A. Obtaining Eggs and Larvae

The collection of a significant number of eggs in the field can be an important element of any study and almost always implies that spawning has actually occurred. Eggs can also be obtained by spawning fish caught from an aggregation, either naturally or artificially. If the eggs are fertile and undamaged, they would permit the description of the egg size and characteristics for species if this information is not already known. The ascent rate (most eggs are positively buoyant) and hatching times at a particular temperature can be determined using these fertile eggs. They can be hatched for larval rearing attempts. Finally eggs can be used for other experimental purposes. As just one example, large quantities of surgeonfish eggs were collected from spawning aggregations to examine the effects of diesel fuel on hatching success (PLC).

Collecting Eggs - Net and Bag Techniques

A number of methods can be used to collect eggs in the field and there is a substantial literature available for reference. Here we discuss methods applicable to spawning aggregation work. The simplest is by straining the water where eggs have been released with a fine mesh net (Colin and Clavijo, 1988; Colin 1983, Colin and Bell, 1991). One of us (PLC) prefers a small hand net about 10 cm across at the mouth and made with plankton netting of about 250 micron mesh (Fig. 48). The net has a short handle, about 15 cm long. This net can be swept through the volume of water where eggs have been released and then the entire net put into a plastic bag, and the netting everted as it is withdrawn, resulting in the eggs being deposited inside the bag. If desired the net can simply be left inside the bag until removal in the lab. Generally it is best to only about half fill the bag with water, then tie a knot in the neck, so the eggs are retained and unlikely to leak away (unless the bag is punctured). They can be returned to the lab in the plastic bags.

Figure 48. (Left) Quantity of reef fish eggs collected by moored plankton net in only one hour, Lighthouse Reef, Palau. The material on the bottom of the jar is almost 100% fish eggs. (Right) Typical fine mesh hand net used by diver for collecting eggs after spawning. Mesh is 220 micron and the net is about 15 cm across.

Similarly Kiflawi et al. (1998) collected eggs of Acanthurus nigrofuscus aggregations using a 100 micron mesh net with a 20 cm diameter frame and a 1 liter plastic bottle on the cod end from gamete clouds released by spawning. The bottle had two sides with 300 micron mesh so water would flow through the bottle. Samples were collected by sweeping the net through the area of gametes for 5-10 sec, then the bottle would be removed, capped and put in a ‘zip-loc’ plastic bag (has a plastic fastener that seals tightly). The net could be cleaned, presumably by everting it, a new bottle installed, and a new sample collected.

Plankton nets moved through the water by divers have also been used to collect eggs. For example, Samoilys (1997b) collected coral trout (Plectropomus leopardus) eggs at Scott Reef, Great Barrier Reef, using a plankton net of 700 micron mesh towed by divers. While this will work and they usually have a larger mouth size than hand nets, they are hard to handle and often have to be brought to the surface with each sample. A diver-operated plankton net is not an efficient way to capture large numbers of eggs. A small net can be used over and over, and each sample deposited in a separate plastic bag.

Shapiro et al. (1994) used large plastic bags (about 67 by 60 cm holding up to 50 l of water) to collect spawns of Thalassoma bifasciatum in an effort to capture all eggs and sperm released by these relatively small fish. Using such large bags for collecting eggs has the advantage of having less effect on the normal process of fertilization, compared to collecting eggs with a net, but such bags are difficult (weights would be approaching 50 kg) to remove from and handle out of the water (Marconato et al., 1997). Given the documented effects of collection methods on fertilization rates (egg damage or collecting eggs too quickly-generally less than one minute after spawn) with a net, investigators are advised to choose their methods carefully with 72 consideration of what they want to do. If the purpose of egg collections is to document egg size or perhaps collect eggs for rearing work, net collection may be preferable, while studies of fertilization rates and sperm numbers would probably benefit from collecting eggs in large plastic bags. Some aspects of these are discussed further below.

The potential for damage to eggs collected by net techniques is real. Petersen et al. (1992) collected eggs using hand nets, and found damage to eggs by this technique. Kiflawi et al. (1998) found some evidence of damage to eggs by the net, but many eggs were not damaged, hence such samples could be used to document the size and characters of eggs. If fertilization rates are being examined, or some other aspect of spawning that requires all eggs be undamaged, then such net damage becomes an issue. The time between gamete release and collection with a hand net may be the critical factor in egg damage by the net. The longer the time elapsed after release, the less egg damage will occur. Also all "plankton mesh" is not the same. The nitex mesh normally used in plankton nets is fairly rough, particularly when new, and may cause abrasive damage to eggs. The mesh used in "brine shrimp nets" in the aquarium trade, is softer and may not damage eggs as much (Colin, 1982)

It is likely that increasing the time between gamete release and net collection of eggs reduces the mechanical damage to eggs during collection. The delay needs to be balanced against the dispersion of the eggs after release. Fertilization must have occurred or at the least the sperm penetrate the egg before the eggs can be collected. This happens very rapidly in most reef fishes and generally if about 1 min has elapsed after spawning, the eggs will be fertilized and can be collected. If you jump the gun too quickly, though, the eggs captured will probably be infertile and fertilization rate can not be assessed. For many aggregation-spawning fishes, the gametes released remain visible, particularly with copious amounts of sperm released, for a minute or more, making it is easy to track the location the eggs. For those species that spawn as pairs out of an aggregation, or that do not have a highly visible gamete cloud, marking the water where the eggs were released with a dye is often useful. We have used both ink and fluorescein dye successfully, dispensing a bit of the marker just after egg release a short distance away from the actual focus of the gamete cloud. After waiting the required time, the hand net is swept through the dyed area and all the volume of water around it. Sweeping in a figure 8 motion is often effective in hitting the egg patch. The eggs are then deposited in a plastic bag as described previously. Fluorescein dye in high concentrations can have a detrimental effect on fertilization (PLC pers. obs.), however, so ideally the dye is released a short distance from the gamete cloud to minimize any effects.

