1.3 TECHNIQUES – BROODSTOCK

1.3.1 Gonadal and muscle indices

The use of gonadal indices as an indication of reproductive activity is a simple, fast and inexpensive procedure in a hatchery. It provides reliable quantitative information required prior to the stimulation of spawning.

The verification conducted at the Bermuda hatchery by comparisons with simultaneous histological analyses, confirmed the usefulness of gonadal index determination. Although there are various definitions of gonadal and muscle indices, all are based on the weight change of the tissue with reproductive maturity. Lucas and Beninger (1985) critically reviewed several of these indices, concluding with that best representing bivalve static condition. In the present work, gonadal and muscle indices are calculated separately; the former for reflecting gonadal development, and the latter as an indication of nutrient mobilization and utilization associated with gonadal development (Gabbott and Bayne, 1973). The step by-step procedure for determining both indices is given in detail below in Protocol–1; a sample data sheet used during analysis is given in Appendix 2.
Gonads and adductor muscles are dissected and dried to constant weight at 80 °C. Indices are calculated as follows:
Dry weight of organ
–––––––––––––––––––– x 100
Empty shell weight
A high gonadic index is indicative of mature gonads, and a lower index reflects the onset of gametogenesis or spent gonads.

For some pectinid species, visual observation of the gonad is sufficient to determine the spawning condition as for Patinopecten yessoensis, the Japanese scallops (Bourne, Hodgson and Whyte, 1989); however, visual observations have been misleading for the calico scallop studied in Bermuda. Although this species exhibits a change in colouration as the gonad matures from whitish-yellow to bright reddish-orange as observed by Roe, Cummins and Bullis (1971), it has been observed in Bermuda that gonads become bright orange prior to maturation and coincidental with sub-maximal gonadic index values. It is therefore advised that gonadic index determinations be made to assess the reproductive status of these species.

PROTOCOL–1

DETERMINING GONADAL AND MUSCLE INDICES

Preparation for procedure
1. Collect 15 scallops from the grow-out site. Record date and site of collection.
2. Place scallops in a tank supplied with flowing filtered seawater. Do not feed and allow 24 hours before dissecting to ensure gut clearance.
3. Use a 2 or 3 decimal point balance, if possible. Switch on and leave to warm up for at least 15 minutes.
4. Bring scallops in a bucket of seawater to laboratory.
5. Set up dissection kit with scissors, forceps and scalpel. You will also need Vernier calipers for measuring scallops and a laboratory book to record all information.
6. Take about 1 meter of absorbent paper and lay it on the bench. Number the paper with the number of scallops for dissection, i.e. if there are 10 scallops, Label 1, 2, 3…10, in equidistant spacing along the paper. Lay each scallop by their number on the paper, and the scallop then becomes this number. In this way, it avoids confusion. Also you will find that as scallops start to gape for air they are much easier to work with!
7. Tear small squares of foil paper (6x6 cm); two for each scallop.
8. Record date and site of collection, and date of dissection in lab book.
9. In the lab book, organize columns for scallop number (see step 6), shell height, total wet weight, gonad dry weight, and muscle dry weight.
The dissection procedure (wet weights)
1. Measure scallop height with Vernier calipers (see Figure 1.3). Record data on the lab book next to appropriate scallop number.
2. Blot dry each scallop with absorbent paper, and place on balance for total wet weight. Record data on the lab book next to appropriate scallop number.
3. As scallops begin to gape open, take one and keep it open with thumb and forefinger. Use scalpel to cut the adductor muscle from shell. This will allow opening the scallop completely, laying it flat for dissection.
4. Take a piece of prepared foil, label as “G” for gonad; write matching scallop number next to it directly on the foil paper. Such that each piece of foil is labelled G1 for scallop no. 1, G2 for scallop no. 2, etc. Tare the balance, and weigh foil. Record as foil weight under proper label.
5. Remove the gonad, using scissors and tweezers. Place the gonad on the matching piece of foil, and weigh. Record as gonad wet weight with foil (gram). Fold the foil around the gonad and place it to one side.
6. Take the next piece of foil for muscle, and label as “M” for muscle; write matching scallop number, such that similar to the gonad, each piece of foil is labelled as M1 for scallop no. 1, M2 for scallop no. 2, etc. Tare the balance and weigh foil. Record as foil weight under proper label.
7. Remove the muscle, carefully removing all the muscle from the ventral shell using a scalpel. Place on foil and record muscle wet weight with foil. Wrap the muscle in the foil and put it to one side.
8. Scrape all other tissues into a waste container.
9. Blot empty shells dry with a piece of absorbent paper. Tare the balance, and weigh and record the weight of the empty shell. Discard shells.
10. Repeat the dissection procedure for all remaining scallops. At least 10 scallops are required for reliable determination of Gonadal Index (GI) and Muscle Index (MI). 11. When all scallops have been dissected, put all labelled gonads and muscles wrapped
in foil into a drying oven at 80 °C. Dry until constant weight; this should take 48 hours.
12. Clean all equipment thoroughly, especially dissecting tools and Vernier calipers to prevent any salt corrosion.

