AU2021201595B1

AU2021201595B1 - Improved method of producing oysters

Info

Publication number: AU2021201595B1
Application number: AU2021201595A
Authority: AU
Inventors: Serean Leigh Adams; Achim Ralph Janke; Nicholas George KING; Julien Daniel Edmond VIGNIER
Original assignee: Cawthron Institute Trust Board
Current assignee: Cawthron Institute Trust Board
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2022-03-31
Anticipated expiration: 2041-03-12
Also published as: AU2022200815A1

Abstract

A method for improving the induction of the triploid state in a species of oyster using 6-dimethylaminopurine (6-DMAP) is disclosed. The method employs a preincubation of oocytes stripped from the gonad of a diploid female to increase the degree of synchrony of meiosis prior to fertilisation and induction. Batches of fertilised ova with an increased degree of triploidy and viability are thereby provided. The method is particularly applicable to the induction of the triploid state in Pacific Oyster.

Description

IMPROVED METHOD OF PRODUCING OYSTERS TECHNICAL FIELD

The invention relates to an improved method of inducing the triploid state in

species of bivalve molluscs. In particular, although not exclusively, the invention relates to an improved method of producing triploid Pacific oysters

(Crassostrea gigas) and batches of triploid Pacific oysters produced

according to this method.

BACKGROUND ART

In some areas of the world the peak market for oysters occurs during the

summer months when naturally occurring fertile oysters are sexually mature

and either "milky" or "spent". This renders the oysters unpalatable and unsuitable for market. Naturally occurring oysters are diploid, i.e., each of

their somatic cells contain two sets (2N) of homologous chromosomes. Haploid (1N) male and female gametes, i.e., spermatozoa and oocytes, develop from

germ cells located in the gonads through the process of meiosis. Divided into

two main stages, meiosis involves phases of chromosomal separation, rearrangements and segregation before the haploid cells are formed. In

bivalve molluscs polar bodies are extruded in each of these stages and serve as biological indicators of the development of meiosis. Whereas in most animals meiosis is completed before fertilisation, the process in many

bivalve molluscs, including oysters, is delayed and only completed after fertilisation. By inhibiting or blocking the release of the polar bodies the

egg can be made to retain its two sets of chromosomes. The fertilised egg will therefore contain three sets of chromosomes (3N).

Triploidy refers to the condition of a cell or organism having three sets of

chromosomes. Such a condition in mammals is lethal, but in many species of

amphibians, fish or molluscs, triploids are fully viable and do not appear morphologically different from diploids. Triploidy can occur spontaneously at

low but consistent frequencies in the Pacific oyster. Inducing triploidy in oysters significantly reduces gonad development and energy that would

normally be used towards reproduction is instead diverted to general tissue

growth and carbohydrates. As a result, triploid oysters have a superior flesh flavour and improved meat quality during summer months enabling the

opportunity to harvest them year-round. Triploid oysters are faster growing and grow larger than their diploid counterparts. Reaching a marketable size

more quickly they are also less vulnerable to particular parasites such as

Perkinsus marinus.

Oyster production is based on both wild-caught and hatchery-produced spat, the vast majority of hatchery-produced spat being triploid. As disclosed in the publication of Stanley et al (1981) triploidy was first established for the Eastern oyster (Crassostrea virginica). As disclosed in the publication of Allen et al (1989) triploidy was then established for the Pacific oyster (Crassostrea gigas) where it was developed for commercial use on the North

American West Coast. Two principal methods are now utilised for the creation of triploid oysters. The first method makes use of tetraploid oysters having

four chromosome sets. As disclosed in the publications of Guo et al (1996)

and Guo and Allen (1998) fertilizing the oocytes from diploids (2N) with the spermatozoa of these tetraploids (4N) results in triploid (3N) progeny.

