The fundamentals of producing monosex fish for

aquaculture

D.J.Martin-Robichaud and Tillmann Benfey

• Monosex stocks of various finfish are commercially produced in Canada

• Salmonids primarily, recently Atlantic halibut (Scotian Halibut Ltd) and research now on Atlantic cod

• Alleviate any misunderstandings regarding physiological and genetic changes involved

• protocols species specific, process of answering questions similar

• Atlantic halibut research as example

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Date (mon-yr)

Wei

ght (

g)

2288 g

3042 g

Why monosex Atlantic cod stocks?

• Mixed sex stocks of cod in cages will release fertilized eggs; genetic implications for wild stocks

• Very likely sexually dimorphic growth characteristics

• Performance and survival of one sex better, both mature prior to harvest

• All-female triploid stocks• New funding to develop techniques

(NSERC & ACRDP)

B) INDIRECT FEMINIZATION. FEMALE HOMOGAMETY

F 0

Androgen

Treatment

XX XY XXF 1

NEOMALE

XXF 2

100% female 50% male

50% female

XX XY

XX XY

A) DIRECT FEMINIZATION. ANY GENETIC SYSTEM

SEXUALLY UNDIFFERENTIATED FISH

ALL- FEMALE STOCK

Estrogen

Treatment

XX

Indirect FeminizationX X/X Y

M D H T

W Z/ZZ

M D H T

* *

*

X X/X Y

M D H T

W Z/ZZ

M D H T

* *

*

Many species specific questions….

1. Genetic mechanism of sex determination (gynogenesis)

2. Timing of gonadal differentiation, labile period

3. Efficacy of direct hormonal sex reversal

4. Reproductive ability of sex reversed fish

5. Differentiating neomales

Gynogenesisuniparental maternal inheritance

(all genetic contribution from female)

1. Exclude paternal genomeUV irradiation of sperm optimum treatment will:

(a) disable sperm’s genomic DNA (b) not affect sperm’s ability to swim

and activate development in eggs

optimum treatment for halibut

1:80 dilution in seminal plasmaexposure to UV at 65 mJ/cm2

yields gynogenetic haploids (non-viable)

2. duplicate maternal genome (1n to 2n)

– pressure treatment of eggs– optimum treatment will:

(a) retain 2nd polar body (final product of meiosis)

(b) not affect survival– optimum treatment for halibut

(a) activate eggs with UV-treated sperm(b) 5 min @ 9500 psi, 15 min post-

activation– yields gynogenetic diploids (viable)

Sufficient numbers of gynogens only need to be produced once to determine the sex ratio

Determine sex ratio of gynogenetic progeny

histology(9 mo, 7 cm)

visual(21 mo, 25 cm)

female maleGynogen halibut 100% females = females are the homogametic (XX) sex

Indirect Feminization to produce all-female Atlantic halibut stocks

XX XX

Hormonal sex reversal

**Neomale Broodstock:Genotypic femalebut phenotypic male

XX All females

XX

masculinization

Steroid hormones and sex differentiation

Testosterone

17ß-Estradiol

11ß-Hydroxytestosterone

11-Ketotestosterone

P450 Aromatase

11ß-Hydroxylase

11ß-HSD

MALE

FEMALE

The key steroids for gonadal differentiation in teleost fishes are 17ß-estradiol and 11-ketotestosterone.

The critical enzymes in the synthesis of these compounds are P450 aromatase and 11ß-hydroxyalase, respectively.

Ovarian development

Testicular development

BipotentialUndifferentiatedgonad

Species specific labile period

Some species temperature (ESD) etc influence gonadal development, or combination

1. Genetic mechanism of sex determination

2. Timing of sex differentiation

3. Efficacy of direct hormonal sex reversal

4. Reproductive ability of sex reversed fish

5. Differentiating neomales

Timing of sex differentiation

1. Histologically determine timing of gonadal differentiation in Atlantic halibut(histology of 338 fish, 0.8 – 23.0 cm)

A 1.0 cm (hatch): germ cells appearB 2.1 cm (end of yolk-sac stage): primordial gonad apparentC 3.8 cm (post-metamorphosis): ovarian cavity formed (‘anatomical differentiation’)D 5.0 cm: oogonia apparent (‘cytological differentiation’)

Therefore the ‘labile’ period (i.e., hormonal sex reversal possible)– begins after 2.1 cm– ends before 5.0 cm– Corresponds to period of metamorphosis and weaning at

about 35 mm FL

A

D

B

C

1. Genetic mechanism of sex determination

2. Timing of sex differentiation

3. Efficacy of direct hormonal sex reversal

4. Reproductive ability of sex reversed fish

5. Differentiating neomales

Efficacy of direct hormonal masculinization

• apply androgen during ‘labile’ period

– incorporate androgen into feed– optimum treatment will:

(a) cause genetic females to develop into functional males (b) not affect fertilization ability

– optimum treatment for halibut(a) 17α-methyldihydrotestosterone

at 1mg/kg in dry feed(b) feed MDHT-diet from 3.0 to 3.8 cm

– results in all phenotypic males (presumably still 50% XX and 50% XY)

Hendry, C.I., D.J. Martin-Robichaud & T.J. Benfey. 2003. Hormonal sex reversal of Atlantic halibut (Hippoglossus hippoglossus). Aquaculture 219: 769-781.