Dry weights
1. After about 48 hours, take gonads and muscles out of the oven. Make sure that constant weight has been reached after this time period.
2. Switch balance on and leave to stabilize according to manufacturer’s instructions (usually about 15 minutes).
3. Weigh each organ with foil. Record on lab book.
4. Repeat until all organs weighed.
5. Transfer data into an Excel Spreadsheet (see Appendix 2). This will allow you to calculate gonad and muscle weight without foil, and gonadic and muscle indices as follows:
To calculate Gonadal Index (GI):
Gonad dry weight –––––––––––––––––––– x 100
Empty shell weight
To calculate Muscle Index (MI):
Muscle dry weight –––––––––––––––––––– x 100
Empty shell weight
Calculate the mean and standard deviation of the GI and MI for each collection date.

6. Keep all gonadal and muscle indices conducted in one spawning season in one Excel workbook. Assess gonad development on a routine basis. Keep yearly records for comparison.

1.3.2 Maintenance and conditioning of broodstock

The main goal in maintaining a broodstock in the hatchery is to ensure a healthy stock of reproductively mature and ripe adults. Depending upon species, this goal may be easily achieved or not. Conditioning broodstock, by manipulating environmental factors, such as temperature, food, photoperiod, allows for control of the gametogenic cycle, or parts of, and provides an aquaculturist with a tool for management of spawning periods and larval rearing and production in the hatchery. Again, depending on species, conditioning may be easily achieved or not. It has also been reported that conditioning of the broodstock affects lipid content of the egg, and subsequently larval survival (Gallager and Mann, 1986); these authors found that variations in broodstock conditioning protocol induced large fluctuations in egg lipid levels of two bivalve species, and suggested that strict attention should be paid to conditioning if optimal culture potential is desired. In other cases, difficulty in obtaining ripe broodstock, has prompted development of hatchery conditioning protocols, as for Pecten fumatus, in Australia (Heasman, O’Connor and Frazer, 1996); these authors found that the rapid conditioning of P. fumatus was possible by controlling water temperature and feeding to satiation; conditioned scallops exhibited a better fecundity than those spawned immediately upon collection from the wild.
For E. ziczac, Velez, Alifa and Perez (1993) found that sexual maturation and spawning could be induced out of the regular spawning season, by maintaining a temperature of 26–29 °C up to a total of 400 °C days; they furthermore found that the number of oocytes released was significantly higher in scallops maintained at a higher temperature for a longer number of day degrees. In Bermuda, no attempt was made to condition scallops out of their regular spawning season; however, efforts were made to ensure ripe broodstock at specific periods, thus optimizing use of the available hatchery and nursery space in this compact facility. As is seen below, working with two different species resulted with two different strategies.
In Bermuda, broodstock was maintained in the tanks described on Page 8 of the technical drawings. It was found that 50 calico scallops of mean shell height 57.2±2.7 mm could be comfortably held in one broodstock tank (equivalent to 80 scallops per m2). Due to the larger shell size and recessing nature of the zigzag scallop, a maximum of 20 scallops only (approx. 75 mm in shell height) were kept in one broodstock tank (equivalent to 30 scallops per m2).

