Liu Xiyin1, Zhu Guanghui1, Zhao Qiang1,
Wang Liang1 and Gu Benxue2
1Yantai Fisheries Research Institute, Yantai, China; 2Penglai De-run Sea-Treasure Hatchery Plant, Penglai, China
In this paper, the authors give an outline of hatchery systems and breeding techniques of the sea cucumber, Apostichopus japonicus, in northern China. The methods for selection and maintenance of broodstock, for spawning induction, procedures for larval rearing and stimulation of settlement, aspects of juvenile growth and management of wintering are presented. The cause of some common diseases observed in the hatchery (such as the rotten-stomach of larva), the low success of metamorphosis and the mortality of juvenile sea cucumbers are discussed together with methods used to prevent such problems.
Keywords: Broodstock, larvae, juvenile, disease, metamorphosis, overwintering
The Chinese have been using sea cucumber as a food item and for treating illness for over a thousand years. There are about 134 species of sea cucumber distributed in Chinas seas. Among them, Apostichopus japonicus is well known as a delicacy and for its medicinal properties. According to Chinese traditional medicine, sea cucumber is a tonic food. In recent years, pharmacodynamic studies have shown that sea cucumber contains mucoitin that could retard aging and act as anti-tumour and blood coagulation agent. Hence, sea cucumber is very popular and A. japonicus is considered to be the most valued of the edible species.
In China, A. japonicus is distributed along the shore of the Yellow Sea and Bohai Sea from 34° of northern latitude, including the provinces of Liaoning, Hebei, Shandong and Jiangsu. The highest biomass is found along the shores of Dalian and Jinzhou in the Liaoning Province and off Qingdao and Yantai in the Shandong Province. The natural stocks of this species have decreased significantly, having been overfished for a long period. Therefore, the Chinese Government has been encouraging research into artificial breeding, farming and enhancement of A. japonicus since the early 1980s.
An outline of hatchery techniques development
Studies on A. japonicus hatchery techniques started in the early 1950s. By 1954, Zhang Fengying and Wu Baoling had produced a number of auriculariae and doliolariae in their laboratory. Two years later, they had managed to produce a number of juvenile sea cucumbers. During the 1960s, Chen Zongrao, who worked at the Shandong Mariculture Research Institute, used the same methods and was also able to produce juveniles. The hatchery production of the species developed rapidly during the 1970s, but it did not become a prosperous activity until the early 1980s. Since then, the Yellow Sea Fisheries Research Institute, the Liaoning Marine Fisheries Research Institute, the Shandong Mariculture Research Institute as well as the Yantai Fisheries Research Institute have successively succeeded in breeding A. japonicus, and have devised a series of protocols for hatcheries. This work has been supported by the Ministry of Agriculture.
As more and more commercial hatcheries emerged along the northern coastal line of China, the rearing techniques were widely adopted and improved during the 1990s, while the scale and dimensions of hatchery facilities for breeding larvae increased rapidly. Presently, a single harvest typically yields 5 000 juveniles (about 1 cm long) per m3 of water in commercial hatcheries, although over 10 000 juveniles per m3 can sometimes be obtained. Juvenile production of A. japonicus has now become a commercially stable activity.
An ideal hatchery should be built on the coast where the seawater is clear, unpolluted and unaffected by freshwater runoff. Hatcheries for scallop can be used for sea cucumber production, either alone or in co-culture. A typical hatchery consists of a series of interrelated systems as described below.
The hatchery is used for conditioning broodstock and rearing larvae. The inner room should be well ventilated, sunlit, but protected against direct sunlight. The illumination should be between 500-2 000 lux.
Tanks, either for rearing larvae or for spawning the broodstock, are commonly built with bricks, blocks or reinforced concrete. They are usually rectangular or elliptical in shape with a capacity of between 10 and 30 m3. Tanks of 15-25 m3 are preferred. The depth of the tanks should be about 1.2 m. The total water volume in commercial hatcheries may vary between 300 and 3 000 m3, though facilities of 500-1 000 m3 are more common.
Microalgae culture room
A microalgae culture room generally has a fibreglass or transparent plastic film roof and is oriented in an east-to-west direction. Tanks for microalgae culture are generally rectangular, 0.7-1 m in depth and with a volume of 1 to 10 m3. The bottom and inner wall of the tanks are lined with white ceramic tiles or white cement in order to reflect sunlight. An ideal microalgae culture facility is well exposed to sunlight. Commonly, the volume of water required for the culture of microalgae is in a ratio of 1:4 to 1:3 of the total hatchery.
Sea cucumber larvae / early juvenile tank(s)
The capacity of the larval culture and settlement tanks should be twice or three times that of the total hatchery water volume. A roof is also necessary to provide protection from rainwater and dust and to provide a dark environment for the sea cucumber larvae to settle on the bottom.
