carp: age 2–3 years, weight 2–4 kg. 3. Silver perch: age 2–3 years, females and males weighing from 0.5 to 0.9 kg and from 0.4 to 0.6 kg, respectively. 4. Palmetto bass: age 2–3 years, females and males weighing from 0.8 to 1.2 kg and from 0.5 to 0.7 kg, respectively.
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5. Black carp: age 6–10 years, females and males weighing 18–24 kg and 15–18 kg, respectively. 2.2. Spawning tank The tank is an octagonal cement tank previously used to raise ayu fish Ž Plecoglossus altiÕelis . and has a bottom area of approximately 140 m2 . The bottom has a 5–10% slope from the sides to the two center drains. One drain serves as a discharge port where a 6-cm diameter underground plastic tube can be attached to facilitate automatic transfer of sinking fertilized eggs, while the other serves as an overflow port where a vertical plastic tube can be inserted. Four inlet waterpipes are mounted on the upper sides of four tank walls. The ports of the pipes are flattened to allow for water be supplied in a jet flow, to cause a rotational movement to the water. 2.3. Incubation tank and hatching nets The incubation tank is rectangular in shape Ž10 = 2.6 = 0.9 m. and is constructed of concrete. The bottom of this tank is 1 m deeper into the ground than the bottom of the
Fig. 1. Schematic diagram of the automated system for egg collection, hatching and larval transfer for some freshwater cultured finfish.
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spawning tank. A tube from the spawning tank runs through the center of the incubation tank at the bottom with 20 manifolds branching out sideways ŽFig. 1.. Each manifold has pipes pointed upward which connect to a round hatching net, mesh size 1.0 mm, diameter 67 cm = 50 cm depth ŽFig. 2.. These hatching nets are used for grass carp, bighead carp and black carp, because the diameters of water-hardened eggs of these species are 5.1, 4.5 and 5.4 mm, respectively. However, those of the silver perch and palmetto bass are 1.8 and 1.0 mm, respectively. In order to incubate these eggs, the hatching net was modified in May 1995 to have a mesh size of 0.4 mm and diameter of 58 cm. Moreover, the funnel slope was changed from 40 to 458. In addition, underneath the inlet port of each hatching net, an aeration hole was created to facilitate flow of air ŽFig. 2.. 2.4. Biological filter A filter Ž3.4 = 2.1 = 1.4 m. built of concrete was constructed outside and above the spawning tank ŽFig. 1.. It consists of a simple pilot filter Žfrom the bottom to the top, the filter is filled with large stones, small stones, pebbles and brush. and a sedimentation pool. Water was recirculated from the incubation tank through the biofilter to the spawning tank.
Fig. 2. Schematic shows the characteristic of hatching nets used for some freshwater fish.
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2.5. Induced spawning and strip method For spawning tests, grass carp and bighead carp were given hormonal treatments at 2000–2100 h, while other species were injected at 1600–1700 h. 2.5.1. Grass carp and bighead carp Five experiments on induced spawning were conducted between April and July 1993 and in June 1994. The females were injected once with LHRH-a ŽSigma, USA. at 15 mgrkg body weight, along with one carp pituitary gland. The males were injected only with half the LHRH-a dosage. 2.5.2. SilÕer perch Two induced spawning experiments were carried out between May and July 1994, with six additional experiments conducted between May and August 1995. Two tests of strip method were also performed during this period. The females were induced with a single injection of HCG ŽChina Chemical and Pharmaceutical, Taiwan. at 200 IUrkg body weight or LHRH-a at 20 mgrkg body weight, whereas the males were administered half these doses. 2.5.3. Palmetto bass Two tests of both induced strip method and induced spawning were performed between March and April 1995. The females were injected with HCG at 300 IUrkg body weight, and the males were injected with half this dosage. 2.5.4. Black carp In April 1998, two experiments on induced spawning were carried out. The females were injected once with a combination of HCG Ž600 IUrkg body weight. and fresh carp pituitary gland Žweight of donor fish: weight of recipient fish is administrated on a 1:2 basis.. The males were injected with half of HCG dose. 2.6. Spawning and automatic egg collection and hatching After hormone treatment, the brooders were transported to the spawning tank. The directional water jets produced a water current that concentrated eggs at the opening of the underground egg transfer tube. Due to the difference in water level, fertilized eggs flowed freely through the underground plastic tube into the nets and were kept in motion for normal hatching. The total numbers of eggs, fertilization rate and hatching rate in each net were calculated. The spawned females were identified by the slight hyperemia of the genital papillae and by the differences in body weight before and after spawning. Detecting spawning in males is less reliable and data are not presented for males. 2.7. Comparing percentage of eggs collected in each hatching net In each of the five experiments conducted on silver perch in 1995, post spawning samples were taken from the 10 hatching nets on each side of the tank. Samples were
