PART III
BREEDING OF ABALONE: PRACTICAL EXERCISES

PRACTICAL I
APPEARANCE AND INTERNAL STRUCTURES OF ABALONE
(Haliotis discus hannai Ino

OBJECTIVE: To be able to describe morphological features of the abalone by observing the appearance and internal structures of the abalone.

PROCEDURE:

1.1 Appearance of abalone

• Place the abalone in a glass tank to observe their behaviour. Allow them to creep on the bottom and walls of the tank.

• A pair of tentacles protrude from the front end of the body. Observe the length and location of the tentacles and touch their tips. Upon disturbance they disappear under the shell, however they reappear after a short interval. These sensitive and chemotactic organs are protrusile.

• Immediately below the tentacles, the pair of eyes are visible. The eyestalk is named after its shape. Observe the tentacles of the mantle protruding from beneath the shell. Identify the location and number of tentacles of the upper foot. Count the number of brown spots on the upper foot.

• Observe the abalone moving its foot in an undulating and gliding fashion. Observe the abalone grazing on benthic diatoms attached to the bottom of the holding tank, with the aid of its calyx lobe and radula. The calyx lobe and radula are located on the anterior part of the body.

1.2 Shape of the abalone shell

• The abalone shell is shaped like an ear and although it is relatively thin it is strong. The shell length is about one-fifth of the width. The spiral is very small. The soft part of the body is broad and wide and occupies most part of the shell.

• As for the respiratory pores, only the last two to three stomas are perforated, while old pores are closed to form bosses. The row of respiratory pores and bosses divides the shell into two parts, the upper part which is wide, and the genital slope which has a rough surface and wave-shaped wrinkles.

• The abalone shell has a narrow and thickly laid growth-line which is commonly used to calculate the age of the specimen. The outer surface of the shell is blue-brown or ash-brown in colour, however occasionally it may also be red.

• The anterior end of the shell is wide and is of an ovate shape. The outer tip is thin, with a sharp edge, while the inner tip is much thicker. The inner tip has a slimy edge.

• The inner part of the shell is blue-white or copper-white in colour, with a nacreous lustre.

1.3 Internal organs

• Remove the shell from the body by cutting the ligament. While separating the shell from the body, take care to avoid injury to the intestine and other soft organs.

  1. Place the abalone on a dissecting plate, with its foot downwards and its head facing forward. Pour seawater on the plate. Observe the overall body of the abalone and note the mantle structure.
  2. Observe the shape of the tentacles, eyes and mouth located on the head at the anterior part of the body. Identify the tentacles of the mantle and observe the feather-shaped gills and anus. Observe the heart (left side), the spiral part of intestine (left side and posterior end), and the liver (right side). Identify also the ramiform tentacles at the margin of the upper foot and separate them for better analysis.
  3. When the above observations have been carried out, draw the organs on paper, making notes on their location, size and colouration.

• Cut off the mantle with the aid of dissecting scissors in order to observe the internal organs.

  1. Observe the shape of the gills and locate the two auricles and atrium.
  2. Separate the gills and observe the ciliary movement under a dissecting microscope.
  3. Separate the muscular mouth and radula. Observe under microscope the pair of calyx lobe and the long radula zone connecting the two lobes.
  4. The radula is a distinctive feature of all gastropods and it is commonly used for their classification. It consists of numerous minute teeth shaped like knives. The radula is usually long and broad in shape. An abalone with a shell length of about 80 mm has a 39 mm-radula with 104 lines of teeth. The middle teeth are rather small with rounded tips and quadrangular bases. The lateral teeth vary in shape while the marginal teeth resemble one another in shape. They are sharp, narrow and relatively long. The uppermost tip is shaped like a surgical spoon with a dentate edge.

• Observe the digestive organs of abalone: the gullet, stomach, intestine and anus.

  1. The digestive organ is in the soft tissue of the intestine capsule and therefore rather difficult to identify its shape. Identify the mouth and anus. Then, using a dissecting needle, locate the digestive tube.
  2. The saccate V-shaped stomach can be seen next to the gullet. Microscopic examination will show that the food in the stomach is mainly composed of seaweed fragments. The vermiform appendix and liver are attached to the stomach and the cavity connecting to the liver is placed at the curve of the stomach.
  3. The liver is located on the left side of the ligament. It is well developed and relatively bulky.

• The liver is surrounded by the genital gland of the abalone. Remove a fragment of the tissue with the aid of a sharp scalpel and place it under a microscope for detailed observation.