1.3.2.1 The sand scallop, Euvola (Pecten) ziczac

Unlike results reported by Velez, Alifa and Perez (1993), where conditioning was conducted successfully out of season, it was found that the sand scallop was not easily maintained in the hatchery in Bermuda, even during spawning season. Keeping apparently ripe animals in a broodstock tank with a daily supplement of algae for any length of time, yielded poor spawns in terms of number of eggs released (Fecundity= 1.4±1.1 million eggs per female as opposed to the norm of 5 million eggs per female) and poor subsequent survival to D-larval stages (1.6 percent as opposed to 60 percent). Moreover, this species was found to spawn spontaneously following any type of stress, such as handling on the boat, transport to the hatchery, etc. The strategy for ensuring ripe zigzag scallops thus became one for collection of scallops when ripe, and prevention of spontaneous spawning. Protocol–2 provides a detailed description of the procedure. In short, scallops are collected directly from the field. Their reproductive state is assessed visually on board the boat. Scallops are considered ripe when the following three criteria apply: 1) well rounded gonads 2) female colour orange 3) digestive tubule invisible, covered by gonads. Ripe scallops are transported to the hatchery with great care. In order to avoid release of gametes due to boat movement, scallops are transported “dry”. Zigzag scallops, as most pectinids, gape open when exposed to the air; in order to avoid dehydration, rubber bands are quickly placed around the two shells upon collection to prohibit them from opening their valves. Spawning is induced upon arrival to the hatchery.

PROTOCOL–2

COLLECTING AND HOLDING OF SAND SCALLOP BROODSTOCK

Preparation for procedure
1. Collect 30 specimens from the grow-out site by SCUBA using collector bags. 2. Assess gonadal state on board for as many animals as possible. Place ripest scallops aside (see criteria Section 1.3.2.1).
3. Quickly fasten rubber bands around shell of ripe scallops to prohibit valves from opening.
4. Place scallops gently in a cooler on a bed of seaweed, or layers of polyethylene mesh previously moistened with seawater.
5. Total exposure out of water for this species should not exceed 45 minutes.
6. Upon arrival at the hatchery, transfer scallops to a cold-water bath previously prepared for spawning induction (see Protocol–4).
7. If not possible to induce scallops on the same day, transfer into one of the broodstock tanks at ambient seawater, and induce the following day.
8. Do not feed 24 hours prior to spawning induction if maintained in hatchery.