Worldwide, this is the method most commonly used on a commercial scale, but it is a complex, time-consuming process and can take 5 to 10 years to produce

harvestable triploids. The second method directly induces the triploid

condition by treating fertilised eggs physically or with a chemical solution that impairs the completion of meiosis. Historically, fertilised eggs have

been exposed to heat shock, hydrostatic pressure or the mycotoxin cytochalasin B to inhibit the release of a polar body during meiosis and

induce the triploid state. More recently, cytochalasin B has been replaced by

6-dimethylaminopurine (6-DMAP) as an agent to induce the triploid state.

The publication of Allen et al (1989) discloses a method of inducing

triploidy in oysters using hydrostatic pressure to inhibit the extrusion of a

polar body from a fertilised egg. An efficiency of 30% is asserted (as determined by cytofluorometric analysis.

The publication of Gerard et al (1994) discloses experiments conducted to optimize the production of triploid Crassostrea gigas by examining the

dosage, timing and duration of embryo treatment with 6-DMAP. An improvement

in the synchronization of the polar body extrusion is attributed to a

decrease in the egg to sperm ratio. The publication concludes with the statement that more experiments should be conducted with the same parameters, and the influence of temperature or spermatozoa concentration should also be

investigated.

The publication of Chuanyuan et al (2001) [machine translation] discloses the

use of 6-DMAP to inhibit the release of the second polar body from a fertilized egg and thereby obtain a triploid Pacific oyster. Before fertilization the eggs are first filtered using a 100 to 250 mesh sieve and

then filtered through a 500 mesh sieve. The fertilized eggs are contacted with the 6-DMAP at a concentration of 30 to 90 mg/L, i.e., 180 to 550 uM, for

ten to fifteen minutes following release of the first polar body from 30 to

50% of the eggs. A triploid induction rate greater than 70% is asserted.

It is an object of the present invention to provide an improved method of producing triploid oysters on a consistent basis. This object is to be read in the alternative with the object at least to provide a useful choice in the selection of such methods.

SUMMARY OF INVENTION

In a first aspect a method of inducing the triploid state in Crassostrea gigas is provided, the method comprising incubating oocytes stripped from the gonad of at least one brood female prior to fertilization. The oocytes are incubated at a temperature and for a time sufficient to provide greater than % (by number) of oocytes in metaphase I of meiosis.

Preferably, the oocytes are incubated at a temperature and for a time sufficient to provide greater than 95% (by number) of oocytes in metaphase I of meiosis. More preferably, the oocytes are incubated at a temperature and for a time sufficient to provide greater than 95% (by number) of oocytes in metaphase I of meiosis.

Preferably the temperature at which the oocytes are incubated is about 25°C. Preferably the oocytes are incubated for a period of time of 90 to 105 minutes.

In a preferred method of inducing the triploid state in Crassostrea gigas the method comprises:

a) Preincubating eggs stripped from the gonad of at least one brood female to provide a batch of matured eggs;

b) Contacting a suspension of the matured eggs with spermatozoa obtained from the gonad of at least one male at a ratio of eggs to spermatozoa sufficient to provide a batch of fertilized eggs; immediately then

c) Incubating the batch of fertilized eggs at a predetermined temperature for a predetermined first period of time; immediately then

d) Contacting the fertilized eggs with an inducing agent at the predetermined temperature for a predetermined second period of time; immediately then

e) Rinsing the fertilized eggs to remove substantially all the inducing agent and provide a batch of rinsed eggs; and then

f) Incubating the rinsed eggs to provide a batch of D-larvae.

Preferably greater than 90% (by number) of the D-larvae are in the triploid state. More preferably greater than 95% (by number) of the D-larvae are in the triploid state. Most preferably greater than 99% (by number) of the D larvae are in the triploid state.

Preferably a portion of the oocytes are discarded following the incubation and prior to fertilization to increase the median volume of the oocytes by %. More preferably a portion of the oocytes are discarded following the incubation and prior to fertilization to increase the median volume of the oocytes by 10%.

Preferably greater than 90% (by number) of the matured eggs are in metaphase I of meiosis. More preferably greater than 95% (by number) of the matured eggs are in metaphase I of meiosis. Most preferably greater than 95% (by number) of the matured eggs are in metaphase I of meiosis.