  1. The male has a milk-white genital organ while the female organ varies in colour depending on the stage of development, fully matured organs being usually greenish-brown in colour. The degree of maturity of the genital glands can be identified and classified as follows:
     1. Stage 1 (Granule stage) Granules are being formed but sperm and ovum cannot yet be identified.
     2. Stage 2 (Green stage) The genital gland is not easily dissolved in seawater with many passive and amorphous elementary particles.
     3. Stage 3 (Maturing stage) The genital gland becomes corpulent and most of the eggs are globular in shape.
     4. Stage 4 (Pre-spawning stage) The genital product are released in small quantities.
     5. Stage 5 (Active-spawning stage) About 50 % of the genital glands are released.
     6. Stage 6 (Spent stage) Remaining genital glands can be seen with the naked eye. The mature eggs and sperm are visible by microscopic examination.

  2. The measurement of maturity has a great significance in abalone culture.

1.4 Nervous system

• Its central nerve ganglion is placed around the mouth. A little scraping will expose the cerebral nerve ganglion. Abalone also has a pair of nerve nodules (placed at the foot, intestine, etc.) and also a collection of nerves connecting them.

EXERCISE:

After the above observations, follow the instructions below:

1. Draw the appearance of abalone and make notes on its moving and feeding behaviour.
2. Draw the shape of the abalone deprived of its shell.
3. Remove the mantle and observe/draw the shape of the circulatory system, alimentary canal, etc.
4. Observe the genital gland and identify its stage of maturity.

PRACTICAL II
PREPARATION OF ADULT ABALONE

OBJECTIVE: To be able to select good abalone adults for seed production.

PROCEDURE:

2.1 Selection of adult abalone from the sea

• The optimum size of abalone for spawning ranges between 7–9 cm.

  1. The best way to collect abalone is through the use of divers or hookmen. Well-trained divers can catch abalone with minimum injury to the specimen.
  2. The seriousness of the injury inflicted on the abalone will depend on the part of the body injured. Mortality increases when the abalones are injured on the head, intestine sac, ligament or around the foot. Injury may be minimized when hooks are placed around the middle part of the abalone. If injury occurs, blood flow may not be easy to control as it does not coagulate. The collected abalones are then placed into a holding tank for few days, after which the undergo a second selection. After 3–4 days in the tank, injured specimens can be easily detected as the injured part usually becomes swollen and therefore visible. At this stage the broodstock is selected from specimens which have no injury and appear healthy.

• The suggested period for catching adult abalone is between March-May and October-November when the water temperature is low. During these periods the abalones are fairly inactive and can be easily caught. In addition, at higher water temperatures injuries inflicted during the catching process infect easily.

  1. At water temperatures above 20 °C, the injured parts become inflamed and serious signs of bacterial infection can be seen at water temperatures between 25–27 °C. At temperatures below 20 °C, bacteria growth is restrained while it increases at temperatures above 20 °C. Compared with that of healthy abalone, the blood of injured abalone when tested will show impurities.
  2. Sulfathiazole is applied in the treatment of a wide range of diseases and contaminations. Animal sulfathiazole is acknowledged for its effectiveness, and is applied in two ways: by reagent soaking and by surface application. When the abalone are soaked in the reagent, the concentration should be 5 % of the filtered seawater. After 2–3 minutes in the reagent, the abalone should be exposed to the air for about 10–15 minutes. The efficiency of the medication depends on the duration of the air exposure. When the temperature is high, exposure time should be shortened. The other method is the application of sulfathiazole on the injured part with the use of a brush or a sprayer. Sulfathiazole may also be injected, but this is not recommended as the needle pore causes bleeding. Treatment should be carried out for one week.

2.2 Transportation of abalone

Frequent transportation is necessary in the culture of abalone. Adult abalone may be transported in buckets containing water or without water. Whichever the method used, the abalone must be in a comfortable state so as to reduce mortality. Care must be given to ensure their firm attachment to the bucket or on the attaching boards during transport.

• If the distance is short or if the quantity of abalone to be transported is small, they may be transported in buckets containing water. On the other hand, when abalone are transported over long distances or the quantity is large, they are usually transported in containers without water.

  1. If the abalone are transported in buckets filled with water, frequent water changes and aeration are necessary to ensure proper water temperature and an adequate supply of dissolved oxygen (not below 4 ml/l). A fine mesh knotless net or polyethylene sheet is usually laid over the bottom of the bucket to prevent injury of the abalones when removed.
  2. In case more than a day of travel by train or truck is required, the abalone must be attached on the bottom or walls of an empty bucket, which should then be covered by a seawater-wet medical cloth or fresh seaweed to provide the necessary moisture. The buckets should be protected from the sun or wind to prevent drying and rise in temperature.

• The transported abalone should be moved to the rearing tank as soon as possible.