Seawater must be filtered through a gravel bottom filter tank before being used for larval rearing and microalgae culture. A gravel bottom filter tank should be filled with cobble, gravel and sand in that order, from bottom to top. The top layer of fine sand (particle diameter: 500 mm) should be more than 60 cm thick. Recently, some newly established hatcheries obtain their water supply from sea wells rather than using sedimentation and filter tanks.
The reproductive seasons of A. japonicus in different areas may vary according to the prevailing water temperatures and abundance of natural food that influence the development of the gonads. The reproductive season of this species occurs in late May in Qingdao and Rizhao, from early to mid June in northern Shandong Province, from late June to the end of July in Changdao Islands and from late June to early August in Dalian and the northern Yellow Sea. Commonly, when the seawater temperature rises to 16-17°C (Liu et al., 2002) the mature individuals will be collected for immediate use. In recent years, individuals farmed in deep water with sufficient water exchange can also be used for broodstock. These may be caught ahead of the season.
The broodstock should be over 250 g in body weight and 20 cm or more in body length with fully developed gonads. Gonadal index (gonad to body weight ratio) should be over 10 %. The broodstock must be captured with care so as to protect it from oil and avoid injuries.
There are two methods for transporting the broodstock: dry and wet. The former is used for short distances within three hours of the destination. The sea cucumbers are packed in a single layer into a foam box, with ice bags to keep the temperature low. Wet techniques are used for long distances. A canvas tank of 50x50x80 cm3 is filled to about a third to a half full with aerated seawater; it can hold 60-80 individuals. It is preferable to transport the broodstock in the morning or at night in order to avoid exposure to direct sunlight. The temperature inside the box or tank should be maintained below 20 °C.
Most of the broodstock will not spawn on the same day after being transported to the destination. Several days of maturation are required. The broodstock should be cultured in tanks at a density of 30-50 individuals/m3 and a water temperature of 18-20 °C.
Daily management operations - The seawater should be exchanged twice a day: a third to a half of the total volume in the morning and the total volume at night, pumping air into the water, gently but continuously, and clearing the deposits. There is no need to feed the broodstock if the breeding period is imminent, but if the sea cucumbers do not spawn after 5-6 days at 19 °C, then they must be fed with brown seaweed (Sargassum thunbergii), mixed feed or sea bottom mud. Daily feeding rate is 5 % of the body weight (Liu et al., 2002).
Broodstock conditioning - When the sea cucumber juveniles are needed ahead of the natural reproductive season in order to maximize their growth during the same year, the broodstock are collected earlier in the season and matured by gradually raising the water temperature. This procedure will induce the animals to spawn in advance. The method is as follows: maintain the ambient seawater temperature for 2 to 3 days after the capture, and then raise it by 0.5 °C/day. When the water temperature reaches 13-15 °C, maintain it again for another 7-10 days prior to spawning and then raise it gradually to 17-18 °C for spawning. Feed the animals daily at a rate of 5-10 % of the body weight (Wang et al., 2001).
Spawning, fertilization and hatching
Three methods can be used for spawning.
1. Natural spawning - Fully matured broodstock will spawn naturally in the tanks between 1900 and 2000 hours.
2. Induced spawning through temperature shock - The water temperature is abruptly raised by 3-5 °C.
3. Induced spawning through water shock - The holding tank is drained completely and the sea cucumbers kept dry for 0.5-1 hour. Following this the individuals are shocked with a flow of seawater for 30 mins. The tank is subsequently filled with fresh seawater. Generally, the animals will spawn 1-2 hours after the shock (Figure 1).
In some cases, a combination of the last two methods is used to induce artificial spawning.
Figure 1. A spawning Apostichopus japonicus.
Artificial fertilization - As the broodstock starts to spawn, the individuals are removed from the tank as soon as possible; males and females are placed separately into fibreglass or plastic containers. While the female is spawning, a small quantity of sperm is added and the water stirred constantly to aid fertilization. Upon microscopic examination, a ratio of 3-5 spermatozoa per oocyte is considered to be suitable.
Natural fertilization in tanks - When the broodstock starts to spawn, the males are removed from the tank. When all the females have ceased spawning, they are removed and the tank is filled with fresh seawater. After about half an hour, the oocytes sink to the bottom. The uppermost half to two-thirds of the volume is gently siphoned out. After repeating this protocol two or three times, the excess spermatozoa are effectively eliminated.
Once the oocytes are fertilized, the eggs are kept in an incubator vessel and maintained at a density of 10-20 per ml. The eggs may also be allowed to hatch directly in the spawning tank, maintaining a density of 1-2 per ml. In both methods, the water must be gently aerated or stirred every half hour. At a temperature of about 20 °C, the gastrula will begin to hatch within 24-26 hours, and the auriculariae will appear within 34 hours.
