Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND SUBSTANCE FOR THE INCUBATION OF EGGS
AND LARVAE OF FISH AND CRUSTACEA
The invention applies to a method and substance for the incubation of
eggs and larvae of fish and crustacea and similar organisms.
BACKGROUND
When traditional incubation methods are used, the mortality rate
among fish and larvae is high due to impact and other mechanical forces. This
is a problem for both pelagic (freely-suspended) and demersal (sea bottom
resident) fish eggs.
Certain types of larvae are extremely sensitive to mechanical
forces. This is particularly the case for halibut, where the incubation of
the larvae prior to start feeding seems to be a problem for the production of
fry.
In some situations, traditional approaches necessitate keeping the
salinity of the water at a high level to prevent the egg and larvae sinking to
the bottom. From an energy perspective, it would have been more rational to
incubate the eggs or larvae at lower salinity levels, since these organisms
would then use less energy for ion-control.
Another substantial problem with traditional incubation systems is
that eggs/larvae are susceptible to fungal and bacterial infection.
A further difficulty with present systems is the maintenance of
eggs/larvae during vaccination and gene transfer. This is a factor which
makes such treatment difficult to implement on a commercial scale.
OBJECTIVES
The main objective of the present invention is to develop a method
for the incubation of eggs/larvae of fish and crustacea, where the eggs/larvae
are protected as much as possible from physical forces such as impact and
blows. A second objective is to prevent the organisms in question from coming
into contact with fungus and bacteria, and find a means to immobilize the
organisms so as to allow injection and vaccination.
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SUMMARY OF THE INVENTION
The invention provides a method of incubating eggs or larvae of fish
or crustacea, wherein the organism is encapsulated in a storage medium
comprising a gel or gel-like substance. The storage medium may be produced
with a relatively hard outer shell and soft inner core and may take a
spherical, tubular/string-like or plate shape. A particularly preferred form
of the storage medium is as plates, wherein the organism is encapsulated
between opposing plates, preferably in hollow depressions provided in the
plates.
The invention also extends to a storage medium for eggs or larvae of
fish or crustacea, comprising plates of a gel or gel-like substance in the
form of a two-part egg tray, wherein hollow depressions are provided in
opposing mating surfaces of the trays.
More particularly, this invention provides a method for incubating
eggs and larvae of fish and crustacea, comprising the steps of:
(a) encapsulating the eggs or larvae in a substance selected from
polysaccharides, agar, alginate, and polymers, in an aqueous gel state;
(b) providing an at least an intermittent supply of water to the
encapsulated eggs or larvae, and maint~ining the encapsulated eggs or larvae
for a time sufficient to allow incubation to occur;
(c) dissolving the aqueous gel encapsulant from the incubated eggs or
larvae.
In another aspect, the invention provides a device for storing eggs
and larvae of fish and crustacea during incubation, comprising a pair of
plates formed of a substance selected from polysaccharides, agar, alginate,
and polymers, in an aqueous gel state, each plate of said pair having a
surface in contact with a surface of the other plate of said pair, at least
one of said plates having a hollow depression on said surface in contact.
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More particularly, the invention provides in a method for incubating live eggs
or larvae of fish or crustaceans in an aqueous environment for a predetermined time and at a
predetermined temperature to obtain an incubated live org~ni~m, the improvement comprising:
a) protecting said eggs or larvae from mechanical forces and infection by casting, using a
mold, a plate of a predetermined shape and dimensions of an aqueous polymeric gel, said
plate having at least one depression in a surface thereof for receiving said eggs or larvae,
depositing at least one said live egg or larvae in said depression, and covering and sealing
said surface and said depression with a porous gas and water permeable membrane; b)
exposing said plate with covered surface to a source of oxygen and at least intermittent flow
of water externally of the membrane for a time and at a temperature sufficient to achieve
incubation of said eggs or larvae with passage of oxygen through said membrane to said eggs,
and passage of excretory matter through said membrane away from said eggs; and thereafter
c) removing an incubated live organism from said membrane and plate.