  1. Attention should be given to differences of temperature between the water in the bucket used for transporting, and that in the rearing tank. The difference of water temperature between the bucket and the rearing tank should not be over 10 °C. If the difference in the water temperatures exceeds 10 °C, the mortality rate will increase. At large temperatures differences the abalones should be gradually conditioned by increasing the temperature by 3–5 °C at intervals of 3–4 hours.
  2. Following the temperature conditioning period the abalone are transferred to tanks specifically designed for rearing abalone broodstock. An abalone with a fleshy foot is usually considered to be a good broodstock and specimens with this characteristic are usually selected to eventually undergo conditioning.

PRACTICAL III
BROODSTOCK CONDITIONING

OBJECTIVE: To know how to obtain sexually mature abalone and to be able to control and ensure spawning as and when required.

PROCEDURE:

3.1 Rearing facilities

To induce maturation in adult abalones, the necessary biological and physical conditions should be met throughout the year, and the rearing system itself should be properly maintained.

• Plastic drums with a holding capacity of 1–2 tons are often used in the rearing and conditioning of the abalone broodstock. However, concrete tanks are sometimes used.

• Corrugated plastic plates (made of vinyl chloride) and/or V-shaped structures (usually made of cement, e.g. roof tiles) are placed in the tanks for the abalones to attach on and seek protection. Abalone may also be matured in plastic netcages which are convenient to maintain

• The netcage size should be for easy handling; they are usually square or oval. A one-ton tank with two netcages is recommended as it is fairly convenient for all farm operations including feeding of adult abalone.

3.2 Rearing conditions

• Adult abalone sexually mature at water temperatures between 17–21 °C, however proper gonadal growth starts at a water temperature as low as 7.6 °C.

• The above temperature minimum is considered the biological zero point in abalone farming, that is, at any temperature above 7.6 °C gonad maturation may be triggered. The sum of the effective accumulated temperature (EAT) is:

  (T - 7.6 °C)

  where:

  T = refers to the average rearing water temperature.

  Laboratory observations and trials indicate that the gonad grows at temperatures between 7.6–21 °C.

  The formula for the linear relationship between the rate of increase of the gonad index (Y) and water temperature (T) is:

  Y = 0.00597T - 0.04537 (T= ≤20 °C)

  At an EAT of 800 °C days, the reproductive organs mature, however the spawning rate still remains low. Spawning rate increases up to 98% when an EAT of 1000 °C days is provided. Spawning time can be effectively planned when applying the EAT technique to condition the broodstock.

• Laminaria and Undaria are a good feed for the adult abalone. Laminaria can be cultured and has a long rearing period.

  1. The average feeding rate should be 7 % above the normal for proper maturation of the abalone with an EAT between 800–1000 °C days. Below this temperature the adult abalones take a long time to mature.
  2. The optimum feeding quantity is 30 % of the body weight; the food should be provided three times a week. At this time, the unconsumed seaweeds should be collected in order to calculate how much food has been consumed. The average feeding rate per day can then be calculated. As the abalones mature, feeding quantity should be reduced.

• During abalone maturation the amount of water change should be ten times the abalone's oxygen consumption. Oxygen stability is normally guaranteed when 100 l/h per kg of abalone are supplied at the rearing water temperature of 20 °C. The formula for calculating the rearing quantity of abalone by the amount of water change is as follows:

  

  where:

  K	= oxygen consumption by the abalone (ml/kg/hr)
  W	= possible rearing quantity
  U	= volume of inflowing water
  V	= tank capacity (m3)
  C1	= dissolved oxygen of inflowing water (ml/l)
  C	= dissolved oxygen of outflowing water (ml/l)
  K1	= coefficient of dissolved oxygen
  K2	= oxygen consumption except abalone (ml/l/hr)

• When the abalones are matured, a sudden change in the environmental conditions of the rearing tank may trigger spawning. It is therefore, essential to carefully pay attention to maintain a constant environment.

  1. The light method is effective to stimulate spawning. Initially the rearing tank is irradiated by artificial light, with a photoperiod of 12:12 hours. Two months before spawning, the day light may be reduced to nine hours in order to obtain higher fecundity. Exposure to light does not greatly affect spawning if the abalones have a high EAT. As the abalone is a nocturnal animal light exclusion is applied and no maintenance is conducted during this period.
  2. During the maturation process, tanks should be cleaned at least once a week. The rearing tank should be totally drained to remove all faeces, and thoroughly washed with a strong jet of water.
  3. Abalone maturity should be investigated once the EAT of 800–1000 °C days is reached. Weekly investigations should be carried out at higher water temperatures. The following formula is used to calculate abalone maturity:
  

  where:

  M =	maturity (%)
  Ti =	thickness of the middle part of the reproductive sac at the time of investigation
  Ts =	thickness of the middle part of the reproductive sac before spawning

  The thickness of the middle part of the reproductive sac prior to spawning varies according to the size of the abalone and the prevailing environmental condition at the culture site. Therefore, prior investigation is conducted a relation formula, to ease calculation, should be formulated. In the case of the local (Korea DPR) abalone, the relation formula Y = -4.3 + 1.28 × is used to measure the thickness of the middle part of the reproductive sac before spawning.