1.3.2.2 The calico scallop, Argopecten gibbus

Gonadal indices are determined monthly to assess the stage of reproduction, as visual observation of the gonad was found deceiving in this species (see Section 1.2). Two strategies for ensuring a ripe broodstock in this species are used in Bermuda.
The first strategy relies strongly on the natural gametogenic cycle of the calico scallop. When a gonadal index reaches 2 or above, a sub-sample of scallops is collected from the grow-out sites and brought into the hatchery. Animals are kept in a broodstock tank at ambient seawater temperature, and fed on a daily basis a mixture of algal species. Diets consist of Isochrysis galbana, Chaetoceros muelleri and Tetraselmis chuii. Algal food is
supplied to the broodstock via a 20-litre carboy fitted with a drip-feed. This carboy is filled in the morning and in the evening, such that scallops receive a continuous flow of food amounting to 40 litres of algae for 50 animals or 14 litres of algae per kg total wet weight. This yields levels of 380 cells.g-1 wet tissue weight. Although, this is a relatively low level of food compared to others (Bourne, Hodgson and Whyte, 1989; Neima and Kenchington, 1997), it seems adequate for development of gonads to maturity as seen in results obtained following a 2–month conditioning period (see below). Feeding is stopped 24 hours prior to spawning induction. Ripe scallops are usually induced to spawn 1 or 2 weeks following collection.
The second strategy involves conditioning of broodstock to accelerate the later stages of gametogenesis, and thus advance the commencement of the spawning period to an earlier date. Several studies were conducted at the hatchery on conditioning of the calico scallop broodstock. Conditioning regime consists of two phases: Phase 1 involves exposure of scallops to a temperature lower than ambient, stimulating the differentiation of gametes; a temperature differential of 3 °C was sufficient. Phase 2 involves transition of scallops to ambient temperature, and exposure to a gradual increase in temperature to 4 °C higher (for the acceleration of gamete maturation). The evaluation of conditioning is done by routine determination of gonadal indices.
A series of conditioning studies were conducted at the BBSR hatchery, and provides the basis for the standard protocol followed for the calico scallop. These studies demonstrate that the required length of the conditioning regime depends on the gametogenic state of the scallops upon collection. For scallops with an initial gonadal index <1, both phase 1 and 2 have a duration of 30 days; this regime yields an index approaching 3 by the end of the 60–day conditioning, with 100 percent ripe oocytes (determined by histological analyses) following Phase 1. For scallops with an initially higher gonadal index (>1), maintaining scallops in lower than ambient temperature has shown to accelerate gametogenesis within the first two weeks, exceeding the developmental rate of scallops from the wild. Gonadal index doubled in this time. However, for these scallops, Phase 2 should probably be shortened as atretia (or resorption) of oocytes was observed during the latter part of this phase. Spawning induction results show an increased fecundity for conditioned scallops, if atretia is avoided.
The conditioning protocol followed at the Bermuda hatchery, therefore, allows for advancement of the spawning period, by acceleration of the later stages of gametogenesis. Use of gonadal indices is found to be a practical tool supplying reliable information on the reproductive stage of the calico scallops, and may be used as a basis for the timing of spawning induction in the hatchery. In Bermuda, it has been found that a gonadal index of 2 is required for successful spawning induction of calico scallops.
The procedure used in Bermuda is given in detail in Protocol–3.

PROTOCOL–3

CONDITIONING OF CALICO SCALLOP BROODSTOCK

Preparation for procedure
1. Collect 50 scallops from the grow-out site, and transport back to the hatchery in coolers filled with ambient seawater.
2. Upon arrival at the hatchery, transfer 40 scallops to pre-chilled broodstock tank (T= 15 °C)
30 Installation and operation of a modular bivalve hatchery
3. Remainder 10 scallops are maintained in holding tank at ambient seawater for 24 hours. Use this sub-sample for assessment of gonadal and muscle indices the following day. 4. Feed broodstock with a mixture of two or three algal species, depending on availability. Clean 20-litre carboy with jet of fresh water and chlorine, taking extra care to clean drip-feed set up (stopcock valve and tubing). Fill 20-litre carboy and open drip-feed. 5. Record temperature before leaving the hatchery.
6. Do daily checks, recording temperature, flow rate, algal ration supplied and record in data sheet as sampled in Appendix 3.
7. Two weeks later, collect a second sub-sample of 10 scallops from conditioned broodstock for gonadic and muscle indices. Keep scallops in holding tank, not fed for 24 hours prior to dissection.
8. If gonadic index assessment results in GI<2, maintain broodstock at T= 15 °C for another two weeks. Skip to 10.
9. If GI>2, transfer broodstock into an ambient seawater tank. Following 24 hours, increase temperature by 1 °C every two days, so as to reach 22 °C within one week. Maintain high temperature for one week and induce scallops to spawn.
10. From scallops in 15 °C tank, collect another sub-sample of 10 scallops after two more weeks of conditioning (or 4 weeks after start of trial) for gonadal index assessment. GI should be close to 2.
11. Transfer the broodstock into an ambient seawater tank. Following 48 hours, increase the temperature by 1 °C every two days, so as to reach 22 °C.
12. Maintain the broodstock at 22 °C for a period of 30 days. Continue the same feeding regime. Following 30 days, collect a sub-sample of scallops for determination of gonadal indices. At this time, gonadal index should be above 2.
13. Stop feeding for 24 hours. Induce spawning.