Preferably the method includes removing a portion of the batch of matured eggs to increase the median volume of the eggs contained in the batch before contacting the eggs contained in the batch with spermatozoa. Preferably, the portion is 5 to 10% (by number) of the eggs contained in the batch.

Preferably the suspension of the matured eggs contains 0.5 x 104 to 0.8 x 104

eggs per litre.

The at least one male is a male in the diploid state.

Preferably the ratio of eggs to spermatozoa sufficient to provide the batch of fertilized eggs is 0.1 x 10-2.

The contacting the fertilized eggs with an inducing agent suppresses the extrusion of the second polar body following extrusion of the first polar body. Preferably the inducing agent is selected from the group consisting of: cytochalasin B and 6-DMAP. More preferably the inducing agent is 6-DMAP. Yet more preferably the 6-DMAP is at a concentration of 425 to 475 pM. Most preferably the 6-DMAP is at a concentration of about 450 pM.

Preferably the predetermined temperature is about 25 °C, the predetermined first period of time is 18 to 19.5 minutes and the predetermined second period of time is about 10 minutes when the species is Crassostrea gigas.

Preferably the yield of D-larvae is greater than 25% (by number) of the rinsed eggs. More preferably the yield of D-larvae is greater than 45% (by number) of the rinsed eggs. Most preferably the yield of D-larvae is greater than 99% (by number) of the rinsed eggs.

An embodiment of the method comprises:

a) Stripping eggs from the gonad of a diploid female to provide a suspension of eggs; b) Preincubating the suspension of eggs at about 25°C for a period of time of 90 to 105 minutes to provide a batch of matured eggs; c) Contacting the matured eggs with spermatozoa obtained from the gonad of at least one diploid male at a ratio of 0.1 x 10-2 eggs per spermatozoon to provide a batch of fertilized eggs; immediately then d) Incubating the batch of fertilized eggs at about 25°C for a period of time of 18 to 19.5 minutes; immediately then e) Contacting the fertilized eggs with 6-DMAP at a concentration of about 450 pM at about 25°C for a period of time of 10 minutes; immediately then f) Rinsing the fertilized eggs to remove substantially all the 6-DMAP and provide a batch of rinsed eggs; and then g) Incubating the rinsed eggs to provide a batch of triploid D-larvae, where greater than 90% (by number) of the rinsed eggs develop into D-larvae and greater than 90% (by number) of the D-larvae are in the triploid state.

All steps of the method are performed in treated seawater.

In a second aspect a batch of fertilized eggs of Crassostrea gigas is provided where greater than 25% (by number) of the eggs are capable of developing into D-larvae and greater than 95% (by number) of the D-larvae are in the triploid state. The batch of claim 5 where greater than 45% (by number) of the eggs are capable of developing into D-larvae. Preferably the batch comprises greater than 2 x 107 fertilized eggs. More preferably the batch comprises greater than 4 x 107 fertilized eggs.

In the description and claims of this specification the following abbreviations, acronyms, terms and phrases have the meaning provided: "batch" means a quantity produced at one time; "CAS RN" means Chemical Abstracts Service (CAS, Columbus, Ohio) Registry Number; "comprising" means "including", "containing" or "characterized by" and does not exclude any additional element, ingredient or step; "consisting essentially of" means excluding any element, ingredient or step that is a material limitation; "consisting of" means excluding any element, ingredient or step not specified except for impurities and other incidentals; "6-DMAP" and "6 dimethylaminopurine" mean N,N-dimethyl-9H-purin-6-amine (CAS RN 938-55-6); "immediately" means without any intervening time; "initiation time" means the time at which a suspension of eggs is first contacted with a suspension of sperm; "oocyte" means a cell in an ovary which may undergo meiotic division to form an ovum; "ovum" means a mature female reproductive cell which can divide to give rise to an embryo after fertilization; "Pacific oyster" means the bivalve mollusc of the Crassostreinae subfamily named Crassostrea gigas or Magallana gigas; "period of time" means a continuous length of time, i.e., without interruption; "polar body" means a cell that separates from an oocyte during meiosis; "predetermined" means decided in advance; spermatozoonn" means a mature motile male sex cell; "synchrony" means the state of developing according to the same time scale; and "triploid" means containing three homologous sets of chromosomes or composed of cells containing three homologous sets of chromosomes. A paronym of any of these defined terms has a corresponding meaning. No distinction between the meaning of the phrases "inducing the triploid state" and "induction of the triploid state" is intended.