EXERCISE:

1. Determine the percentage maturity of the adult abalone.
2. Calculate the effective accumulated temperature (EAT).

PRACTICAL IV
SPAWNING

OBJECTIVE: To be able to perform the technique of controlled spawning.

MATERIALS:

• Eight adult abalone
• Tanks for egg collection (20, 200–500 l)
• One UV-sterilizer
• Photo-electric colorimeter
• Microscope
• Glass tube (diameter 10–20 mm, length 500–600 mm)
• Light exclusion cloth

PROCEDURE:

A number of stimulating methods can be applied to spawn mature abalone.

4.1 UV light method

The artificial spawning method should be selected taking into consideration the maturity of adult abalone.

• During the stimulation process the female-male ratio should be 4:1. As spawning starts the male and female abalone should be separated and allowed to continue the emission of the genital product.

• An abalone with a mature gonad may release its gametes when the UV-sterilized seawater stimulating method is applied. The volume of water to be supplied at the time of ultra-violet exposure is 1/20 of normal sterilizing capacity. Ultra-violet lamps with various capacities and with outputs between 15W-2KW are used. Low-pressure sterilizing lamps are more effective as they emit a great amount of short-waves between 275–180 mm.

• The number of adult abalone to be used should be limited according to the number of eggs required. The number of spawned eggs is about 1.5 million per 100 g of gonadal mass of a mature abalone.

EXERCISE:

Select a number of adult abalone and separate them by sex. Measure the shell length and mass, and calculate the gonad index. Remove the abalone from the water and allow them to dry under the shade; the time of air exposure will vary depending on the maturity level of the broodstock, from 30 minutes to 2 hours.

After air-drying, clean the abalone in UV-treated seawater always keeping the sexes separated. Then place them in a tank filled with UV-irradiated seawater. One to two adult abalone are placed in a 20-litre plastic tank while a larger number can be collected in tank with a larger capacity (500–1000 l). When the above operations have been completed the following ones should be carried out.

• When the abalone have been released into the tank, the tank should be covered so as to simulate night time. This is because the amount of the genital product released will be at its highest when light exposure is reduced. After one hour check the abalones at intervals of 15–30 minutes and note the beginning of the spawning activity as well as record the number of specimens which have been induced.

• When an abalone begins spawning, observe the dispersion of the gametes in the water; siphon out the genital product by using a glass tube cleaned with filter-sterilized seawater to avoid contamination of the eggs and sperms. At this stage the fertilization process can be clearly observed under the microscope. Observe the spawning behavior of the male and female abalone and note the frequency of release.

• When the male abalone have ejaculated sperms for an hour or so, move the sperm suspension to another container to estimate its concentration. The sperm concentration can be observed under microscope using a haemocytometer. If the concentration is too thick to be calculated, proceed to dilute the suspension. Another way of evaluating the concentration of sperms is through the use of photo-electric colorimeter. This method is very simple and practical.

• When the female abalone have released eggs for one hour or so, transfer the egg suspension to another container and calculate the number of eggs. The number of eggs can be calculated through the volumetric method.

PRACTICAL V
EGG FERTILIZATION AND EARLY EMBRYO HANDLING

OBJECTIVE: To acquire the techniques of artificial egg fertilization and early embryo handling.

MATERIALS:

• Fertilization and early rearing tanks (20, 100–500, 1000 l)
• Glass beakers (Diameter 10–200 mm, length 500–600 mm)
• Graduated cylinders (100, 250, 500 ml)
• Glass pipettes
• Simplified cell counter
• Microscope

PROCEDURE:

5.1 Fertilization

• Fertilization rate is increased when it is carried out within one hour from egg release. The quantity of semen required for fertilization is calculated based on its concentration. The recommended egg density during the fertilization process is a maximum of 40 eggs/ml, while the ratio of eggs and sperm should be 1:1000. At a higher ratio, polyspermy usually occurs.

• Place the calculated amount of sperm into the bucket containing the freshly released eggs and stir gently in order to facilitate dispersion of the sperm and aid egg fertilization. Examine under the microscope the number of sperms sticking on the membrane of one egg. Three to five sperm heads on one egg is an indication of correct egg:sperm ratio. Too many sperms cause egg shrinkage and consequent abnormal development. The normal rate of fertilization is above 95 %.