Where there is a distinct current associated with spawning and the fish are releasing eggs near the surface, it is possible to use a moored plankton net some distance down current from the aggregation to collect eggs (Fig. 49). Colin and Hamner (in prep) did this at Lighthouse Reef, Koror, Palau and were able to capture many thousands of eggs about 80 m down current of the spawning area (Fig. 48). Disadvantages of moored nets include not being able to collect specific spawns and often the eggs taken have a mix of many species.

Obtaining Eggs - Artificial Fertilization - Spear and Strip Technique

It is also possible to strip eggs and sperm from fish speared or otherwise caught from the aggregated fish at the time of spawning, mix them and obtain fertile eggs (Fig. 50). Colin et al. (1987) used this "spear and strip" technique to acquire fertile eggs from red hind, Epinephelus guttatus, assembled in an aggregation. Fish were initially stripped underwater after spearing by placing them inside plastic bags, squeezing to expel the gametes and mixing them. Fish were also brought to the surface and stripped shortly thereafter. Finally they were held at room temperature

Figure 49. Moored plankton nets on Lighthouse Reef, Koror, Palau used to collect eggs of fishes spawning upcurrent (from Colin and Hamner, in prep). A. Plankton net moored over a shallow reef flat. B. Net moored over deep water. The frame has a four-point bridle, with the upper portion supported by buoys so that the net fishes at the surface.

for a few hours after death, and successfully stripped and fertilized. All variations were successful, but stripping immediately produced a high fertilization rate with reduced rates after longer delays. Colin and Clavijo (1988) stripped gametes from a number of species of speared reef fishes, including aggregating acanthurids, to obtain fertile eggs, while others (Randall and Randall 1963) have done additional species.

Rimmer et al. (1994) obtained eggs and sperm from coral trout, Plectropomus leopardus, speared from an aggregation site at Scott Reef off Cairns, Australia. The sex of fish could be determined by color differences and the selected sex speared and brought to the surface. Gametes were stripped using firm hand pressure to the ventral surface of the fish. Milt was collected in disposable syringes and stored at 5-10o C for up to 2 hours. If milt was not readily flowing, the testes of the fish were removed and macerated to obtain milt. Eggs were stripped into a plastic bowl and their volume measured in a graduated cylinder. Milt was added and sperm activated by the addition of 100 ml of seawater. The eggs, milt and seawater mixture was swirled for 15 min. Afterwards the eggs were placed in a clean plastic tray with a 120 micron screen base and washed in clean seawater to remove excess sperm. The clean eggs were then placed in a 2 l transfer container and aerated during transfer (3 h) to the hatchery. Fertile eggs were obtained on several occasions with these methods, with fertilization rates of 5-85%.

Obtaining Eggs - Artificial Fertilization Techniques - Live Fish

Kiflawi et al. (1998) also obtained fertile eggs by stripping gametes from live fish captured at an aggregation. They found that stripped eggs may be "aged" (not exposed to sperm) a few minutes without losing their ability to be fertilized, as long as fresh sperm were added. If both eggs and sperm are allowed to "age", fertilization capacity drops greatly 20-30 sec after stripping. For field collected samples, they found eggs collected from the water less than 20-30 sec after spawning had low fertilization rates (60-90%), while those taken after 25-30 sec had high success, typically near 100%.

When ripe fish are not available, or are needed for mariculture work, it is often expedient to induce maturation of gonads by hormone injections. While not generally within the scope of the present manual, there have been numerous instances where fishes caught from spawning aggregations have been artificially spawned. Since such fish are usually in an advanced state of readiness for spawning, it takes little effort to fully induce them to gonadal maturation. Colin (1992), Colin et al. (1996), Tamaru et al. (1996), and Head et al. (1996) used such techniques to obtain fertile eggs.

Figure 50. Stripping of gametes from Nassau grouper caught from spawning aggregation and held in tanks on board a vessel at the site. (PLC)

If fish are obtained from fishermen or by fishing, and you wish to retain them alive, it is often necessary to release gas from the swim bladder if the fish have been brought up from 10 m or more. This is best done with a large gauge hypodermic needle stuck into the swim bladder, releasing the excess gas pressure. In the Bahamas fishermen often simply stuck a thin bladed knife into the sides of Nassau grouper in the area of the swim bladder and twisted the blade 90o , releasing the gas pressure. The fish were then put into live wells and survived for at least a few days. No matter what method is used, once stable, the fish can be held in tanks. For females, the disturbance of being captured, deflated and put into a tank often stops the process of egg maturation. It has been our experience with Nassau grouper and others that if nothing is done, the eggs will not mature and females will become "egg bound" and often die. A series of hormone injections are generally used to push the final maturation of the eggs. This might include hormone injections for both species, but often running ripe males need no further inducement, if stripped within a few days, while using hormones to cause females to mature ova. Testes removed from ripe males (such as those obtained from a fisherman who wishes to sell the catch) can often be stored for up to several days in a refrigerator or cryopreserved using liquid nitrogen for use when hydrated eggs are available. The testes of male red grouper, Epinephelus morio, obtained from fishermen during the spawning season were stored in plastic bags on ice for up to several days. When needed, a portion of the testes was macerated and mixed with seawater, then used to fertilize freshly stripped eggs from hormone-injected females (Colin et al. 1997).

The appropriate levels of hormones need to be injected and if excessive, can significantly shorten the life of the fish, an important consideration for mariculture broodstock. There is a large mariculture literature for appropriate materials and dosages that should be consulted for more details on hormonal induction of spawning.