ARTICLE ON FISH EGG MANAGEMENT (E-book)

Fertilized seabass and gilthead seabream eggs float in water with 35 to 37 ppt salinity. With lower salinity egg buoyancy decreases and a strong aeration is advisable to prevent their sinking to the bottom, which would pose a big risk in terms of physical stress and bacteriological contamination.
Because egg quality represents a crucial factor for hatchery success and for production of fry of good quality, any stress to fertilised eggs should be avoided, such as:

• physical shock during egg collection, transport, disinfection, weighing and incubation;
• thermal and salinity shocks. It is recommended to maintain in the incubation tanks the same environmental conditions at which eggs have been exposed during spawning. The same precaution should be adopted when packing eggs for sale.

Before dealing in detail with egg management and larval rearing of seabass and gilthead seabream, the following paragraphs give a brief description of the development stages from egg to juvenile.
What follows takes place as close as possible to spawning temperature, characteristic of each species, up to completion of swimbladder primary inflation and at 17-18°C afterwards. Larval length indicated is equivalent to total length, age in days starts from the hatching day, considered day zero.

Gilthead seabream eggs and larvae development

• Gilthead seabream eggs are spherical with a diameter range of 0.94 - 0.99 mm and have a single large oil droplet. Hatching starts approximately 48 hours after spawning at 16 - 17 °C (common range) and the newly hatched larvae have a total length of around 3 mm.
• At hatching the yolk sac occupies approximately 1/3 of the larvae length. The eyes are not pigmented, the mouth is closed and the larvae have very few melanophores on their body. The pectoral fins develop two days after hatching.
• Within the following three to four days the body pigmentation increases, the mouth opens and as the yolk sac is consumed its volume is progressively (more than 50 %) reduced. The larvae length is around 3,8 mm and the eyes are completely pigmented. The swimbladder starts inflating when larvae reach 4 mm, at which length the yolk sac is completely reabsorbed.
• By day 15 (at 5 mm length), the swimbladder primary inflation is completed; by this time the cauda fin develops and extensive body pigmentation appears.
• By day 20 the anal fin and the stomach develop. By the day 35 a complete pigmentation of visceral organs appears.
• Between day 45 and day 50, the dorsal and ventral fins develop and scales appear. The swimbladder expands progressively to its definitive size.

Seabass eggs and larvae development

Fig.41.01 Seabass eggs and larvae development (photo Barnabè)

• Eggs are spherical with a diameter range of 1.1 - 1.25 mm. Hatching starts approximately 72 hours after spawning at 13 - 14 °C (common range) and newly hatched larvae have a total length of around 4 mm. The yolk sac is almost 1/2 of the whole body length and the oil droplets are smaller than in seabream and frequently two or more are present. Eyes are not pigmented, the mouth is closed and larvae have evident melanophores on their body. The pectoral fins develop two days after hatching.
• Within the following three to four days the body pigmentation increases, the mouth opens and the yolk sac size progressively reduces (more than 60 %) its volume. The length is around 5 mm and the eyes are now completely pigmented. The swimbladder starts inflating when larvae reach 5,5 mm, at which stage the yolk sac is completely reabsorbed and the oil droplets only partly.
• By the day 16 (6,5 mm length) the swimbladder primary inflation is completed; by this time the oil droplets of the yolk-sac have been completely reabsorbed. Extensive body pigmentation is observed.
• Between days 20 and 35 the caudal and anal fins, the stomach, and the teeth develop. The swimbladder expands progressively to its final shape.
• Between days 40 and 45 the dorsal and ventral fins develop. Scales appear at 70-80 days.