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repeatedly collected from five points in each net Žwith one sample taken at the central area and four samples taken at the rim area, each sample amounting to 250 ml.. The number of eggs in each net was estimated volumetrically, and the percentage of eggs collected in each net after each spawning was calculated. 2.8. Automatic transfer of the larÕae In July 1995, the hatchery system was improved by extending the tube from the egg incubation nets to the larviculture ponds. This allowed automatic transfer of larvae. The larvae of silver perch hatched on July 6, 1995 were incubated in the nets for about 5 days until absorption of the yolk sac was completed. At this time, automatic transfer of the free swimming larvae began. First, the master valve to the spawning tank tubeway was turned off; then, all the inlet ports of the nets were closed, the tubeway was opened for connection to the larviculture pond. At this point, the valve for inlet ports of the right side nets could then be slowly opened. Due to the difference in water level between spawning tank and incubation tank as well as between incubation tank and larviculture pond, water carrying the larvae flowed out naturally and ran through the tubeway into the larviculture pond, which contained food organisms. The surface of the pond had to first be equalized to the level of the larvae transfer port in case the flushing water cause physical damage to the larvae. The same procedure was also performed for the nets on the left side to bring the larvae to the other larviculture pond. 3. Results 3.1. Spawnings In 1993 and 1994, three experiments were conducted with a total of 18 female and 27 male grass carp given hormonal injections. After approximately 10–12 h at water temperature of 23–268C, 12 females spawned providing a mean spawning rate of 67% ŽTable 1.. In June of 1994, a total of 9 female and 12 male bighead carp were hormone treated at water temperature of 23–258C and about 10–12 h later, six females spawned, yielding a mean spawning rate of about 67%. In April 1998, 5 female and 11 male black carp were induced to spawn after 11–13 h at water temperature of 23–258C with a spawning rate of 100%. Fertilization and hatching rates ranged were high for the black carp ŽTable 1.. In May 1994, two experiments were conducted with 15 female and 23 male hormone treated silver perch. After about 33–36 h at a water temperature of 28–31.58C, 14 females were successfully induced to spawn with a mean spawning rate of 93% and fertilization rate of 75%. The original system was modified in May 1995. Six induced spawning experiments were performed between May and August using silver perch. A total of 46 out of 65 females spawned, with an average spawning rate of 71%, fertilization rates of 73% and hatching rate of 52%. Two experiments with the induced strip method were also conducted with 10 female and 9 male silver perch. Silver perch showed a range of 20–75% in spawning rate, 38–73% in fertilization rate, and 0–15% in hatching rate ŽTable 1..
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Table 1 Experimental results of induced tank spawning and the automation in egg transfer and hatching of five freshwater cultured finfish Date
Species
No. of female injected
No. of fish spawned
Spawning success Ž%.
Fertilization rate Ž%.
Hatching rate Ž%.
April 30, 1993 July 2 June 2 June 5 June 22, 1994 May 12 July 14 March 9, 1995 April 11 May 24 May 30 June 5 July 6 July 17 August 8 April 13, 1998 April 29
Grass carp
6 4 4 5 8 10 5 6 10 10 10 11 10 11 13 2 3
4 2 2 4 6 9 5 6 10 9 9 5 5 8 10 5 6
67 50 50 80 75 90 100 100 100 90 90 45 50 73 77 100 100
87.2"2.2 89.2"1.5 90.5"2.3 86.4"1.2 85.3"1.6 80.7"1.1 69.1"0.9 91.9"3.1 82.4"1.3 75.9"1.0 82.2"1.2 81.8"1.4 87.3"2.1 60.5"1.1 52.8"0.8 83.6"1.7 87.8"2.2
– 35.6"0.8 65.3"1.1 – 30.7"0.9 11.5"0.5 – – – 50.8"1.2 62.0"0.9 78.6"1.5 33.6"1.3 51.7"0.8 35.6"0.7 75.1"1.6 78.4"1.3
Bighead carp Grass carp Silver perch Palmetto bass Silver perch
Black carp
In March 1995, an induced spawning experiment with 6 female and 12 male palmetto bass was conducted. After 26–32 h at 21–238C water temperature, all six females spawned, showing a 100% spawning rate and an 82% fertilization rate. This achievement was the first success toward reproduction of the second generation of palmetto bass in Taiwan. A second experiment was performed in April with 10 female and 12 male. All 10 females spawned, resulting in a spawning rate of 100% and a fertilization rate of 92%. Unfortunately, though, all fertilized eggs in both cases failed to hatch ŽTable 1.. Two trials with the induced strip method were also carried out with eight females and five males. Spawning rate was 50 and 70%, fertilization rate was 68 and 92%, and hatching rate was 0 and 17%. 3.2. Comparison of egg distribution in different nets Nets either closer to or distant from the spawning pond contained slightly more eggs than the others. Data on number of eggs collected from 17 spawns from various fish were analyzed and there were no significant differences in the number of eggs collected according to net location Že.g., silver perch spawned in July 1995 s 34,700 " 3200 eggsrnet, n s 20.. 3.3. LarÕal transportation After a 5-day incubation, the number of silver perch larvae hatched in July 1995 was estimated to be approximately 174,100. The larvae were automatically transferred and
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distributed evenly in the two larviculture ponds based on the different water levels. After 1 1r2 months, 36,500 and 39,500 juveniles were harvested in the two ponds, indicating survival of 41% and 45%, respectively.