The terms "first", "second", "third", etc. used with reference to elements, features, integers or other limitations of the matter described in the

Summary of Invention, or when used with reference to alternative embodiments are not intended to imply an order of preference. Preferments for elements,

features, integers or other limitations described in the Summary of Invention

are identified in order of preference by "preferably...", "more preferably...", "yet more preferably...", etc. Preferments of any combinations of these

elements, features, integers or other limitations are similarly identified in order of preference.

Where concentrations or ratios of reagents are specified the concentration or

ratio specified is the initial concentration or ratio of the reagents.

Similarly, where a pH or pH range specified, the pH or pH range specified is the initial pH or pH range. Where values are expressed to one or more decimal places standard rounding applies. For example, 1.7 encompasses the range

1.650 recurring to 1.749 recurring.

Where a parameter is expressed as being "about" a specified range or value

the term is used to indicate tolerance for some variation of the specified range or value (provided the parameter dependent effect is still achieved). In the absence of any other qualification the term "about" should be

understood to indicate a tolerance of no greater than 5% above or below the upper and lower limits, respectively, of the specified range or plus or minus

5% of the specified value.

A first essential feature of the method of inducing the triploid state is the combination of temperature at which and period of time for which the batch of

fertilized eggs is incubated before contacting the fertilized eggs with an inducing agent. Any addition or alteration to this combination is a material

limitation.

A second essential feature of the method of inducing the triploid state is the combination of temperature at which and period of time for which, the batch of fertilized eggs is contacted with the inducing agent 6-DMAP before rinsing the fertilized eggs to remove substantially all the inducing agent.

Any addition or alteration to this combination is a material limitation.

Throughout the description and claims of this specification, where the term

"eggs" is used, the term is being used to encompass the stripped oocytes, the fertilized oocytes or ova, and the embryos and early-stage larvae

subsequently developing from the ova. The terms "egg" and "oocyte" may also

be used interchangeably.

The invention will now be described with reference to examples and the

figures of the accompanying drawings pages. The examples provided are

intended to demonstrate the practice of the invention and are not intended to limit the scope of protection to which the invention is entitled.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. Photomicrographs of oocytes in prophase I (A) and metaphase I (B)

of meiosis.

Figure 2. Observed correlation between induction of the triploid state (3N)

and maturation (prophase I to metaphase I) by preincubation of oocytes

stripped from the gonad.

Figure 3. Observed correlation between preincubation of oocytes stripped from

the gonad of a diploid female and yield of D-larvae following rinsing of the

fertilized oocytes. Broken line at a yield of 45% (by number) of D-larvae.

Figure 4. Observed correlation between period of time fertilized eggs are

contacted with 6-DMAP at a concentration of about 450 M and yield of D larvae following rinsing. Broken line at a yield of 25% (by number) of D larvae.

Figure 5. Schematic representation of the steps of the method of inducing the

triploid state in Pacific oyster at a temperature of 25°C and the corresponding periods of time (A - 90 to 105 minutes; B - 10 to 15 minutes; C - less than 2 minutes; D - 18 to 19.5 minutes; E - 10 minutes).

