The fundamentals of producing monosex fish for aquaculture

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The fundamentals of producing monosex fish for
aquaculture

• D.J.Martin-Robichaud
• and Tillmann Benfey

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• 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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3042 g
2288 g
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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)

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Indirect Feminization
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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

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Gynogenesisuniparental maternal inheritance(all
genetic contribution from female)
1. Exclude paternal genome UV irradiation of
sperm optimum treatment will (a) disable sperms
genomic DNA (b) not affect sperms ability to
swim and activate development in
eggs optimum treatment for halibut 180 dilution
in seminal plasma exposure to UV at 65
mJ/cm2 yields gynogenetic haploids (non-viable)
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• 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 _at_ 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
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• 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
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Indirect Feminization to produce all-female
Atlantic halibut stocks
Neomale Broodstock Genotypic female but
phenotypic male
XX
XX
masculinization
Hormonal sex reversal
All females XX
XX
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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
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• Genetic mechanism of sex determination
• Timing of sex differentiation
• Efficacy of direct hormonal sex reversal
• Reproductive ability of sex reversed fish
• Differentiating neomales

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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
D
C
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• Genetic mechanism of sex determination
• Timing of sex differentiation
• Efficacy of direct hormonal sex reversal
• Reproductive ability of sex reversed fish
• Differentiating neomales

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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) 17a-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.
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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.
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Differentiating neomales

• Sex offspring produced by each male.
• Sex-reversed females (XX) will produce 100
  female offspring.

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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

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Acknowledgments Chris Hendry, Harald Tvedt Mike
Reith, Tim Jackson, Darrin Reid Scotian Halibut
Ltd NSERC, Aquanet, ACRDP
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Sex-linked Markers
Micro array
Accomplishments
Gynogens/ X/Y Sex
Linkage Map ESTs
Pedigree Analysis
QTL
Mapping
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
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Part 1 Summary of the Problem and General
Scientific Principles
Hormonal regulation of sex differentiation
Genotypic Master gene (e.g., dmy), minor sex
determining genes, autosomal genes
Environmental factor (e.g., temperature)
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, figa
amh, sox9
Aromatase
dmrt1
Sex differentiation
Estrogen
Testis differentiation
ER
11ß-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.
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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
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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.
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Atlantic halibutEffect of Sex on Growth
Females on average 750 g larger than males at
Nov-08 sampling.