4. Discussion There were high spawning, fertilization and hatching rates with our induced spawning approach compared with the traditional strip method for all species, with the exception of the hatching rate for palmetto bass ŽTable 2.. The automated system for egg transfer, hatching, and larval transportation in our finfish hatchery is superior to conventional artificial propagation ŽTable 3.. It is clear that the automated system has some favorable attributes such as higher egg production and hatching rates, higher survival of spawners, labor savings, and water-conservation through recirculation. The collection and transfer of eggs is labor intensive ŽKumarasiri and Seneviratne, 1988; Chang and Hsueh, 1989.. However, our system avoids a manual transfer procedure, as fertilized eggs flow automatically into the incubation nets, thus achieving substantial saving of time and labor. Juario et al. Ž1984. reported that the resultant fertilization of strip method in milkfish is relatively low. It has also been demonstrated by Watson Ž1987. that a lower fertilization rate was obtained with hand-stripped batches of Atlantic tomcod Ž Microgadus tomcod .. Liao Ž1993a; b. pointed out that induced spawning involves the use of well-balanced and sound approaches to hatchery practice. It has several advantages, such as spawner-saving, water-saving, with high fertilization and hatching rates. Kuo Ž1995. also concluded that the induced spawning method has a consistently high fertilization rate and, more importantly, reduces damage to broodstock. Although both silver perch Žas of 1994. and palmetto bass Žas of 1995. had been successfully induced to spawn using the system, the hatching rate was very low or virtually nil. Using grass carp hatching nets, the small size of eggs or newly hatched larvae of silver perch and palmetto bass were easily blocked and injured when they passed through the large mesh. In addition, inadequate flotation together with insufficient angle of the funnel web base kept eggs from rolling sufficiently and eventually caused mass mortality of eggs. For this reason, we changed the mesh size to 0.4 mm,
Table 2 Spawning efficiency of induced strip method ŽSM. and induced spawning ŽIS. in some freshwater cultured finfish. All data are presented as percentage Ž%. Grass carp or bighead carp
Spawning success Fertilization rate Hatching rate a
Silver perch
Palmetto bass
SM a
IS
SM
IS
SM
IS
11–57 10–95 5–65
50–80 85–90 36–65
20–75 38–73 0–15
45–100 53–82 34–79
50–70 68–92 0–17
100 82–92 –
According to the results of some researchers ŽLiu, 1964; Tang et al., 1964; Kuo, 1965..
Hatchery parameter
Conventional method
New system
Maturity checking interval Spawning success rate Fertilization rate Hatching rate Fertilization and incubation
Broodstock health after stripping Žspawning.
periodic checking Low Low Low Induced stripped method; fertilized eggs are washed and transferred manually to incubator Larvae are manually handled and shipped to larviculture ponds Serious injury, sometimes causing death
Water system used for incubation
Flow through
No need for checking ŽAutomated. High ŽEfficient. High ŽEfficient. High ŽEfficient. Induced spawning, fertilized eggs are conveyed automatically to incubator Žautomated, labor-saving. Larvae are conveyed automatically to larviculture ponds Žautomated, labor-saving. Broodstock could be used several times due to reduced injures Žbroodstock-saving. Recirculated water Žwater-saving.