1.3.3 Spawning induction of scallops

To date, thermo-stimulation is agreed to be the most efficient method for inducing sperm and ova release (Monsalvo-Spencer, Maeda-Martinez and Reynoso-Granados, 1997), especially in pectinids where a critical temperature range or minimum threshold temperature has been most frequently implicated in the initiation of spawning (Moyer and Blake, 1986; Barber and Blake, 1983). This induction of gamete release by temperature proved most efficient for the calico scallop in Bermuda; it yields a relatively rapid (90 minutes following induction) and efficient (70–90 percent) response to release gametes, compared to responses seen in other species Monsalvo-Spencer, Maeda-Martinez and Reynoso-Granados, 1997). This same method proved even more efficient when applied to the sand scallop, when 90 percent of the animals released both male and female gametes within 20 minutes induction.
The protocol utilized at the Bermuda hatchery differs slightly for both scallop species. Differences lie mainly in the degree of thermal shock provided, and length of shock required. E. ziczac is more sensitive to stress, and hence requires a lower thermal differential than A. gibbus. For both species however, the strategy is the exposure to an initial cold shock, followed by an exposure to a warm shock. Both species release gametes when exposed to warmer seawater temperatures.
As mentioned in the previous section, scallops are induced to spawn when observed to be ripe or close to ripe following methods outlined above. It must be added in this section that the sand scallop responds readily to a thermal shock by releasing gametes; although this response is caused by stress, and release of gametes may occur at times when gametes are not mature. On the other hand, the calico scallop is found to be more tolerant of stress, such that although gametes may be determined to be ripe (GI>2), release does not always occur following thermal stimulation.
This was seen on several occasions in the hatchery over a 4-year period.
Figure 1.8 outlines the number of attempts made from February 20 to April 22 in 2002, with scallops showing a GI>2. As can be seen, only 2 out of 6 attempts resulted in release

Figure 1.8: Spawning attempts with calico scallops, A. gibbus, collected from the grow-out sites.

of eggs within 2 hours of induction. It has also been observed that response can also be very slow with the calico scallop, and that a time of up to 5 hours in a warm water immersion may be necessary for gamete release. Nonetheless, this lack of consistent response for animals known to be ripe renders further investigating of what cues initiate spawning, worthwhile. This is especially true for commercial aquaculture applications, where control of timing of spawning events is necessary.
Protocol–4 outlines in point form, the procedure followed for spawning induction of both species in Bermuda. Generally, extreme care is taken to ensure cleanliness of all equipment and seawater lines used during spawning and larval rearing. All equipment is washed with commercial grade bleach, rinsed abundantly with fresh water, and given a final rinse with filtered seawater (1 µm). Seawater used for spawning baths and for collection of gametes is filtered twice to 1 µm. Set up of the heating system is primordial, as it may take approximately 2 hours to obtain heated seawater for filling of rearing tanks. A saltwater table and bench space are prepared for collection of gametes and counting of eggs prior to distribution into tanks. In both species, sperm release usually occurs first, appearing as a milky white stream. Care must be taken when spawning a hermaphroditic species to avoid self-fertilization. For this reason, attributing a number to a scallop, and keeping the same number as it switches from male to female is important. Scallops are labelled as they begin to spawn; such that the first male is labelled as male 1 on beaker. As soon as egg release is noted (orangey-pink in colour), the scallop is removed from the beaker, the contents of which are discarded, the scallop rinsed, and transferred to a new beaker, labelled as female 1. In this way, sperm from other scallops is taken to fertilize this female. Scallops are changed regularly into new beakers with clean seawater as they spawn, as solutions become very cloudy, and it becomes difficult to observe a change in gamete release (Figure 1.9). As animals are transferred to new beakers, care must be taken to also transfer the label. Sperm or egg solutions are pooled into a larger beaker or bucket for use at a later date.
As the spawn continues, initial sperm may be discarded, as it is advised that sperm <30 minutes old be utilized for fertilization. Two or three males may be pooled, and such a pool may be

