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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

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the fundamentals of producing monosex fish for aquaculture

The fundamentals of producing monosex fish for aquaculture

D.J.Martin-Robichaud

and Tillmann Benfey

slide2
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
slide3

3042 g

2288 g

why monosex atlantic cod stocks
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)
slide5

A) DIRECT FEMINIZATION. ANY GENETIC SYSTEM

SEXUALLY UNDIFFERENTIATED FISH

Estrogen

Treatment

ALL- FEMALE STOCK

B) INDIRECT FEMINIZATION. FEMALE HOMOGAMETY

F

XX

XY

0

Androgen

Treatment

XX

XY

XX

F

XX

1

NEOMALE

F

XX

XX

XY

2

100% female

50% male

50% female

many species specific questions
Many species specific questions….
  • Genetic mechanism of sex determination (gynogenesis)
  • Timing of gonadal differentiation, labile period
  • Efficacy of direct hormonal sex reversal
  • Reproductive ability of sex reversed fish
  • Differentiating neomales
gynogenesis uniparental maternal inheritance all genetic contribution from female
Gynogenesisuniparental maternal inheritance(all genetic contribution from female)
  • 1. Exclude paternal genome
    • UV 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 plasma
      • exposure to UV at 65 mJ/cm2
    • yields gynogenetic haploids (non-viable)
slide9
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

slide10
Determine sex ratio of gynogenetic progeny

histology

(9 mo, 7 cm)

visual

(21 mo, 25 cm)

female male

Gynogen halibut 100% females =

females are the homogametic (XX) sex

slide11

Indirect Feminization

to produce all-female Atlantic halibut stocks

**NeomaleBroodstock:

Genotypic female

but phenotypic male

XX

XX

masculinization

Hormonal sex reversal

All females

XX

XX

steroid hormones and sex differentiation

11-Ketotestosterone

MALE

11ß-HSD

11ß-Hydroxytestosterone

11ß-Hydroxylase

Testosterone

P450 Aromatase

17ß-Estradiol

FEMALE

Steroid hormones and sex differentiation

Testicular development

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.

Species specific labile period

Bipotential

Undifferentiated

gonad

Some species temperature (ESD) etc

influence gonadal development, or combination

Ovarian development

slide13
Genetic mechanism of sex determination
  • Timing of sex differentiation
  • Efficacy of direct hormonal sex reversal
  • Reproductive ability of sex reversed fish
  • Differentiating neomales
timing of sex differentiation
Timing of sex differentiation
  • 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 appear

B 2.1 cm (end of yolk-sac stage):

primordial gonad apparent

C 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

B

C

D

slide15
Genetic mechanism of sex determination
  • Timing of sex differentiation
  • Efficacy of direct hormonal sex reversal
  • Reproductive ability of sex reversed fish
  • Differentiating neomales
efficacy of direct hormonal masculinization
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

      • 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.

reproductive ability of sex reversed females neo males
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.

differentiating neomales
Differentiating neomales

Sex offspring produced by each male.

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

technology transfer to industry
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
slide20

Acknowledgments:

Chris Hendry, Harald Tvedt

Mike Reith, Tim Jackson, Darrin Reid

Scotian Halibut Ltd

NSERC, Aquanet, ACRDP

slide21

Sex-linked

Markers

Micro

array

Accomplishments

Gynogens/

X/Y Sex

Linkage

Map

ESTs

Pedigree

Analysis

Mapping

QTL

Hormonal

Sex Reversal

Microsatellites

Light

Shifted

Broodstock

All-Female

Broodstock

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

NSERC Strategic

CBS

AquaNet

DFO ACRDP

Funding

Pleurogene

Scotian AIF

slide22

Part 1: Summary of the Problem and General Scientific Principles

Hormonal regulation of sex differentiation

Environmental

factor

(e.g., temperature)

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

genes, autosomal genes

Sex

determination

Bipotential gonad

Sex differentatiation involves similar

or the same players across vertebrates, with the steroidogenic enzyme aromatase and the transcription factor dmrt1

playing a central role

Germ cell proliferation

Entry into meiosis

Mitotic arrest

Proliferation

sf1, sox,

foxl2, figα

amh, sox9

Aromatase

dmrt1

Sex

differentiation

Estrogen

Testis

differentiation

ER

11β-hydroxylase

Estrogen-regulated

genes

Androgen

Ovarian

differentiation

AR

F. Piferrer & Y. Guiguen (2008). Fish Gonadogenesis. Part 2. Molecular Biology and Genomics of Sex Differentiation. Rev. Fish Sci., 16 (S1): 33-53.

Androgen-regulated

genes

Male

Female

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

slide23

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

slide24

Endocrine Sex Control Involved in Practical Aquaculture

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)

Scottish Rainbow Trout Production

Information provided by

Dr. B. McAndrew,

Univ. Stirling, Scotland

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

slide25

Atlantic halibutEffect of Sex on Growth

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

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