Larval transportation for stocking
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Table 3 Comparison of conventional artificial propagation techniques with the new system for Chinese carps in egg transfer, hatching and larval transportation
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and increased the angle of the funnel web to 458. We also installed an additional aeration hole under the inlet port of each net in order that aeration would help to enhance egg rolling. Thus, even if the water circulation stopped, for one reason or another, the eggs would keep rolling to prevent them from dying. After this change, from May 1995 onwards, the hatching rate of silver perch in spawning experiments increased to between 34 and 79%. There have been studies on egg incubation system for various fish species with respect to egg characteristics: such as the buoyant eggs of certain marine fish like sea bream Ž Sparus aurata., sea bass Ž Dicentrarchus labrax . and sable fish Ž Anoplopome fimbria. ŽDevauchelle et al., 1986; Alderdice et al., 1988.; and the large, demersal eggs of salmon, walleye Ž Stizostedion Õitreum. and trout Ž Oncorhynchus mykiss . ŽMiller, 1992; Blacklidge and Bidwell, 1993; Tabachek et al., 1993.. These studies utilized small-scale, indoor designs, which involved mostly mechanical and power-driven structures. These systems are therefore more energy-consuming than our system. Moreover, due to costly materials used, they incurred higher initial costs. Our system is an important breakthrough for large-scale, outdoor use and includes savings in labor, water, and cost. There has been a successful artificial propagation of silver perch in Australia ŽRowland et al., 1983; Rowland, 1984.. However, the manual transfer of fertilized eggs from indoor induced spawning into an aquarium or circular tank for aerated incubation is less efficient than this system. Likewise, the seed production technology striped bass has been established for years in USA, either using the strip or induced spawning method. The fertilized eggs must be reaped manually for transfer to indoor incubation flasks or tanks for aerated incubation with flowing water ŽHenderson-Arzapalo and Colura, 1987; Harrell et al., 1990.. Similar egg collection methods for manual transfer to incubation tanks are used for sole Ž Solea solea., turbot Ž Scophthalmus maximus . and milkfish Ž Chanos chanos . ŽDevauchelle et al., 1987, 1988; Garcia et al., 1988; Marte, 1988.. Thus, it is clear that no design similar to ours for automatic egg transfer for incubation is available outside Taiwan. An additional advantage of this system lies in the formation of two pipeways down the center of the spawning tank, with one outlet port in the bottom fit for the collection and transfer of demersal or semi-pelagic nonadhesive eggs, such as the species used in this study. The same system may use an overflow pipe Žthe outlet port can be adjusted according to the water level. with adaptable whirlpool flow to convey and collect buoyant eggs from marine fish such as red seabream Ž Pagrus major ., grouper Ž Epinephelus tauÕina. and grey mullet Ž Migil cephalus .. So, this design should make it possible for the seed production of marine species to be more automated and labor-saving than ever before. The system reduces handling stress and mortality by the automatic transfer of free swimming larvae from the hatching nets to the larviculture ponds. This is a distinct improvement over systems which require manual collection and transfer of larvae to larviculture ponds ŽEmata et al., 1994; Fermamdez-Palacios et al., 1994.. In conclusion, the designed system has a number of advantages over existing systems. It is hoped that the use of the automated system described here will substantially benefit the hatchery industry and accelerate development of automation techniques for mass seed production of freshwater finfish and even for marine species.
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Acknowledgements F.-G. Liu wishes to thank Dr. H.C. Chen, Professor of Institute of Zoology, National Taiwan University, for his valuable advice and discussion. We would like to thank Mr. C.F. Huang, S.C. Jen and all technicians of Chupei Branch, Taiwan Fisheries Research Institute, for their technical assistance. We are also grateful to Miss Y.H. Hsieh, for her help in the manuscript preparation. The patent of automated system described in this study has been granted by the National Bureau of Standards, Ministry of Economic Affairs, R.O.C., with the patent number: New Invention No. 080097 and patent period: From Aug. 1, 1996 to Oct. 29, 2015.
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An automated system for egg collection, hatching, and transfer of larvae in a freshwater finfish hatchery Fu-Guang Liu a,b,) , Tain-Sheng Lin c , Der-Uei Huang c , Meei-Ling Perng c , I Chiu Liao b a
c
Institute of Zoology, National Taiwan UniÕersity, Taipei 107, Taiwan b Taiwan Fisheries Research Institute, Keelung 202, Taiwan Chupei Branch, Taiwan Fisheries Research Institute, Chupei 302, Taiwan Accepted 7 July 1999
Abstract An automated system for egg collection, hatching, and transfer of larvae of several cultured freshwater finfish has been developed. The system consists of a spawning tank, a filter, an incubation tank, hatching nets, and larviculture ponds. Using this system, grass carp Ž Ctenopharyngodon idellus ., bighead carp Ž Aristichtys nobilis ., silver perch Ž Bidyanus bidyanus ., palmetto bass Ž Morone saxatilis= M. chrysops . and black carp Ž Mylopharyngodon piceus . were successfully induced to spawn with a single hormone injection ŽLHRH-a at 15 mgrkg for grass carp and bighead carp; HCG at 200, 400 and 600 IUrkg for silver perch, palmetto bass and black carp, respectively.. Using this system, fertilized eggs flowed freely into nets for normal hatching, and the hatched larvae were conveyed automatically into larviculture ponds. The new automated system reduces labor. It achieved relatively high rates of spawning, fertilization and hatching. The filtration and reuse of culture water conserves water. This system offers an improvement to the existing finfish hatchery technology. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Spawning; Eggs; Incubation; Hatchery; Automated