DESCRIPTION

The improvements provided by the present invention are both the increased proportion of fertilised eggs developing into larvae and the increased

proportion of the larvae that develop that are in the triploid state. These improvements are attributed at least in part to the maturation of the eggs

stripped from the gonad during the preincubation period. The eggs are in prophase I of meiosis while held in the gonad. Once stripped from the gonad a spontaneous maturation of the released eggs to metaphase I of meiosis occurs. It is believed the incubation of the eggs prior to fertilization results in greater synchrony and an increased proportion of the fertilized eggs thereby being induced to enter the triploid state. The proportion of these triploid eggs developing into viable larvae is believed to be further increased by discarding a portion of the eggs in metaphase I of meiosis to increase the median volume of the eggs that are subsequently fertilized. Although the aim of the experiments disclosed in the publication of Gerard et al (1994) was to maximize the triploid percentage and the larval survival for each treatment, preincubation of the stripped eggs and the median volume of the eggs were not considered as parameters to be optimised to maximise induction of the triploid state and viability of the fertilized eggs. These parameters have now been found to be very significant in optimizing the induction of the triploid state in species of bivalve mollusc such as the Pacific oyster (Crassostrea gigas). Although demonstrated primarily for use with oysters it is believed the method may be similarly applicable to other species of bivalve molluscs such as geoduck (Panopea generosa) and green lipped mussels (Perna canaliculus). In both these species oocytes may be stripped from the gonads of brood females and incubated prior to fertilization to increase the degree of synchrony. This improved method of inducing the triploid state allows for the rapid introduction of desirable traits from selectively bred lineages of bivalve molluscs.

Treated seawater

Seawater containing 12 pm EDTA and a pH adjusted to between 8.36 to 8.40 with sodium carbonate (Na 2 CO 3 ) is exposed to ultraviolet light (100 MJ/cm 2 ) and filtered (1 pm) before being stored with vigorous aeration for at least 25 hours at a temperature of 24 °C.

Broodstock

Mature (2- to 4-year-old) diploid Pacific oysters are used as brood stock. Oysters collected from the field during the winter months are transferred to a hatchery. The oysters are conditioned by raising the water temperature to 23°C plus or minus one degree Celsius over a period of time of eight to 10 weeks. During the conditioning period the oysters are fed a mixed diet of phytoplankton from artificially fertilised eutrophic ponds and continuously cultured species. Individual mature oysters are randomly selected during the conditioning period, opened and their gender and gamete quality determined microscopically.

Harvesting of eggs

Eggs stripped from a single female brood oyster are collected in a volume of

800 mL of the treated seawater in a 1 L glass beaker and debris removed by

filtering through a 55 um screen. The suspension of eggs is maintained at a temperature of 25 °C for a period of time of around 20 minutes until a

stratification between an upper layer of glycogen and eggs and a lower layer of sedimenting eggs is observed. The upper layer is discarded by decanting

and the sedimenting eggs resuspended in a total volume of 900 mL of the

treated seawater to provide a suspension of eggs with an increased median volume. Following determination of the density of eggs a volume of 100 mL of

the suspension is transferred via repeated (x 3) gentle washing with the

treated seawater on a 15 um screen to a container containing the treated seawater to provide an estimated density of no greater than 2.3 x 106 of the glycogen-free sedimenting eggs per litre. Up to 40 million eggs may

conveniently be processed in a 20 L container. The container is periodically

gently agitated while continuing to maintain the suspension at a temperature

of 25 °C.

Harvesting of sperm

Sperm are stripped from individual male oysters and collected in a volume of 10 mL of the treated seawater 15 to 20 minutes before being used to fertilize

a batch of synchronized eggs. Following determination of density of sperm,

the suspension is diluted to a total volume of around 900 mL in a 1 L glass

beaker and filtered through a 55 um screen to remove debris. Motility of the sperm in the diluted and filtered suspension is confirmed microscopically. The suspension of sperm is maintained at 8 °C prior to use.

Maturation and fertilization

The suspension of eggs prepared according to the method described under the heading 'Harvesting of eggs' is maintained at a temperature of 25 °C for a

period of time of 90 to 105 minutes prior to fertilization. This maturation period increases the degree of synchrony of the eggs prior to fertilization,

thereby contributing to the efficiency of induction of the triploid state

following fertilization. Microscopic observation (Figure 1) may be used to confirm the synchrony of the matured eggs before fertilization.

