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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. 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

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  1. The fundamentals of producing monosex fish for aquaculture D.J.Martin-Robichaud and Tillmann Benfey

  2. 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

  3. 3042 g 2288 g

  4. 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)

  5. 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

  6. Indirect Feminization

  7. 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

  8. 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)

  9. 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

  10. 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

  11. 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

  12. 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

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

  14. 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

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

  16. 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.

  17. 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.

  18. Differentiating neomales Sex offspring produced by each male. Sex-reversed females (XX) will produce 100% female offspring.

  19. 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

  20. Acknowledgments: Chris Hendry, Harald Tvedt Mike Reith, Tim Jackson, Darrin Reid Scotian Halibut Ltd NSERC, Aquanet, ACRDP

  21. 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

  22. 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.

  23. 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

  24. 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.

  25. Atlantic halibutEffect of Sex on Growth Females on average ~750 g larger than males at Nov-08 sampling.

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