Egg harvest

As fertilised seabass and gilthead seabream eggs float in full seawater, most Mediterranean hatcheries have adopted the automatic egg collectors described below. If well dimensioned and properly placed, these devices harvest only the floating eggs, while the dead or unfertilised ones sink to the bottom. A few important precautions should be taken into consideration. The presence of eggs in the collectors should be checked rather frequently in the case of seabass, as its spawning produces a large amount of eggs in a very short time and there is risk of clogging the collectors or of mechanical stress to the eggs. Due to the waste produced by spawners in their tanks, egg collectors have to be kept properly cleaned and should be replaced at least daily with sterilised ones. The water flow should be adjusted so as to gently transfer eggs from the spawning tank into the egg collector without harmful mechanical shocks.
The overflow collector is placed outside the spawning tank. It consists in a screened container that receives the water of the spawning tank by overflow and that is placed inside another container. The collector is usually a PVC cylinder with large lateral and bottom openings screened by a 400 µm mesh size nylon net. A flexible hose connects the spawning tank with the open top of the screened cylinder, assuring that the surface water from the spawning tank flows into the egg collector. The water level inside the collector tank is maintained by another overflow outlet in a way that it is only few centimetres below the level in the spawning tank. Eggs retained by the screen are kept floating by water flow and a gentle aeration.
The airlift collector is a device placed inside the spawning tank. It is basically a screened box or bucket equipped with floaters and small airlifts. These airlifts are PVC pipes that transfer the surface water of the spawning tank into the collector by means of an air flow. They are connected to the low-pressure air distribution system of the hatchery, and their flow is adjusted to gently lift the floating eggs, sparing them from any mechanical stress.
To take out the eggs from both types of collectors, the aeration and water flow have to be stopped. Viable eggs are allowed to float freely in still water. In this way a first separation between sinking dead eggs and viable ones takes place. To minimise the presence of poor-quality eggs, which usually float deeper in the water, it is advisable to collect only the eggs found at the water surface, after a period of settling without aeration, that in any case should not exceed 10 minutes to avoid risk of anoxia. Then with a 1-l jar scoop the eggs from the water surface and place them directly in a temporary stocking vessel (that could be a bucket or a conical container). This vessel should have been previously filled with sterile water at the same temperature and salinity and should be provided with a gentle aeration through a fine air stone. A floating layer of eggs thicker than one cm should be avoided. A thicker layer may reduce oxygen supply to the eggs, leading to possible anoxia after a short time. When in the temporary container, eggs must be thoroughly examined to assess their quality, number and development stage.
Table 3.7 Comparative development of seabass and gilthead seabream larvae and postlarvae

	Seabass		Gilthead seabream
Age	Tot. length	Observations	Tot. Length	Observations
Days	mm		mm
1	4	Hatching	3	Hatching
2	4,5	Appears pectoral fins	3,5	Appears pectoral fins
3			3,8	Exotrophy starts
4				Eyes pigmentation
				60% of yolk sac reabsorbed
				40% of drop reabsorbed oil droplet
5	5	Exotrophy starts	4	Primary swimbladder inflation
		Eyes pig mentation		100% of yolk sac reabsorbed
		60% of yolk sac reabsorbed		70% of oil droplet reabsorbed
		40% of oil droplets reabsorbed
7	5,5	Primary swimbladder inflation
		100% of yolk sac reabsorbed
		60% of oil droplets reabsorbed
15			5	End of primary swimbladder inflation
				100% of oil droplet reabsorbed
				Caudal fin
16	6,5	End of primary swimbladder inflation
		100% of oil droplet reabsorbed
17			7	Anal fin
20	8	Caudal fin	7,5	Stomach starts developing
25	12	Anal fin
35	14	Teeth
		Stomach starts developing
40	15	Second dorsal fin
45 50	20	First dorsal and ventral fins	11 15	Second dorsal fin First dorsal and ventral fins
60-70			20	Scales
70-80	30	Scales
		Definitive morphology
90			30	Definitive morphology

Quality controls

A reliable egg control quality needs usually just a few dozens of eggs, which are placed under a microscope (10 to 100x magnifications), or a transmitted-light stereomicroscope. With a pipette they should be taken from the floating egg layer in the temporary container, and should be placed on a watch-glass or on a Petri dish, making sure that the eggs form a single layer.
Check for the following egg characteristics:

• absence or occasional presence of dead (opaque, whitish) or unfertilised (transparent, but without evidence of cell divisions) eggs in the sample (less than 5% of the batch normally),
• regular rounded shape and size (diameter in gilthead seabream: 980-990 µm, in seabass: 1 000 -1 250 µm), regular cell division that can be observed only in the first blastomers;
• regular shape of vitellus (it should occupy the egg volume entirely, without perivitelline space),
• crystal clear transparency (no superficial spots and dark areas),
• proper number and size of oil droplets (in seabass from 1 to 6 of a 400-440 µm diameter; in gilthead seabream one droplet with a diameter of 230-240 µm),
• absence of parasites or associated micro-organisms on the chorion surface.