Immediately following the maturation period, the suspension of eggs is drained through a 15 um screen and the collected synchronous eggs returned to

the same container in a reduced volume of around 5 litres. An aliquot of the

sperm suspension prepared according to the method described under the heading

'Harvesting of sperm' is left at ambient temperature for 10 minutes and the motility of sperm reconfirmed before being added to the reduced volume of the suspension of eggs and immediately mixed by plunging. An egg to sperm ratio of 0.001 (1/1000) is sought in the mixture of eggs and sperm. Within a period of time of 20 seconds a sample of the mixture of eggs and sperm is examined under a microscope to confirm an egg to associated sperm ratio of 0.1 (1/10) to 0.2 (1/5). (When necessary an additional aliquot of the sperm suspension may be added and mixed to achieve the desired egg to associated sperm ratio, but only where these additional steps are completed within 60 to 120 seconds of the initial addition of the sperm suspension (the 'initiation time'). While continuing to maintain the mixture of eggs and sperm at a temperature of 25 °C the suspension is maintained by gentle mixing for a period of time of 7 to 8 minutes from the initiation time, whereupon the suspension is diluted to a total volume of 17 to 18 litres with treated seawater at a temperature of 25 °C.

Induction of triploidy and incubation

The diluted suspension is maintained at 25 °C until a period of time of 18 minutes from the initiation time has elapsed. The diluted suspension of eggs is then drained through a 15 pm screen kept submerged in flowing treated seawater at 25 °C. The screen retaining the eggs is then transferred to a pre-prepared induction treatment bath when a period of time of 19 minutes and seconds from the initiation time has elapsed. The induction treatment bath consists of a volume of 4 litres of a solution in treated seawater of 450 pm 6-DMAP at a temperature of 25 °C in an 8-litre container. The eggs are immersed in the treatment bath with gentle agitation for a period of time of minutes, i.e., until a period of time of 29 minutes and 30 seconds from the initial addition of sperm has elapsed. The eggs are then washed by transferring the screen to a first rinse bath consisting of treated seawater at a temperature of 25 °C. The eggs are gently agitated before transferring the screen to a second rinse bath also consisting of treated seawater at a temperature of 25 °C. The washed eggs are then transferred from the surface of the screen to a 170-litre incubation tank filled with treated seawater and maintained in suspension by gentle aeration. The stocking density for each incubation tank should not exceed 40 to 50 million eggs.

Determination of viability

Yields of D-larvae are determined by a comparison of the density of eggs in a sample volume of a uniform suspension immediately following rinsing and the density of D-larvae in the same sample volume following incubation. The sample volume is selected to contain at least 103 eggs.

Determination of triploid state

The ploidy of the larvae developing from the fertilized eggs is determined at the D-larval stage. Around 20 hours after fertilisation approximately 10,000 veliger D-larvae are collected from each incubation tank for flow cytometric (FCM) analysis. The percentage of triploids (3N) are determined according to a protocol adapted from that disclosed in the publication of Allen and Bushek (1992). Briefly, larvae are washed with cold freshwater on a 40 pm screen, concentrated into a 1.5 mL suspension, and placed on a glass petri dish using a disposable pipette. Larvae were then crushed/disintegrated by means of a glass slide, and 1.5 mL of CYSTAIN m DNA 1 step (Sysmex, Sysmex Flow Cytometry, USA) added to the homogenised larval suspension and left for 1 minute. The cell suspension was then passed through a sterile CELLTRICS m 30 pm filter (Sysmex, Sysmex Flow Cytometry, USA) and immediately analysed on a T PARTEC PA m ploidy analyser. Samples of diploid larvae were also analysed concurrently for calibration of ploidy peaks. The proportion of cells in each ploidy class was calculated relative to the proportion of observations in all ploidy classes.