Well developed auricularia are selected from the hatching tanks by siphoning them into a fine mesh screen (NX103) or are hand collected using a purposely designed trawl net. If the egg concentration is less than 1 egg/ml and the hatching rate of the auricularia is high, the larvae do not have to be transferred to other tanks. In this event debris, unfertilized oocytes and malformed eggs should be removed and the tank filled with fresh seawater. A rearing density of 0.5 auricularia/ml should be maintained (Zhang et al., 1998).
Feeding regime - The following microalgae species are suitable for the development of the sea cucumber larvae: Dunaliella sp., Chaetoceros muelleri, Nitzschia closterrium, Phaeodactylun tricornutun, Isochrysis zhanjiangensis, and Isochrysis galbana 3011. Diatoms and Dunaliella sp. can be used as the main food, supplemented with Isochrysis sp. Other feeds such as yeast and finely grounded and filtered Sargassum thunbergii can also be used.
Daily feeding rate - Microalgae should be supplied 4-8 times a day in order to maintain a concentration of 2-5 x 104 cell/ml in the culture tank. Observation through a microscope should clearly show that about half of the larvas stomach is filled with food.
Water exchange - The water should be changed twice a day, either by replacing a third to a half of total volume at once, or using a siphon to gradually exchange 1-1.5 times the total volume. When exchanging the water a mesh screen (NX103) should be placed on the water outlet to avoid loosing any larvae.
Cleaning - All debris should be siphoned from the bottom of the rearing tanks every 2 to 3 days; these include uneaten food particles, faeces, dead larvae and protozoa. If the water quality has deteriorated and the larvae are growing very slowly, the water must be completely replaced, even though the larvae might be injured during this operation.
During larval rearing, the water should be aerated continuously or gently stirred every 30 minutes.
Monitoring the physico-chemical factors of the water
The optimum water temperature ranges from 18 to 22 °C, the dissolved oxygen should be maintained above 5 mg/l, the salinity between 26.2 and 36.7, the pH value between 8.1 to 8.3, the illumination between 500 and 1 500 lux, the concentration of non-ion ammonia less than 0.02 mg/l and the turbidity should not be extremely low (Liu, 2000).
Two different setups are used for the settlement of the larvae. The first setup is made of flexible polyethylene film sheets measuring 50x60 cm. About 10 to 15 sheets are fixed on a metal frame at a distance of 5 cm from one another (Figure 2). Each cubic metre of water contains 30-40 sheets. The second setup uses corrugated polyethylene plates measuring 42x33 cm and 1 mm thick. Each holding frame supports 10-20 plates (Figure 3). Each cubic metre of water holds 60-80 plates (Figure 4).
Benthic diatoms must be inoculated on the polyethylene sheets and plates 7-10 days before larval settlement commences in order to supply the pentactulae with an adequate starter food. The settlement plates are placed into the rearing tanks when 10-20 % of the larvae have metamorphosed into doliolariae.
Figure 2. Polyethylene filmstrips.
Figure 3. Corrugated polyethylene plates.
Figure 4. Installation of settlement plates.
An optimum density of 1-3 individuals/cm2 should be maintained for early juvenile sea cucumbers; if the density is too high, the juveniles must be dispersed onto other plates within 10 days.
There is no need to feed early juveniles with artificial food as they will consume the benthic diatoms present on the settlement plates. However, usually 3-5 days after settlement, it is important to supply fresh sea bottom mud and/or a mixed microalgae diet, while grounded and filtered Sargassum thunbergii becomes necessary as the juveniles develop. Based on the feeding rate and growth of the sea cucumber juveniles, yeast, fishmeal, sea kelp powder, as well as Spirulina platensis powder can be further added. Grounded and filtered Sargassum thunbergii, for example, can be used at an early stage, feeding 20-50 g/m3 of water a day, and increasing the quantity to 50-100 g when the body length is 2-5 mm, and to 100-150 g as the juveniles continue to grow.
Water exchange and aeration
A rearing technique using flow through water was recently adopted. If this system is not used, then the water should be completely changed 2-4 times a day. Filtered air should be injected continuously into the water throughout the juvenile rearing period in order to maintain good levels of dissolved oxygen and optimise water quality.
The optimum seawater temperature range for rearing the juvenile sea cucumbers is 24-27 °C. The dissolved oxygen should be above 5 mg/l and the minimum salinity adjusted according to body length as follows: 0.4 mm individuals, salinity 20-25; 5 mm individuals, salinity 10-15; larger individuals, salinity 15-20. The pH should be kept between 7.9 and 8.4, and the light intensity under 2 000 lux.
Both hatchery and microalgae tanks can be used for wintering. The optimal density of juveniles is shown in Table 1.
Table 1. Stocking density of juvenile sea cucumbers.