1. Introduction Three approaches are used for finfish seed production. They are natural, induced and strip spawning. In natural spawning, fish mature and spontaneously spawn in their )
Corresponding author. Taiwan Fisheries Research Institute, 199 Hou-Ih Road, Keelung 202, Taiwan. Tel.: q886-2-24622101; fax: q886-2-24629388 0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 5 5 - 0
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natural habitat or in captivity. In induced spawning, either wild or captive broodstocks are induced to spawn through the use of a hormone or without hormone treatment but with environmental manipulation. The strip method is the manual stripping of eggs and sperm, and artificial fertilization, after hormonal treatment. Natural or induced spawning dominates the fish hatchery industry, as it is more effective than the strip method ŽJuario et al., 1984; Watson, 1987; Liao, 1993a.. In Taiwan, reproduction of the major freshwater cultured finfish, i.e., Chinese carps, such as grass carp Ž Ctenopharyngodon idellu., bighead carp Ž Aristichthys nobilis . and black carp Ž Mylopharyngodon piceus ., has for a long time followed the traditional strip method. It is time-consuming and relatively ineffective, and mortality of spawners often occurs, particularly with the large black carp ŽLiu et al., unpubl. data.. Although the induced spawning method has been used in most hatcheries in Taiwan and other countries, transporting fertilized eggs and hatched larvae by manual labor remains a bottleneck in larval fish production ŽLin et al., 1986; Leu, 1994.. We, therefore, conducted a study to improve the efficiency in hatcheries through the use of automation in egg collection and incubation, and transfer of larvae. In 1993, a prototype design for automation in egg collection and hatching was constructed and the first successful induced tank spawning of grass carp and bighead carp was obtained in Taiwan ŽLiao, 1996.. In 1994, the same facility was used successfully for induced tank spawning, egg collection, hatching and larval rearing of silver perch Ž Bidyanus bidyanus .. In March 1995, the first successful induced spawning of palmetto bass Ž Morone saxatilis= M. chrysops . in Taiwan was also achieved using the system ŽLiu et al., 1998.. Thereafter, the system was further improved in May 1995. In April 1998, the first case of induced spawning and automatic egg collecting and hatching of black carp was also recorded in Taiwan through the use of this facility. This paper describes this automated system for egg collection and hatching and for transfer of larvae.
2. Materials and methods Components of the automated system included broodstock, spawning tank, incubation tank, hatching nets, and biological filter. 2.1. Broodstock Broodstock were cultured in the freshwater ponds at Chupei Branch, Taiwan Fisheries Research Institute. They consisted of the following: 1. Grass carp: age 3–5 years, weight 3–4 kg. 2. Bighead carp: age 2–3 years, weight 2–4 kg. 3. Silver perch: age 2–3 years, females and males weighing from 0.5 to 0.9 kg and from 0.4 to 0.6 kg, respectively. 4. Palmetto bass: age 2–3 years, females and males weighing from 0.8 to 1.2 kg and from 0.5 to 0.7 kg, respectively.
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5. Black carp: age 6–10 years, females and males weighing 18–24 kg and 15–18 kg, respectively. 2.2. Spawning tank The tank is an octagonal cement tank previously used to raise ayu fish Ž Plecoglossus altiÕelis . and has a bottom area of approximately 140 m2 . The bottom has a 5–10% slope from the sides to the two center drains. One drain serves as a discharge port where a 6-cm diameter underground plastic tube can be attached to facilitate automatic transfer of sinking fertilized eggs, while the other serves as an overflow port where a vertical plastic tube can be inserted. Four inlet waterpipes are mounted on the upper sides of four tank walls. The ports of the pipes are flattened to allow for water be supplied in a jet flow, to cause a rotational movement to the water. 2.3. Incubation tank and hatching nets The incubation tank is rectangular in shape Ž10 = 2.6 = 0.9 m. and is constructed of concrete. The bottom of this tank is 1 m deeper into the ground than the bottom of the
Fig. 1. Schematic diagram of the automated system for egg collection, hatching and larval transfer for some freshwater cultured finfish.
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spawning tank. A tube from the spawning tank runs through the center of the incubation tank at the bottom with 20 manifolds branching out sideways ŽFig. 1.. Each manifold has pipes pointed upward which connect to a round hatching net, mesh size 1.0 mm, diameter 67 cm = 50 cm depth ŽFig. 2.. These hatching nets are used for grass carp, bighead carp and black carp, because the diameters of water-hardened eggs of these species are 5.1, 4.5 and 5.4 mm, respectively. However, those of the silver perch and palmetto bass are 1.8 and 1.0 mm, respectively. In order to incubate these eggs, the hatching net was modified in May 1995 to have a mesh size of 0.4 mm and diameter of 58 cm. Moreover, the funnel slope was changed from 40 to 458. In addition, underneath the inlet port of each hatching net, an aeration hole was created to facilitate flow of air ŽFig. 2.. 2.4. Biological filter A filter Ž3.4 = 2.1 = 1.4 m. built of concrete was constructed outside and above the spawning tank ŽFig. 1.. It consists of a simple pilot filter Žfrom the bottom to the top, the filter is filled with large stones, small stones, pebbles and brush. and a sedimentation pool. Water was recirculated from the incubation tank through the biofilter to the spawning tank.
Fig. 2. Schematic shows the characteristic of hatching nets used for some freshwater fish.