Although the invention has been described with reference to embodiments or examples it should be appreciated that variations and modifications may be made to these embodiments or examples without departing from the scope of the invention. Where known equivalents exist to specific elements, features or integers, such equivalents are incorporated as if specifically referred to in this specification. Variations and modifications to the embodiments or examples that include elements, features or integers disclosed in and selected from the referenced publications are within the scope of the invention unless specifically disclaimed. The advantages provided by the invention and discussed in the description may be provided in the alternative or in combination in these different embodiments of the invention. It is contemplated that the invention may be applied to other species belonging to the family Ostreoidea, more specifically species of the genus Crassostrea, such as Crassostrea virginica (Eastern Oyster).

INDUSTRIAL APPLICABILITY

A method of increasing the efficiency of production of oysters in the triploid state is provided.

INCORPORATION BY REFERENCE

For the purposes of 37 C.F.R. 1.57 of the United States Code of Federal Regulations the disclosures of the following publications (as more specifically identified under the heading 'Referenced Publications') are incorporated by reference: Creswell et al (2008) and Helm and Bourne (2004).

REFERENCED PUBLICATIONS

Allen and Bushek (1992) Large-scale production of triploid oysters, Crassostrea virginica (Gmelin), using "stripped" gametes Aquaculture, 103(3

4), 241-251.

Allen et al (1989) Inducing polyploidy in oysters United States patent no.

4,834,024.

Chuanyuan et al (2001) Breeding method using 6-DMAP to induce oyster triploid Chinese patent application no. 01107849.9 [publ. no. CN1308856A].

Creswell et al (2008) Better management practice for bivalve molluscan

aquaculture Environmental Best Management Practices for Aquaculture Edited by Craig S. Tucker and John A. Hargreaves, John Wiley & Sons, Inc. ISBN: 978-0

813-82027-9, p. 427.

Gerard et al (1994) Optimization of triploid induction by the use of -6DMAP for the oyster Crassostrea gigas (Thunberg) Aquaculture and Fisheries

Management, 25, 709-719.

Guo and Allen (1998) Tetraploid shellfish United States patent no. 5,824,841.

Helm and Bourne (2004) Hatchery culture of bivalves - a practical manual FAO Fisheries Technical Paper 471 Food and Agriculture Organization of the United

Nations.

Stanley et al (1981) Polyploidy induced in the American oyster, Crassostrea virginica, with cytochalasin B1 Aquaculture, 23, 1-10.

Claims

1) A method of inducing the triploid state in Crassostrea gigas comprising:

(a) Stripping eggs from the gonad of a diploid female to provide a suspension of eggs;

(b) Preincubating the suspension of eggs at about 25°C for a period of time of 90 to 105 minutes to provide a batch of matured eggs;

(c) Contacting the matured eggs with spermatozoa obtained from the gonad of at least one diploid male at a ratio of about 0.1 x 10-2 eggs per spermatozoon to provide a batch of fertilized eggs; immediately then

(d) Incubating the batch of fertilized eggs at about 25°C for a period of time of 18.0 to 19.5 minutes; immediately then

(e) Contacting the fertilized eggs with 6-DMAP at a concentration of about 450 pM at about 25°C for a period of time of 10 minutes; immediately then

(f) Rinsing the fertilized eggs to remove substantially all the 6-DMAP and provide a batch of rinsed eggs; and then

(g) Incubating the rinsed eggs to provide a batch of D-larvae,

where greater than 25% (by number) of the rinsed eggs develop into D larvae and greater than 90% (by number) of the D-larvae are in the triploid state.

2) The method of claim 1 where greater than 45% (by number) of the rinsed eggs develop into D-larvae.

3) The method of claim 1 or 2 where greater than 95% (by number) of the D larvae are in the triploid state.

4) The method of any one of claims 1 to 3 where greater than 99% (by number) of the D-larvae are in the triploid state.

) The method of any one of claims 1 to 4 where the number of eggs in the batch of matured eggs is greater than 2 x 107.

6) The method of claim 5 where the number of eggs in the batch of matured eggs is greater than 4 x 107.

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A B FIGURE 1

FIGURE 2

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FIGURE 3

FIGURE 4

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Initiation

Preincubation of stripped eggs

Preparation of sperm Induction

Fertilization

Incubation

Growth of larvae

Washing

A B C D E

FIGURE 5

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