100 - 300
200 - 1 000
300 - 1 000
1 000 - 2 000
1 000 - 2 000
2 000 - 4 000
2 000 - 3 000
4 000 - 6 000
3 000 - 4 000
6 000 - 8 000
4 000 - 5 000
over 8 000
5 000 - 10 000
Feeding of juveniles
Juveniles should be fed with seaweed powder or a mixed feed twice a day, in the morning and evening, at a rate of 1-2 % of their body weight.
Water temperature and exchange
The water must be heated during the cold winter months. Some of the methods used include a water tube boiler, a deep well, and a winter house with a plastic film roof. The optimum temperature range is between 10 and 12 °C whilst the absolute lower limit is 5 °C.
Water exchange and aeration
The water should be completely replaced once or twice a day, the bottom cleaned once a week. One air-stone should be added for every 2-3 m2 in order to gently and continuously inject air into the water column.
Rotten-stomach disease of auricularia
Early symptoms show a thickened stomach wall, which becomes rough and withered as the auriculariae assume an abnormal shape. Diseased larvae display a slower growth rate than unaffected larvae. Larvae suffering from this disease also have a lower survival rate. In order to control this problem, several actions can be taken as described below.
1. Using healthy broodstock and conditioning them properly are the basic prerequisites to producing healthy larvae, especially in a temperature conditioned hatchery. The water temperature should remain below 20 °C during the broodstock rearing. If the temperature is too high, the gonads will degenerate and auto evisceration may occur. Even after successful spawning under sub optimal conditions the auricularia larvae do not develop properly.
2. Feeding with high quality food without overfeeding. A proper feeding regime should be followed, feeding small quantities frequently, rather than all at once. Old or contaminated food must be avoided.
3. Using proper stocking density. Overcrowding induces slow growth rates and a great variability in body length, as well as causing deformities, rotten-stomach and low metamorphosis rates (Liu et al., 2002).
4. Maintaining good water quality. Among all the environmental factors that influence the growth and development of the larvae, water temperature and salinity are the two main parameters that require special attention, especially since the natural reproductive season occurs in the summer months when it is hot and rainy. Rainfall may cause a rapid decrease of the salinity in inshore waters and the physico-chemical parameters of the seawater may become non-ideal. High water temperatures (above 23 °C) and/or rainfall can cause slow growth rates and can be responsible for the outbreak of diseases. In order to avoid any problems, deep wells located near the hatchery have been used in recent years. Well water has the advantage over open seawater of being cleaner also in terms of microbes and predators. Furthermore, gravel filters are not required for well water so water can be pumped directly into the rearing tanks.
Mortality of juvenile sea cucumbers
In recent years, many hatcheries have experienced mortality of juveniles during the early growth phase on the settlement plates, especially in individuals under 5 mm in body length. The symptoms are that the body remains contracted, adherence is lost and the body changes from translucent to milky-white. The juveniles then become whiter, start rotting, drop from the plates and eventually die. Mortality may reach 90 %. Solutions proposed include:
1. Ensuring the quality of the broodstock. There will be no healthy juveniles and no high survival rates without healthy broodstock.
2. Maintaining a proper density. Experience shows that overcrowding increases mortality. Overcrowding reduces available space and food availability, causing malnutrition, slow growth rates and size variability. Therefore, it is necessary to reduce the density as the juveniles develop to reach a proper density of 1-2 individuals/cm2 prior to body colour change.
3. Designing a good feeding regime. Feeding a single variety of food for a long period should be avoided; instead, a high quality pellet feed must be used to ensure a proper nutrient balance.
4. Maintaining a proper water temperature. Mortality is usually observed after the juveniles have been exposed to a water temperature above 27 °C for a long period. In summer the rearing water temperature must be reduced by: (a) covering the roof and the settlement tank with straw screens to minimize the amount of sunshine, (b) ventilating efficiently and pumping water during the night, (c) increasing water exchange to at least three times a day, and (d) pumping water from a deep well (Liu et al., 2002).
5. Protecting the juveniles from predators. The main predators are copepods, especially Harpactorda sp. They not only compete for food and living space with juvenile sea cucumbers, but also bite them and sometimes kill them. Copepods are particularly dangerous for juveniles under 5 mm in body length. Large numbers of Harpactorda may cause a rapid decrease in the number of juvenile sea cucumber. To eliminate them, crystal dipterex can be used, at a dosage of 2-5 g/m3 of water, followed by a water exchange after 2-3 hours.
The authors would like to thank Professor Chen Jiaxin, former director of the Yellow Sea Fisheries Research Institute of the China Academy of Fisheries Sciences for commenting on the early drafts of the paper and also Professor An Bangchao, the Director of Yantai Fisheries Research Institute for his technical support. Sincere thanks must also be given to Drs J.-F. Hamel and A. Mercier for helping the authors improve the final version of the paper.
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