An early estimate of irregular or aborted eggs can be made at this stage. Any irregular egg will soon develop into an abortive embryo or an abnormal larva. Spots on the external chorion account for physical or bacterial damages. Sometimes, the vitellus colour is yellowish, apparently due to broodstock husbandry/feeding regimes. In such case, eggs with a yellowish vitellus are considered to be normal.
As a general rule, good egg batches have usually less than 10% abnormal eggs. Any batch containing more than 20% abnormal eggs should be discarded.
A batch of eggs with an abnormality rate between 10 and 20% may be accepted only if there is a severe egg shortage, or when eggs are imported from other hatcheries. In this case, the management has to take into account a higher mortality rate at hatching and during the early larval stages. Otherwise, such poor egg batches should be replaced as soon as possible by better ones.
Dead and unfertilized eggs are thus discarded twice, first in the collector where only floating eggs are scooped up, and then in the temporary container before counting and disinfection. But it is also advisable to select only good eggs. Poor-quality eggs usually float deeper in the water. Collect only the eggs found at the water surface, after a repeated decantation without aeration.
In case of evidence of severe infestation by parasites or undesirable micro-organisms (ciliates, flagellates, nematodes, etc.) the eggs should be discarded to avoid contamination risks in the larval rearing sector.

Fig.42.01 Overflow collector (photo STM Aquatrade)

Weighing, disinfecting and counting eggs

Prior to stocking eggs, either into the hatching facilities or directly into the larval rearing tanks, three more steps are required: weighing, estimation of their quantity and disinfection.
Even a rough estimate of the egg numbers allows the person responsible of the larval rearing sector to properly plan the stocking of the larval tanks, to optimise production routines and to coordinate the work of related sectors (live feeds and weaning). It does also allow a proper evaluation of the final survival rate to be expected.
The procedure to weigh the eggs consists in dividing them in many sub-samples, taking each of them out of the temporary container in a plastic filter removing quickly the excess of water, and weighing them on a balance calibrated for the egg filter tare. They are immediately returned to the temporary container (or disinfected, see below). During the collection of the eggs for this operation, only floating, viable eggs are picked. Dead and unfertilized eggs are thus discarded twice, a first time in the collector and then on this occasion.
Egg disinfection is the very first effective barrier against transmission of fish diseases, and is therefore highly recommended for all batches of eggs, both those produced in the hatchery and those brought from other hatcheries. This important operation is usually conducted just after the weighing, when the filter containing the egg sub-sample is dipped in the disinfecting bath for a short period of time prior to being put into the incubation tank. The most commonly used egg disinfectants are Penicillin G, Streptomycin sulphate and active iodine. Even if these antibiotics are commonly used, due to the undesirable side-effects they have and the risks to induce bacterial resistance, active iodine is suggested as the preferred disinfectant.
Table 3.8 Disinfectants used for seabass and gilthead seabream eggs

Active substance	Dosage	Time	Usage
PENICILLIN G	80 I.U./ml	1 min	500/10 l of sea water for 100-200 g of eggs at a time
STREPTOMYCIN-SO4	50 mg/ml	1 min	500/10 l of sea water for 100-200 g of eggs at a time
ACTIVE IODINE	50 ppm/litre	10 min	8 litres for: 1 x 106 seabass eggs or 1.5 x 106 gilthead seabream eggs

The assessment of egg numbers can be made in two ways: by relating number to weight or by counting. In the first case the total egg weight is divided by the average individual egg weight, assessed from a small sample. The second method contemplates counting the eggs present in a few 1-l sub-samples and multiplying the average value by the total tank volume. While the water taken with the egg samples biases the first method, the latter requires a uniform egg distribution in the tank to be statistically correct. This method can also be applied to count freshly hatched larvae, which gives a better estimate of the initial population.
The protocol to weigh, count and disinfect eggs is given in Annex 12.

Incubation of eggs

Egg incubation can take place either in dedicated incubation tanks or directly in the larval rearing tanks. The latter choice has some drawbacks that fully justify the inclusion of a separated hatching sector as the ideal solution. After hatching, only the hatched larvae are moved to the clean larval tanks, whereas the hatching facilities are easily emptied, washed, disinfected and refilled for the next egg batch. Moreover, in this way the management of egg batches with poor hatching rates is facilitated by the smaller size of the hatching facilities.