5.2 Egg washing

• Successfully fertilized eggs sink to the bottom of the tank. The sinking speed of the eggs is 2 cm/sec. Unfertilized and abnormal eggs sink two to three times slower than the fertilized ones.

• The upper water layer is removed by suction or by slightly tilting the tank to one side after the eggs have sunk to the bottom. This procedure is carried out to remove unwanted matter such as abnormal and unfertilized eggs. When drawing out the water, at least 15 cm from the bottom should be left in order the prevent the eggs from being sucked out or damaged.

• At this stage freshly filtered and sterilized seawater is added to the tank containing the developing embryos. The newly added seawater should have the same temperature as water and the fertilized eggs should soon start floating uniformly. When pouring the additional seawater, attention should be taken not to pile the eggs on one side of the tank as more than one egg layer may induce some eggs to develop abnormally.

• The eggs should be washed five to six times until they hatch. If fertilized eggs are piled on the bottom of the tank for more than three hours, their growth is affected as oxygen supply is generally not sufficient. Care should be taken when washing the eggs as handling may affect the subsequent survival of the abalone larvae.

• Examine the developing embryos under the microscope. Observe normal and deformed embryos as well as the unfertilized eggs. Measure the diameter of the normal growing eggs and calculate the rate of fertilization. When the eggs have been thoroughly washed add fresh seawater. At this stage fill a 100 ml graduated cylinder by dipping it in five different places of the tank, to be fixed under formalin.

• When fixing with formalin, the upper 15–20 ml in the cylinder is removed after the eggs have sunk to the bottom and the same volume of formalin added. The water in the cylinder is stirred and a 0.5–1.0 ml sample is collected by using a pipette for observation under the microscope using a simplified cell counter. At this stage the number of normally developed, unfertilized and deformed eggs can be observed and counted. The count is carried out five times to obtain the average value. The ratio of successful egg fertilization and the ratio of normal egg development can be calculated as follows:

  Ratio of fertilization (%)

  Ratio of normal cleavage (%)

• It is important to maintain a constant water temperature during egg cleavage, as even small differences may cause abnormal egg development. The most suitable temperature level for culturing abalone eggs is above 30 °C.

PRACTICAL VI
REARING OF FREE-SWIMMING LARVAE

OBJECTIVE: To acquire the techniques of optimizing the culture conditions for rearing free-swimming larvae.

MATERIALS:

• Rearing tank (1000 l)
• Cotton gauze for larval collection
• Microscope
• Graduated cylinders (250, 500 ml)
• General larval rearing equipment

PROCEDURE:

The free-swimming period of larvae is usually between 4–10 days, however this may vary under specific conditions.

6.1 Larval appearance

• In rearing free-swimming larvae it is important to provide the appropriate environmental conditions to reduce to the minimum the free-swimming period. Newly hatched larvae swim freely with the help of the velum and move to the upper layers of the rearing water as they are actively phototrophic.

  1. Study the activity of newly hatched larvae under the microscope. The health condition of larvae can be judged from their appearance (i.e. body shape), mobility and ability to swim vertically up the water column.
  2. Larvae which swim actively, moving clockwise or counterclockwise by the constant movement of the velum, and normal in shape and size tend to grow and perform well.
  3. Observe larvae with a poor and weak activity. These specimen are usually abnormal in shape or small in size.

6.2 Rearing parameters

• Transfer all swimming larvae in the upper layers of the water column to the rearing tank by siphoning with a hose.

  1. A larval rearing density of 300/l usually causes no particular problem, however in case of higher densities (1000/l) good management should be ensured, particularly with regard to water change, oxygen supply, etc. Rearing density is closely related to the quality of the rearing equipment and knowledge of management techniques.

  2. Constant water temperature is an important factor in the rearing of free-swimming larvae.

     1. Water temperature suitable for larval growth and survival is around 20 °C.
     2. Salinity should be above 30 ‰.
     3. The amount of dissolved oxygen (DO) in the rearing water should be more than 4 ml/l. Oxygen deficiency lasting for half an hour causes the larvae to sink to the bottom of the tank due to a weakened state. Air should be supplied regularly to maintain the DO at the standard value.

     4. The volume of water to be regularly changed varies according to the larval rearing density. At densities above 1000/l, the entire water volume should be changed once every 6–8 hours, while at 300/l, the entire water volume should be changed daily.

  3. Observe on a regular basis the developmental stages of the larvae and note their activity, shape and mobility as well as calculate the survival rate.

EXERCISE:

a. Observe and compare the activity and behaviour of normal and abnormal larvae.

PRACTICAL VII
REARING OF JUVENILE ABALONE

OBJECTIVE: To acquire the techniques of rearing juvenile abalone.

