the fundamentals of producing monosex fish for aquaculture d.j.martin-robichaud and tillmann benfey
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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
• Alleviate any misunderstandings regarding physiological and genetic changes involved
• protocols species specific, process of answering questions similar
• Atlantic halibut research as example
0
500
1000
1500
2000
2500
3000
3500
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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3000
3500
Dec-0
5
Jun-
06
Dec-0
6
Jun-
07
Dec-0
7
Jun-
08
Date (mon-yr)
Wei
gh
t (g
)Atlantic halibut
Effect of Sex on Growth
Females on average ~750 g larger than males at Nov-08 sampling.
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