Egg incubation in dedicated facilities

To incubate eggs, plastic or fiberglass round tanks with a conical bottom and a 100 to 250 l capacity represent the most common technical solution adopted by Mediterranean hatcheries. The cylindro-conical shape gives a good water circulation pattern, provided that a central aeration source is placed near the tip of the conical bottom, and a better separation of not viable eggs and hatching debris. Their inner surface is smooth to prevent any damage to eggs and newly hatched larvae.
Before being stocked with eggs, these tanks are carefully cleaned and disinfected with a hypochlorite solution (following the same procedure used for rotifer or artemia tanks), including the inlet/outlet pipe system and the submerged aeration devices (diffusers and tubes). Each tank is then filled with filtered and sterilized sea water, taking care to check that the temperature and salinity are the same as in the spawning tank from which the eggs originate. The incubation tanks should be part of a flow through (open) water system, i.e. the outlet water should not re-enter the tank, even if filtered, in order to eliminate the hatching by-products and also potentially dangerous micro-organisms, frequently associated with eggs (see previous chapter). Water exchange rate depends on egg density and has to be adjusted so as to provide enough dissolved oxygen (at 100% of saturation) to the floating eggs without crowding them against the outlet screen.
The outlet is usually screened with a removable 400-µm mesh nylon net. A continuous aeration creates a gentle current all around the screen to prevent its being clogged by eggs or larvae pushed against the net by the outflowing water. Besides that, additional aeration is provided from the bottom of the tank mainly to keep eggs and larvae in suspension and to avoid water stratification. The photoperiod is the same one applied to the larval rearing sector. Recommended stocking density ranges from 10 000 to 15 000 eggs per litre. Water turnover is maintained at one total renewal per hour during the incubation period, a rate that doubles during and shortly after the hatching time.

Fig. 43.00 Fiberglass cylindroconical tank used for eggs incubation (photo STM Aquatrade)

Egg incubation in the larval rearing tanks

When eggs are incubated directly into the larval rearing tanks, two methods can be followed:

1. direct stocking of eggs into the rearing tank, where they occupy the entire water volume;
2. egg stocking into screened floating containers, placed inside the rearing tank.

The first method requires a density not exceeding 200 eggs/litre, it has an easy follow-up and does not need additional equipment, but it is not free from drawbacks. After hatching, the tank bottom must be carefully cleaned using a siphon to remove hatching debris, as they represent a good substrate for bacterial growth. This cleaning process is a time consuming matter, a hard to complete procedure that also causes the loss of many larvae. Moreover, if incubation or hatching is unsuccessful, the entire larval tank has to be emptied, washed, disinfected and refilled. In addition to this waste of time, one should also consider the few days lost when the tank was occupied by the batch of eggs discarded.
The second method allows a better control over the incubation process and could be seen as a partial adaptation of the above mentioned incubation tanks. However, the inconvenients of the first method are not entirely eliminated.
A floating incubator can be easily made with a 30-l screened plastic bucket. A 400 µm mesh size nylon net is glued to side and bottom openings. Buoyancy is provided by sealed LDPE pipes or by pieces of polystyrene foam. The incubator is equipped with an aeration device made with a couple of fine diffusers connected to the aeration system to keep the eggs moving gently in the water. Inside each bucket water renewal is assured by tank water passing through the mesh. If needed, it can be increased by a pair of airlifts attached to the outside of the bucket. Water circulation inside the larval rearing tank should be adjusted to provide a gentle current through all incubators.

Fig.43.01 Large incubation facilities in Nuova Azzurro hatchery (photo STM Aquatrade)
Once hatched, the yolk-sac larvae are easily counted and then released into the same rearing tank, by gently tilting the floating bucket, having stopped the aeration to facilitate their release. In this way the hatching debris remain inside the incubating bucket and can be then disposed of easily. Egg density is maintained at 6 000 to 10 000 eggs/litre, and though still linked to the water exchange rate inside the bucket, it far exceeds the density foreseen for the same rearing tank in absence of incubators.

Fig.43.02 An outdated hatchery layout were eggs incubates directly in the tanks (photo M. Caggiano)
Incubation lasts for about 50 h at 18°C and 36 h at 22°C.This time varies also according to the egg development stage at stocking (see Annex 13).