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2.5. Induced spawning and strip method For spawning tests, grass carp and bighead carp were given hormonal treatments at 2000–2100 h, while other species were injected at 1600–1700 h. 2.5.1. Grass carp and bighead carp Five experiments on induced spawning were conducted between April and July 1993 and in June 1994. The females were injected once with LHRH-a ŽSigma, USA. at 15 mgrkg body weight, along with one carp pituitary gland. The males were injected only with half the LHRH-a dosage. 2.5.2. SilÕer perch Two induced spawning experiments were carried out between May and July 1994, with six additional experiments conducted between May and August 1995. Two tests of strip method were also performed during this period. The females were induced with a single injection of HCG ŽChina Chemical and Pharmaceutical, Taiwan. at 200 IUrkg body weight or LHRH-a at 20 mgrkg body weight, whereas the males were administered half these doses. 2.5.3. Palmetto bass Two tests of both induced strip method and induced spawning were performed between March and April 1995. The females were injected with HCG at 300 IUrkg body weight, and the males were injected with half this dosage. 2.5.4. Black carp In April 1998, two experiments on induced spawning were carried out. The females were injected once with a combination of HCG Ž600 IUrkg body weight. and fresh carp pituitary gland Žweight of donor fish: weight of recipient fish is administrated on a 1:2 basis.. The males were injected with half of HCG dose. 2.6. Spawning and automatic egg collection and hatching After hormone treatment, the brooders were transported to the spawning tank. The directional water jets produced a water current that concentrated eggs at the opening of the underground egg transfer tube. Due to the difference in water level, fertilized eggs flowed freely through the underground plastic tube into the nets and were kept in motion for normal hatching. The total numbers of eggs, fertilization rate and hatching rate in each net were calculated. The spawned females were identified by the slight hyperemia of the genital papillae and by the differences in body weight before and after spawning. Detecting spawning in males is less reliable and data are not presented for males. 2.7. Comparing percentage of eggs collected in each hatching net In each of the five experiments conducted on silver perch in 1995, post spawning samples were taken from the 10 hatching nets on each side of the tank. Samples were
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repeatedly collected from five points in each net Žwith one sample taken at the central area and four samples taken at the rim area, each sample amounting to 250 ml.. The number of eggs in each net was estimated volumetrically, and the percentage of eggs collected in each net after each spawning was calculated. 2.8. Automatic transfer of the larÕae In July 1995, the hatchery system was improved by extending the tube from the egg incubation nets to the larviculture ponds. This allowed automatic transfer of larvae. The larvae of silver perch hatched on July 6, 1995 were incubated in the nets for about 5 days until absorption of the yolk sac was completed. At this time, automatic transfer of the free swimming larvae began. First, the master valve to the spawning tank tubeway was turned off; then, all the inlet ports of the nets were closed, the tubeway was opened for connection to the larviculture pond. At this point, the valve for inlet ports of the right side nets could then be slowly opened. Due to the difference in water level between spawning tank and incubation tank as well as between incubation tank and larviculture pond, water carrying the larvae flowed out naturally and ran through the tubeway into the larviculture pond, which contained food organisms. The surface of the pond had to first be equalized to the level of the larvae transfer port in case the flushing water cause physical damage to the larvae. The same procedure was also performed for the nets on the left side to bring the larvae to the other larviculture pond. 3. Results 3.1. Spawnings In 1993 and 1994, three experiments were conducted with a total of 18 female and 27 male grass carp given hormonal injections. After approximately 10–12 h at water temperature of 23–268C, 12 females spawned providing a mean spawning rate of 67% ŽTable 1.. In June of 1994, a total of 9 female and 12 male bighead carp were hormone treated at water temperature of 23–258C and about 10–12 h later, six females spawned, yielding a mean spawning rate of about 67%. In April 1998, 5 female and 11 male black carp were induced to spawn after 11–13 h at water temperature of 23–258C with a spawning rate of 100%. Fertilization and hatching rates ranged were high for the black carp ŽTable 1.. In May 1994, two experiments were conducted with 15 female and 23 male hormone treated silver perch. After about 33–36 h at a water temperature of 28–31.58C, 14 females were successfully induced to spawn with a mean spawning rate of 93% and fertilization rate of 75%. The original system was modified in May 1995. Six induced spawning experiments were performed between May and August using silver perch. A total of 46 out of 65 females spawned, with an average spawning rate of 71%, fertilization rates of 73% and hatching rate of 52%. Two experiments with the induced strip method were also conducted with 10 female and 9 male silver perch. Silver perch showed a range of 20–75% in spawning rate, 38–73% in fertilization rate, and 0–15% in hatching rate ŽTable 1..
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Table 1 Experimental results of induced tank spawning and the automation in egg transfer and hatching of five freshwater cultured finfish Date
Species
No. of female injected
No. of fish spawned
Spawning success Ž%.
Fertilization rate Ž%.
Hatching rate Ž%.