Figure 1.9: Isolating sand scallops, E. ziczac, once gamete release is initiated.

used for fertilization, ensuring that the sperm utilized does not correspond to females of same individual. Calico scallops can continue releasing sperm for as long as 3 hours, but normally switch as females after approximately1 hour.
In order to further avoid self-fertilization, addition of sperm to an egg solution is done quickly; as soon as release of eggs is constant and solution in the beaker appears pink. This precaution is advised as scallops may switch back to male spawning unexpectedly, and result in self-fertilization. The volume of sperm added must be noted, as a lower ratio of sperm to eggs has been found favourable to subsequent fertilization and development rate (Gruffyd and Beaumont, 1972). Fertilization rate is enhanced by gentle mixing, using a homemade plunger. In Appendix 8 details of plungers used are shown. Egg counts are made using a Sedgewick-rafter cell, on pools of fertilized eggs. Development to D-larval stage is optimized, by eliminating debris from the egg solutions and distributing the egg solution to larval tanks prior to multi-division stage. As seen in Figure 1.10 a fertilized egg can be recognized by the presence of a fertilization membrane surrounding the entire egg. When fertilized, the egg undergoes meiotic division, at which time two polar bodies are released. For both the zigzag and calico scallop, a round membrane showing successful fertilization is seen approximately 15 minutes after the addition of sperm. The sequence of events was timed for E. ziczac. The first polar body is seen approximately 25 minutes after fertilization. The second polar body is observed 15 minutes later. Cell division, two- and three-celled stages, occurs approximately 75 minutes after the addition of sperm (Figure 1.10). From here on, division continues rapidly. This sequence and timing of events is similar to that reported by Costello et al. (1973) for A. gibbus, and concurs with the observations made at the BBSR hatchery for this species.
Distribution of eggs in the culture tanks is preferably done at the time of the two celled division stage. Fertilized egg solutions are passed through a 150 µm sieve prior to suspension to eliminate debris and/or large clumps of eggs. A more complete procedure for removal of debris is the rinsing of eggs within 20 minutes of fertilization; eggs are passed through a 105 µm screen and collected on a 35 µm screen. The eggs are rinsed with filtered seawater while on the smaller screen to remove additional debris and excess sperm. The smaller screen should be submerged in a tray of water, so that the eggs are not pressed against the screen, as they are collected. After the fertilized eggs are rinsed, they are re-suspended in a known volume of filtered seawater. Sub samples are collected to estimate the number of eggs collected. Care must be taken that the distribution of eggs into the larval tanks must be done prior to the multi

Figure 1.10: Sequence of events following fertilization of E. ziczac eggs.

cellular division stage. At this point, eggs become more fragile, and development may be hindered if eggs are passed through a sieve. It is therefore advised that should distribution occur later on, the process of passing eggs through a sieve be eliminated.
Finally, the keeping of records for all procedures, including transfer times between baths, first release of sperm, egg counts, etc., is important in understanding any problems arising at a later date.