MATERIALS:

• Rearing tank
• Rearing boards
• Microscope
• Cotton gauze

PROCEDURE:

7.1 Major rearing technical and management problems

• The physiological and morphological changes in the abalone larvae, such as the growth of the foot and the appearance of the tentacles, take about four days under optimum culture conditions. During this period the larvae search for a suitable surface to initiate their benthic life. If they are unable to locate a bio-chemically or physically suitable surface, they will continue their planktonic life, and the larval period may last up to 10–20 days.

• When a suitable surface is found the larvae move from the free-swimming to the creeping stage. This process is timed with the disappearance of the velum. After several metamorphic stages, they become juvenile abalones.

• The major management and technical problems at this stage involve the provision of rearing conditions suitable for survival and growth after metamorphosis.

7.2 Preparation of rearing boards

• Plastic materials such as polyethylene, polyesterol, acrylic, and PVC are commonly used for manufacturing the rearing boards.

• The size and shape of the boards differ according to the rearing facilities available, however wave-shaped plastic plates are usually used. The size of a rearing board is about 30×30 or 40×40 cm. The boards are assembled in frames that hold 10–20 boards, arranged at 3–5 cm intervals in order to facilitate their handling.

• The provision of a biologically suitable surface to the benthic larvae is an important factor in terms of their survival and growth. In addition, live food of suitable size and appropriate density should be cultured on the surface of the rearing boards in order to provide nutrients to the creeping larvae.

  1. Properly assembled rearing boards should be placed in the larvae rearing tank which should be in the dark or not directly illuminated.
  2. A continuous flow of filtered seawater should be supplied for about ten days. Particles above 10 um in size should be removed through filtration, while protozoans and bacteria of smaller size should be left in the filtered seawater. When the rearing boards are in contact with the seawater for about ten days they become coated with a layer of microorganisms at an appropriate density. Boards with such a surface induce the abalone larvae to set and metamorphose. These also provide good feeding conditions to the creeping larvae during the first 10–20 days. The distance between single microorganism cells on the board should be in the range of 10–50 um, which provides the suitable density.
  3. Place the diatom fluid containing 1–2 species of Navicula, of less than 10–20 um in size, in the rearing tank a day before introducing the larvae. The diatoms successfully become attached to the rearing boards when the water is static for more than 8 hours.
  4. The water temperature should be between 19–21 °C throughout the rearing period. In addition, ammonia should be below 50 ppb, while pH and salinity above 7.9 and 30 ‰ respectively. The level of DO should be maintained in the range of 70–100 % of saturation. The level of nutritive salts per litre of rearing water should be as follows:
     
     

     Nitrogen	=	5 ug - nitrogen atom weight
     Phosphorus	=	1 ug - phosphorus atom weight
     Silicon	=	5 ug - silicon atom weight

7.3 Rearing of larvae

• Collect and transfer free-swimming larvae at the point of metamorphosis, into the rearing tank in which the diatom coated boards have been placed, and adjust the density to about 300 larvae/l.

• At this stage the rearing water should be static, as the time taken by the larvae to settle on the boards ranges between few hours to one whole day.

• Following the completion of metamorphosis into a creeping larvae, the water should be regularly changed and aeration supplied.

  1. The water should be changed three times a day, in order to maintain its quality as described earlier.
  2. It is preferable to supply air at intervals of 15–20 minutes rather than provide a continuous supply.

• Between 10–20 days after setting, the abalone larvae begin to feed on diatoms about 20 um in size. If feed deficiency on rearing boards is determined through observations under the microscope, the above-mentioned diatom fluid should be added and the water allowed to be static for about eight hours. Diatom propagation in the rearing tank takes place when the light intensity is about 3000 lux.

• Attention should also be drawn to the biological management of the rearing tank.

  1. The larvae on the rearing boards grow to about eight times in biomass in the first ten days and accordingly produce more excretion.
  2. Ammonia and carbon dioxide are also produced from the dead larvae and decomposition of unconsumed food. Ammonia is detrimental to the larvae even at a concentration as low as 50 ppb. Undesirable water quality causes larvae to be less resistant to the intrusion of bacteria and other pathogens. The larvae thus tend to grow rather slowly.
  3. Oxygen deficiency may occur as a result of bacterial growth on dead tissue and excretion.
  4. The removal of contaminants is carried out by regular water change and proper air supply. On the other hand, contamination at the bottom and on the walls of the rearing tank should be removed with the aid of a hose or washed manually, after having removed the rearing boards.
  5. Light intensity should be regulated in order to enhance diatom growth which is responsible for the assimilation of ammonia, carbon dioxide, etc., as well as being the main food for the abalone larvae.
  6. Rearing yields are usually high when the density of bacteria remains below 100 cells/ml. The level of bacteria is usually regulated by treating the rearing water with an ultra-violet sterilizer.