Fig.44.01-2-3 Sea bream larvae development within 12 and 36 hours (photo STM Aquatrade)

Hatching

A few hours before the estimated hatching time, water renovation in the incubation tank should be increased to two complete exchanges per hour, paying attention to avoid the clogging of the outlet filter by the eggs.
As soon as hatching begins, environmental parameters should be reset according to the indications contained in Annex 13. Care should be taken to flush out or remove hatching debris and by-products. Amongst the latter of particular importance are proteolithic enzymes, active during egg opening, which may damage the freshly hatched larvae, not protected any more by the chorion.
One day after hatching the viability of young larvae is assessed. In the good batches it remains above 80%. If possible, discard egg batches with a viability rate lower than 80%.

Viability of newly hatched larvae

Before being stocked in the larval rearing tanks, the newly hatched larvae are checked to assess their viability and condition. The evaluation criteria listed below refer to the shape of the larva and to its behaviour.
From each batch, at least 10 to 20 specimens are sampled (the procedure is described in detail in Annex 14), and are then placed on a watch glass in a water drop and observed under the stereomicroscope at low magnification (5 and 10x). They are checked for normality in respect of:

• overall shape and dimensions;
• integrity of the larval (primordial) fin, that should not present malformations and/or erosions;
• absence of external parasites;
• disposition of internal organs;
• proper pigmentation.

In their very first days, the larvae of both species show a typical behaviour and its observation contributes to evaluate their viability. Examination of this behaviour can take place directly in the hatching tank, as well as on a sample of at least 50 larvae taken in a transparent container.
The larval motion is a sort of passive floating with sudden, and infrequent body movements without assuming any clear posture. Typically they sink slowly, head first, and then, every few seconds, swim upwards for two to three seconds. A difference in buoyancy can be noticed between gilthead seabream and seabass larvae, the latter being more buoyant due to their bigger oil droplet in the yolk sac. See Annex 15 for an example of recording data sheet for observations.
Larval fish showing a different pattern (being totally passive or hyperactive), reveal poor viability and should better be eliminated. Such different behaviour should also not be dismissed as being only the result of a poor larval quality, in fact it may also point out the presence of a toxic pollutant in the water circuit. If the same abnormal pattern is observed in several batches of larvae produced one after another, it is probably a sign of pollution problems.

Larval transfer to the rearing facilities

The larvae hatched in the incubation tanks are easily transferred to the larval rearing tanks. To make this operation easier and faster, it is advisable to stock the incubator with enough eggs so as to get the right amount of larvae to stock one larval rearing tank. In this way the larvae produced by an incubator are transferred into one larval rearing tank.
For the transfer proceed as follows:

1. stop water exchange and aeration in the incubator;
2. after a few minutes (5 to 10 maximum), drain the settled hatching debris through the bottom valve;
3. restart a gentle aeration and lower the water level keeping the outlet screen in place;
4. harvest the larvae either via the bottom valve into several partially filled buckets (avoiding any mechanical stress, minimising the difference between inner water level and water level in the bucket), or using a 5 litres jug directly dipped in the incubator. Mechanical stress should be minimised by avoiding splashing.

No special operation is needed when eggs are incubated directly in the larval rearing tanks, except that hatching debris must be carefully siphoned out as soon as the hatching ends and the aeration and water exchange are adjusted to the new situation.
Starting soon after hatching, seabass larvae school in dense clouds at the water surface because of the early development of a continuous swimming activity. Gilthead seabream larvae, on the contrary, have a more pronounced tendency to sink, and their almost complete passivity accounts for their more uniform dispersion in the water body.
Such irregular swimming pattern evolves progressively towards the more active and continuous movements characterizing first-feeding larvae, coinciding with the appearance of fully developed visual and digestive organs. As a matter of fact, and at similar temperature, seabass larvae develop a correct predatory position earlier than gilthead seabream larvae. Due to their being relative passive, significant samples can be easily collected to carry out quantitative analyses to determine, for example, their total number and the percentage of deformities present in the batch. Abnormal larvae often show irregular swimming, an irregular shape or a twisting of the body.
Soon after hatching the eyes are not yet pigmented, the mouth is still closed and the digestive tract is still incomplete. During this period the larvae survive on the reserves of their yolk sac.

Fig.45.01-2 Seabass larval development (from Barnabè)