PROTOCOL–4

SPAWNING INDUCTION

1. Set up heating tank (see Appendix 4).
2. Install a second 1 µm filter cartridge and housing inline with 20 mm ID reinforced hose for ease of supply to seawater baths and receiving beakers.
3. Install a 1 µm filter cartridge and housing inline to larval tanks.
4. Fill three trays with double filtered 1 µm seawater.
5. Prepare one cold-water bath (14±1 °C for sand scallop, 11±1 °C for calico scallop). Fill 1 litre zip-lock bags with ice cubes, and place in seawater tray. Once temperature is reached, remove all bags except one to maintain temperature constant.
6. Prepare two warm water baths by filling with 1 µm filtered seawater and immerging 1 250 W aquarium heater in each tray (see Figure 1.11).
7. Prepare beakers for collection of gametes. Fill as many trays as possible (at least 4) to 1/3 with heated 1 µm filtered seawater. Place one heater per tray so as to maintain temperature at 25 °C.
8. In each tray, place a maximum number of 2 and 3 litres beakers filled with heated seawater.
9. 4 litres beakers and/or 1 000 ml graduated cylinders are kept empty for pooling of gametes.
10. Once trays and beakers are ready, start filling larval tanks with double filtered 1 µm seawater.
11. 1st cold-water shock – 30 scallops are selected for spawning. All scallops are placed in the cold-water bath for a period of 30 minutes. Record time and temperature
12. 1st warm-water transfer – Scallops are divided into two and transferred to a warm water bath for a period of 1 hour. (For sand scallop, response usually occurs within 20 minutes of this first warm water bath).
13. 2nd cold-water shock – Following 1 hour, transfer animals back to cold-water bath. Make sure that water is clean (free of detritus or faeces), and temperature is same to initial cold-water bath. If necessary, prepare a new bath while scallops are in warm water. Record time and temperature. Similarly this cold-water shock only lasts 30 minutes.
14. 2nd warm-water shock – Transfer the batch of scallops to the warm-water bath for a second time, and leave for a period of at least 2 hours. Record time and temperature. Calico scallops usually respond following the first hour of warm-water bath. Should no response be seen, attempt a 3rd cold-water shock, and a 3rd warm water shock; start flow of water in warm-water bath, and leave scallops. Check for gamete release for next 5 hours.
15. Once a scallop is seen to release gametes, leave for a few minutes in water bath. This may trigger spawning in other individuals. Remove the scallop, rinse with 1 µm filtered seawater to clean off sperm or eggs from shell, and thus preventing self-fertilization. Place scallop in a beaker, label and allow spawning to continue.
34 Installation and operation of a modular bivalve hatchery
16. Once solution is cloudy, transfer scallop to new beaker. Keep sperm until no longer required.
17. Once scallop switches to female, rinse scallop and transfer to new beaker. Discard previous solution if suspecting self-fertilization. Label female with same number as initially given to male.
18. When solution becomes orange-pink, add a mixture of sperm using a Pasteur pipette to egg solution – 1 ml of sperm: 1 litre of eggs.
19. Mix sperm with egg, by a gentle up and down motion with plunger (see Appendix 8 for details).
20. Remove scallop from egg solution, and place into new beaker when solution becomes too thick.
21. Pool two or three egg solutions into a 10 litres bucket.
22. Count eggs from pools – Take a known volume of egg solution with an Eppendorf pipette preferably; 100 µl aliquot is usually sufficient and place on a Sedgewick Rafter cell. Count using a compound microscope. Start at one end of the cell, scanning up and down, to avoid counting the same egg twice. Do triplicate counts (see Figure 1.12).
23. Calculate number of eggs as follows: (number of eggs.ml-1 x volume (l) of beaker or bucket) x 1 000.
24. Distribute calico eggs at 15 eggs.ml-1 and zigzag eggs at 10 eggs.ml-1 to larval tanks. No aeration is required for the first 24 hours.
25. For distribution to the rearing tank pass fertilized eggs gently through a 150 µm sieve before they reach the multi-division stage.
26. Once all eggs are distributed in tanks, transfer spawning animals to outdoor holding tank. Ensure a high water flow. Allow complete release of gametes.
27. Wash all equipment with commercial bleach solution and rinse with fresh water. 28. Heating tank unit and hatchery pipelines are cleaned following the procedure in Appendix 7.

Figure 1.11: Warm water bath set-up for spawning induction of scallops.

Figure 1.12: Measuring eggs or larvae on a Sedgewick-Rafter cell.