April 30, 1993 July 2 June 2 June 5 June 22, 1994 May 12 July 14 March 9, 1995 April 11 May 24 May 30 June 5 July 6 July 17 August 8 April 13, 1998 April 29
Grass carp
6 4 4 5 8 10 5 6 10 10 10 11 10 11 13 2 3
4 2 2 4 6 9 5 6 10 9 9 5 5 8 10 5 6
67 50 50 80 75 90 100 100 100 90 90 45 50 73 77 100 100
87.2"2.2 89.2"1.5 90.5"2.3 86.4"1.2 85.3"1.6 80.7"1.1 69.1"0.9 91.9"3.1 82.4"1.3 75.9"1.0 82.2"1.2 81.8"1.4 87.3"2.1 60.5"1.1 52.8"0.8 83.6"1.7 87.8"2.2
– 35.6"0.8 65.3"1.1 – 30.7"0.9 11.5"0.5 – – – 50.8"1.2 62.0"0.9 78.6"1.5 33.6"1.3 51.7"0.8 35.6"0.7 75.1"1.6 78.4"1.3
Bighead carp Grass carp Silver perch Palmetto bass Silver perch
Black carp
In March 1995, an induced spawning experiment with 6 female and 12 male palmetto bass was conducted. After 26–32 h at 21–238C water temperature, all six females spawned, showing a 100% spawning rate and an 82% fertilization rate. This achievement was the first success toward reproduction of the second generation of palmetto bass in Taiwan. A second experiment was performed in April with 10 female and 12 male. All 10 females spawned, resulting in a spawning rate of 100% and a fertilization rate of 92%. Unfortunately, though, all fertilized eggs in both cases failed to hatch ŽTable 1.. Two trials with the induced strip method were also carried out with eight females and five males. Spawning rate was 50 and 70%, fertilization rate was 68 and 92%, and hatching rate was 0 and 17%. 3.2. Comparison of egg distribution in different nets Nets either closer to or distant from the spawning pond contained slightly more eggs than the others. Data on number of eggs collected from 17 spawns from various fish were analyzed and there were no significant differences in the number of eggs collected according to net location Že.g., silver perch spawned in July 1995 s 34,700 " 3200 eggsrnet, n s 20.. 3.3. LarÕal transportation After a 5-day incubation, the number of silver perch larvae hatched in July 1995 was estimated to be approximately 174,100. The larvae were automatically transferred and
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distributed evenly in the two larviculture ponds based on the different water levels. After 1 1r2 months, 36,500 and 39,500 juveniles were harvested in the two ponds, indicating survival of 41% and 45%, respectively.
4. Discussion There were high spawning, fertilization and hatching rates with our induced spawning approach compared with the traditional strip method for all species, with the exception of the hatching rate for palmetto bass ŽTable 2.. The automated system for egg transfer, hatching, and larval transportation in our finfish hatchery is superior to conventional artificial propagation ŽTable 3.. It is clear that the automated system has some favorable attributes such as higher egg production and hatching rates, higher survival of spawners, labor savings, and water-conservation through recirculation. The collection and transfer of eggs is labor intensive ŽKumarasiri and Seneviratne, 1988; Chang and Hsueh, 1989.. However, our system avoids a manual transfer procedure, as fertilized eggs flow automatically into the incubation nets, thus achieving substantial saving of time and labor. Juario et al. Ž1984. reported that the resultant fertilization of strip method in milkfish is relatively low. It has also been demonstrated by Watson Ž1987. that a lower fertilization rate was obtained with hand-stripped batches of Atlantic tomcod Ž Microgadus tomcod .. Liao Ž1993a; b. pointed out that induced spawning involves the use of well-balanced and sound approaches to hatchery practice. It has several advantages, such as spawner-saving, water-saving, with high fertilization and hatching rates. Kuo Ž1995. also concluded that the induced spawning method has a consistently high fertilization rate and, more importantly, reduces damage to broodstock. Although both silver perch Žas of 1994. and palmetto bass Žas of 1995. had been successfully induced to spawn using the system, the hatching rate was very low or virtually nil. Using grass carp hatching nets, the small size of eggs or newly hatched larvae of silver perch and palmetto bass were easily blocked and injured when they passed through the large mesh. In addition, inadequate flotation together with insufficient angle of the funnel web base kept eggs from rolling sufficiently and eventually caused mass mortality of eggs. For this reason, we changed the mesh size to 0.4 mm,
Table 2 Spawning efficiency of induced strip method ŽSM. and induced spawning ŽIS. in some freshwater cultured finfish. All data are presented as percentage Ž%. Grass carp or bighead carp
Spawning success Fertilization rate Hatching rate a
Silver perch
Palmetto bass
SM a
IS
SM
IS
SM
IS
11–57 10–95 5–65
50–80 85–90 36–65
20–75 38–73 0–15
45–100 53–82 34–79
50–70 68–92 0–17
100 82–92 –
According to the results of some researchers ŽLiu, 1964; Tang et al., 1964; Kuo, 1965..