The membrane may comprise the same aqueous polymeric gel as the plate.
The membrane may also comprise a gel and structural reinforcement. The aqueous polymeric
gel may be acrylamide, ~lgin~te or kappa-carrageenan.
In the method disclosed, the aqueous polymeric gel may comprise alginate, and
the plate and membrane may be separated from the incubated egg or larva by dissolving the
gel. In this circumstance, the ~lgin~te may be m~int~ined as a gel by stabilizing, nontoxic,
divalent cations, and dissolved by removing the divalent cations.
In a preferred form of the invention the gel may be polyacrylamide cross linked
with methylene bisacrylamide. The gel may be formed from a solution cont:~ining 5-10% by
weight acrylamide, and may haves pores of a diameter 2.4-3.6 nm. The gel preferably has a
water flux of 0.02-1.32/m~2 hour~l. In another form of the method, the plate and membrane
are immersed in water for the incubation period.
The intermittent flow of water may comprise spraying water onto the
membrane continuously or intermittently during the incubation period.
In another form of the method, the depressions are provided in opposite
surfaces of the plate, and each of the opposite surfaces are covered and sealed with a
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membrane. The depressions may extend through the entire thickness of the plate.
Additionally, sterilizing the aqueous polymeric gel prior to depositing the egg or larva is
preferred. Additionally, the egg or larva is disinfected prior to incubation. Finally, it is also
possible to vaccinate the egg or larva by injection of a vaccine solution into an associated
depresslon.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In accordance with the invention, when a suitable gel and
encapsulation technique is employed, such organisms will be able to lie stored
in a capsule or inside a plate which hinders damaging impact and other
unwanted physical forces
Water diffusion can continue more or less unhindered and thereby
bring the necessary amount of oxygen in and transport the excretory matter
away
The gel can be dissolved either by temperature changes or by flushing
specific ions out of the structure.
The encapsulation can be done in a number of ways One method is
making spherical capsules/gel-beads, where the object is placed in the centre
of the sphere. If alginate gel is used, the object can first be stirred into
an alginate solution of a predetermined consistency and then dripped through a
tube/pipette into a bath for hardening. This will produce almost spherical
capsules where the size and physical characteristics can be suited to specific
requirements.
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A second solution is incapsulating the organisms into gel plates.
These plates can either be cast together with the eggs in one operation, or
the plates can be cast alone before the organisms are placed on them. The
plates are then joined together, possibly under pressure. These plates can
for example be cast into a shape such as an egg tray, wherein hollow
depressions are provided for the organisms in opposing mating surfaces of the
trays.
A further possibility is laying the organisms in a gel between two
dialysis membranes. In certain cases the dialysis membrane can replace the
gel completely, or be combined with one or another viscous mixture, such as a
protein solution or the like.
A fourth approach is incapsulating the organisms in a tube or string
of gel.
The gel structure can be kept in water or placed on a suspended
surface and be continuously or periodically sprayed with water.
Such methods allow the incapsulated larvae/eggs to be stored from
fertilization until hatching, and possibly even later until the yoke-sack
stage.
It is possible to add chemicals to the gel that will hinder fungus
and bacteria~ in order to prevent infection.
The gel can be built up in layers with different physical
characteristics such as viscosity and hardness. During the incubation of fish
eggs/larvae it could, for example, be advantageous to have a relatively hard
shell and a soft inner core to allow the larvae room to move and stretch,
whilst the gel as a whole has the mechanical strength required.
During the injection/vaccination, the physical characteristics of the
gel can change to meet specific requirements. This can either be done by
altering the structure of the gel or by changing the temperature.
The approach described in the present invention allows different
types of gel to be adjusted to various requirements, incubation media,
temperature, species etc. The term "gel" must be considered relatively
generally, so as to include gel-like substances such as viscous mixtures of
proteins and other macro-molecules that are not usually defined as a gel.
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Examples of gels which could be useful include polysaccharides, agar and
alginate (these include polygalacturonates, carrageenates, polyuronides) or
other polymers such as polyacrylamide.