EXERCISE:

1. Observe metamorphosis and the various developmental stages of the cultured larvae, and estimate the survival rate by counting the number of abalones on at least 20 rearing boards.

PRACTICAL VIII
INTERMEDIATE REARING

OBJECTIVE: To acquire the technique of intermediate abalone rearing.

MATERIALS:

• Netcage for intermediate rearing (60x80x30 cm)
• Rearing boards
• Pair of scales

PROCEDURE:

8.1 Rearing facilities and conditions

• Land-based intermediate rearing facility

  1. Abalone juvenile are reared in netcages which are placed in land-based cement tanks coated with synthetic resin or waterproof cloth. Rearing can be carried out by using a water circulation or a flow-through system, depending on the source of seawater supply.
  2. A rearing farm should be supplied with seawater with the following quality parameters:

     1)	ammonia	=	<50 ppb
     2)	pH	=	>7.9
     3)	salinity	=	>30 ‰
     4)	DO	=	70–100 % saturation.

• Sea-based intermediate rearing facility

  1. A sea-based intermediate rearing facility usually consists of a series of floating netcages located in protected sites and kept in position by a system of floats, ropes and weights. When using the above facility it is important to pay particular attention to the selection of the site, as the entire rearing facility may be destroyed and lost during bad weather conditions.

8.2 Intermediate rearing

• Rearing density. The rearing density varies according to the initial size of the abalone juvenile, as well as on the type of the rearing device. However, it is normal to stock 5 kg and 3 kg per m³ in land- and sea-based facilities, respectively. Higher stocking densities usually cause the growth rate of abalone to drop, as well as the piling of abalones on top of one another, which then need to be manually separated. When the mass of the cultured abalones duplicates, thinning should be carried out by dividing the abalones in half. Thinning is easily achieved by removing half of the rearing boards and replacing them with new ones on which the abalone will soon settle.

• Feed supply. The amount of feed supplied varies according to the size of the abalone juvenile as well as on the prevailing seawater temperature. Feeding rate increases with increase in water temperature and it is usually high among small abalone specimens. The food is usually provided at intervals of 5–10 days, and the amount should be twice the actual feeding capacity of the stocked abalones. Major seaweeds fed to abalone include species such as Laminaria, Undaria and Ulva. Seaweeds used as feed should be cut into small pieces the size of the abalone, cleaned and removed of their roots and fouling organisms.

  Abalones of different sizes are usually fed as follows:

  1. Specimens measuring 6–10 mm in shell length are fed with finely chopped Ulva spread over the bottom of the rearing facility, as well as with diatoms attached on the rearing boards.
  2. Young Laminaria and Undaria plants mixed with a certain amount of Ulva are fed to abalone 10–13 mm in size.
  3. Abalones above 13 mm are fed with Undaria and Laminaria. At this stage, attention should be paid to the rate and feeding quantity, as leftover feed easily becomes fouled.

• If intermediate rearing is carried out on land-based facilities, abalone faeces and leftover feed should be removed regularly. Cleaning of the rearing facilities should also be carried out on a regular basis. When the seawater temperature is above 20 °C, cleaning should be done every 6–7 days while once every 10–15 days at temperatures below 20 °C. Fouling organisms on sea-based rearing installations should be removed as they hinder water flow. Netcages should be regularly cleaned or changed at the time of feeding.

• On land-based rearing facilities, a 30 mm abalone may double in size within one year, while on sea-based installations, abalone growth occurs mainly from May to November. During the intermediate rearing period, the major factors influencing growth rate are water quality, mechanical disturbance (eg. wave action in sea-based facilities), excessive rearing density or unsuitable rearing structures.

• It is important to bear in mind that regular investigation on environmental conditions and on the performance of the abalone is needed. The survival rate of abalones during intermediate rearing is usually about 80 %, attaining a shell length of 30 mm.

PUBLICATIONS AND DOCUMENTS OF THE
REGIONAL SEAFARMING DEVELOPMENT AND DEMONSTRATION PROJECT
RAS/90/002 (RAS/86/024)

Working Papers
RAS/86/024

NACA-SF/WP/87/1. Lovatelli, A. Status of scallop farming: A review of techniques. 22 pp.

NACA-SF/WP/88/2. Lovatelli, A. Status of oyster culture in selected Asian countries. 96 pp.

NACA-SF/WP/88/3. Lovatelli, A. and P. B. Bueno, (eds). Seminar report on the status of oyster culture in China, Indonesia, Malaysia, Philippines and Thailand. 55 pp.

NACA-SF/WP/88/4. Lovatelli, A. Status of mollusc culture in selected Asian countries. 75 pp.