Hatchery parameter
Conventional method
New system
Maturity checking interval Spawning success rate Fertilization rate Hatching rate Fertilization and incubation
Broodstock health after stripping Žspawning.
periodic checking Low Low Low Induced stripped method; fertilized eggs are washed and transferred manually to incubator Larvae are manually handled and shipped to larviculture ponds Serious injury, sometimes causing death
Water system used for incubation
Flow through
No need for checking ŽAutomated. High ŽEfficient. High ŽEfficient. High ŽEfficient. Induced spawning, fertilized eggs are conveyed automatically to incubator Žautomated, labor-saving. Larvae are conveyed automatically to larviculture ponds Žautomated, labor-saving. Broodstock could be used several times due to reduced injures Žbroodstock-saving. Recirculated water Žwater-saving.
Larval transportation for stocking
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Table 3 Comparison of conventional artificial propagation techniques with the new system for Chinese carps in egg transfer, hatching and larval transportation
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and increased the angle of the funnel web to 458. We also installed an additional aeration hole under the inlet port of each net in order that aeration would help to enhance egg rolling. Thus, even if the water circulation stopped, for one reason or another, the eggs would keep rolling to prevent them from dying. After this change, from May 1995 onwards, the hatching rate of silver perch in spawning experiments increased to between 34 and 79%. There have been studies on egg incubation system for various fish species with respect to egg characteristics: such as the buoyant eggs of certain marine fish like sea bream Ž Sparus aurata., sea bass Ž Dicentrarchus labrax . and sable fish Ž Anoplopome fimbria. ŽDevauchelle et al., 1986; Alderdice et al., 1988.; and the large, demersal eggs of salmon, walleye Ž Stizostedion Õitreum. and trout Ž Oncorhynchus mykiss . ŽMiller, 1992; Blacklidge and Bidwell, 1993; Tabachek et al., 1993.. These studies utilized small-scale, indoor designs, which involved mostly mechanical and power-driven structures. These systems are therefore more energy-consuming than our system. Moreover, due to costly materials used, they incurred higher initial costs. Our system is an important breakthrough for large-scale, outdoor use and includes savings in labor, water, and cost. There has been a successful artificial propagation of silver perch in Australia ŽRowland et al., 1983; Rowland, 1984.. However, the manual transfer of fertilized eggs from indoor induced spawning into an aquarium or circular tank for aerated incubation is less efficient than this system. Likewise, the seed production technology striped bass has been established for years in USA, either using the strip or induced spawning method. The fertilized eggs must be reaped manually for transfer to indoor incubation flasks or tanks for aerated incubation with flowing water ŽHenderson-Arzapalo and Colura, 1987; Harrell et al., 1990.. Similar egg collection methods for manual transfer to incubation tanks are used for sole Ž Solea solea., turbot Ž Scophthalmus maximus . and milkfish Ž Chanos chanos . ŽDevauchelle et al., 1987, 1988; Garcia et al., 1988; Marte, 1988.. Thus, it is clear that no design similar to ours for automatic egg transfer for incubation is available outside Taiwan. An additional advantage of this system lies in the formation of two pipeways down the center of the spawning tank, with one outlet port in the bottom fit for the collection and transfer of demersal or semi-pelagic nonadhesive eggs, such as the species used in this study. The same system may use an overflow pipe Žthe outlet port can be adjusted according to the water level. with adaptable whirlpool flow to convey and collect buoyant eggs from marine fish such as red seabream Ž Pagrus major ., grouper Ž Epinephelus tauÕina. and grey mullet Ž Migil cephalus .. So, this design should make it possible for the seed production of marine species to be more automated and labor-saving than ever before. The system reduces handling stress and mortality by the automatic transfer of free swimming larvae from the hatching nets to the larviculture ponds. This is a distinct improvement over systems which require manual collection and transfer of larvae to larviculture ponds ŽEmata et al., 1994; Fermamdez-Palacios et al., 1994.. In conclusion, the designed system has a number of advantages over existing systems. It is hoped that the use of the automated system described here will substantially benefit the hatchery industry and accelerate development of automation techniques for mass seed production of freshwater finfish and even for marine species.
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Acknowledgements F.-G. Liu wishes to thank Dr. H.C. Chen, Professor of Institute of Zoology, National Taiwan University, for his valuable advice and discussion. We would like to thank Mr. C.F. Huang, S.C. Jen and all technicians of Chupei Branch, Taiwan Fisheries Research Institute, for their technical assistance. We are also grateful to Miss Y.H. Hsieh, for her help in the manuscript preparation. The patent of automated system described in this study has been granted by the National Bureau of Standards, Ministry of Economic Affairs, R.O.C., with the patent number: New Invention No. 080097 and patent period: From Aug. 1, 1996 to Oct. 29, 2015.
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