4. Reproductive ability of sex reversed females (neo-males)

• All males exposed to MDHT spermiated normally at maturation and were crossed with normal females.

No morphological abnormalities

Sperm motility and fertilization rates good

Problem:Which are neomales (genotypic females) and which are genotypic males.

5. Differentiating neomales

Sex offspring produced by each male.

Sex-reversed females (XX) will produce 100% female offspring.

Technology transfer to industry

• 2005 DFO loaned 12 putative neomales and 2 confirmed neomales to Scotian Halibut Ltd.

• 2007 first stocks (world-wide)of all-female halibut produced

• Continue to confirm neomale status (3 now)

• Continuing to produce new sex reversed broodstock using androgen treatments

Acknowledgments:Chris Hendry, Harald Tvedt

Mike Reith, Tim Jackson, Darrin ReidScotian Halibut Ltd

NSERC, Aquanet, ACRDP

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

NSERC Strategic

CBS

AquaNet

DFO ACRDP

Pleurogene

Scotian AIF

Funding

Accomplishments

LightShifted

Broodstock

MicrosatellitesPedigreeAnalysis

Gynogens/X/Y Sex Linkage

Map

ESTs

All-Female Broodstock

HormonalSex Reversal

QTL

Sex-linkedMarkers Micro

array

Mapping

Hormonal regulation of sex differentiation

Genotypic: “Master” gene (e.g., dmy), minor sex determining

genes, autosomal genes

Environmental factor

(e.g., temperature)

Sexdetermination

ER

Estrogen-regulated genes

AR

Androgen-regulatedgenes

sf1, sox, foxl2, figα

Ovariandifferentiation

Testisdifferentiation

Aromatase dmrt1

Bipotential gonad

Estrogen

Germ cell proliferationEntry into meiosis

Mitotic arrest

Proliferation

Androgen

11β-hydroxylase

amh, sox9

Sex differentiation

Female Male

F. Piferrer & Y. Guiguen (2008). Fish Gonadogenesis. Part 2. Molecular Biology and Genomics of Sex Differentiation. Rev.

Fish Sci., 16 (S1): 33-53.

The Future Prospects for Aquaculture Breeding in Europe. Professional and Scientific Workshop. Paris, October 1-3, 2008.

Part 1: Summary of the Problem and General Scientific Principles

Sex differentatiation involves similar or the same players across

vertebrates, with the steroidogenic enzyme aromatase and the transcription factor dmrt1

playing a central role

Current problems in European fish farming due to skewed sex ratios- Increased size dispersion and thus more need for size-gradings

- Less produced biomass within a given production unit

- Lower product quality if one sex is more valuable than the other

- Precocious maturation brings several additional problems to fish farming

- Depreciated product when release of sperm

Species for which one sex is more valuable and why- Trout – maturation, flesh quality

- Sea bass – highly skewed sex ratios, precocious maturation

- Senegalese sole – highly skewed sex ratios

- Turbot – highest sex-related growth differences in favor of females

- Sturgeons – only females for caviar production

- Tilapias – males are usually larger than females

- Trout, Sea bass, Sea bream, etc. – Only female triploids do not develop gonads

Year

1986 1988 1990 1992 1994 1996 1998

Per

ce

nta

ge

of

ova

ty

pe

s

0

20

40

60

80

100All-female Triploid Mixed sex

Rainbow Trout (France, Scotland, Japan)

Brown Trout (France)

Atlantic Salmon (Canada)

Coho Salmon (Canada, Japan)

Amago Salmon and Masu Salmon (Japan)

Ayu and Hirame (Japan)

Channel Catfish (USA)

Nile Tilapia (China, Fiji, Philippines,

Thailand, USA, Vietnam)

Jordan tilapia (Israel)

Silver Barb (Thailand)

Hulata, G. (2001). Genetica, 111: 155-173.

Scottish Rainbow Trout Production

Endocrine Sex Control Involved in Practical Aquaculture

Information provided by Dr. B. McAndrew,

Univ. Stirling, Scotland

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Date (mon-yr)

Wei

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t (g

)Atlantic halibut

Effect of Sex on Growth

Females on average ~750 g larger than males at Nov-08 sampling.