NACA-SF/WP/88/5. Lovatelli, A. and P. B. Bueno, (eds). Seminar report on the status of seaweed culture in China, India, Indonesia, ROKorea, Malaysia, Philippines and Thailand. 79 pp.

NACA-SF/WP/88/6. Lovatelli, A. and P. B. Bueno, (eds). Seminar report on the status of finfish culture in China, DPRKorea, Indonesia, ROKorea, Malaysia and Singapore. 53 pp.

NACA-SF/WP/88/7. Lovatelli, A. Seafarming production statistics from China, Indonesia, ROKorea, Philippines, Singapore and Thailand. 37 pp.

NACA-SF/WP/88/8. Lovatelli, A. Site selection for mollusc culture. 25 pp.

NACA-SF/WP/88/9. Lovatelli, A. and P. B. Bueno, (eds). Seminar report on the status of finfish netcage culture in China, DPRKorea, Indonesia, ROKorea, Malaysia, Philippines, Singapore and Thailand. 56 pp.

NACA-SF/WP/86/10. Chong, K. C. Economic and social considerations for aquaculture site selection: an Asian perspective. 17 pp.

NACA-SF/WP/89/11. Chen J. X. and A. Lovatelli. Laminaria culture - Site selection criteria and guidelines. 30 pp.

NACA-SF/WP/89/12. Chen J. X. Gracilaria culture in China. 18 pp.

NACA-SF/WP/89/13. Seafarming Project, RAS/86/024. Site selection criteria for marine finfish netcage culture in Asia. 21 pp.

NACA-SF/WP/89/14. Lovatelli A. Seafarming production statistics from China, India, Indonesia, ROKorea, Philippines, Singapore and Thailand. 47 pp.

NACA-SF/WP/89/15. Chong K. C. and D. B. S. Sehara. Women in aquaculture research and training. 20 pp.

Working Papers
RAS/90/002

SF/WP/90/1. Chen J. X. Brief introduction to mariculture of five selected species in China. 32 pp.

Bibliography

NACA-SF/BIB/88/1. Selected bibliography on seafarming species and production systems. 20 pp.

NACA-SF/BIB/88/2. Selected bibliography on seafarming species and production systems. 52 pp.

NACA-SF/BIB/89/1. Selected bibliography on seafarming species and production systems. 49 pp.

Training Manuals

Manual on seaweed farming: Eucheuma spp. (Training manual No. 1). 25 pp.

Culture of the pacific oyster (Crassostrea gigas) in the Republic of Korea. (Training manual No. 2). 64 pp.

Culture of the seabass (Lates calcarifer) in Thailand. Training manual No. 3. 90 pp.

Training manual on marine finfish netcage culture in Singapore. (Training manual No. 4). 275 pp.

Culture of Kelp (Laminaria japonica) in China. (Training manual No. 5). 204 pp.

Training manual on Gracilaria culture and seaweed processing in China. Training manual No. 6. 155 pp.

Meeting Reports

Report of the First National Coordinators' Meeting of the Regional Seafarming Development and Demonstration Project, 27–30 October 1987, Bangkok, Thailand. 71 pp.

Report of the Second National Coordinators' Meeting of the Regional Seafarming Development and Demonstration Project, 20–23 September 1988, Singapore. 102 pp.

Report of the Third National Coordinators' Meeting of the Regional Seafarming Development and Demonstration Project, 24–27 August 1989, Qingdao, China. 103 pp.

Workshop Reports

Report of the FAO Asian Regional Workshop on Geographical Information Systems: Applications in Aquaculture, 5–23 December 1988, Bangkok, Thailand. FAO Fisheries Report No. 414, FIRI/R414. 13 pp.

Report of the Workshop and Study Tour On Mollusc Sanitation and Marketing, 15–28 September 1989, France. FAO/UNDP Regional Seafarming Development and Demonstration Project RAS/86/024. 212 pp.

General Reports

Progress report on the 1988 Regional Training/Demonstration Courses organized under the Regional Seafarming Development and Demonstration Project (RAS/86/024). 26 pp.

Report of the Seafarming Resources Atlas Mission. Regional Seafarming Project RAS/86/024, July 1989. 74 pp.

Audio-visual Materials

Culture of the Pacific Oyster (Crassostrea gigas) in the Republic of Korea. 71 slides.

Culture of the seabass (Lates calcarifer) in Thailand. 40 slides.

Marine finfish netcage culture in Singapore. 37 slides.

Culture of Kelp (Laminaria japonica) in China. 30 minutes video.

Seafarming Atlas Series

Regional Seafarming Resources Atlas. FAO/UNDP Regional Seafarming Development and Demonstration project (RAS/86/024), January 1990. (Atlas series No. 1.). 83 pp.