Problems with early sexual maturation in on-growth farms

Transcription

1 Utrecht University PUBERTIMING Photoperiod control of puberty in farmed fish: Development of new techniques and research into underlying physiological mechanisms G.L. Taranger 1, Eva Andersson 1, S.O. Stefansson 2, N.R.Bromage 3, H. Migaud 3, O.Kah 4, J.J.Lareyre 5, M. Carillo 6 and R.W.Schulz 7 1 Institute of Marine Research, Bergen, Norway. 2 University of Bergen, Bergen, Norway. 3 Institute of Aquaculture, University of Stirling, Scotland. 4 Centre National de la Recherche Scientifique, Rennes, France. 5 Institut National de la Recherche Agronomique, Rennes, France. 7 University of Utrecht, Utrecht, The Netherlands. 6 Instituto de Acuicultura de Torre de la Sal, Spain. Problems with early sexual maturation in on-growth farms Loss of growth Reduction of harvest quality in particular in salmonids Increased risk for diseases and mortality Restricted harvest season and size Sexually mature male salmon 1

2 Continuous light (LL) can delay puberty in Atlantic salmon (% sexually mature) ** *** 4 2 *** Males Females NL LL March LL Jan (Taranger et al. 1998) *** How effective is the LL treatment? Widely used in salmon farms - preliminary positive results with trout, sea bass and sea bream Not always fully effective Dependent on timing of LL treatment Interactions with age, genetics, nutrition, temperature and more...? Rearing systems More effective in in-door/covered tanks? In out-door situations light intensity and spectral quality matters? 2

3 PUBERTIMING applied objective To identify environmental and physiological keyfactors responsible for initiating puberty in fish along with the development of photoperiod protocols to reliably control puberty in farmed fish. Atlantic Salmon Sea bass Basic aspects 1. Improve knowledge on the mechanisms of activation of the brainpituitary-gonad (BPG) axis during puberty in fish, including development of new tools to study the BPG axis in salmon, rainbow trout and sea bass. 2. Improve understanding of the importance of differences in light intensity and spectral quality in affecting the BPG axis and the initiation/postponement of puberty, by assessment of pineal melatonin production in vitro and in vivo in salmon and sea bass. 3. Improve understanding of the interactions between photoperiod protocols, genetic background and adiposity in arresting/promoting puberty in salmon and sea bass 3

4 Improve understanding of the importance of differences in light intensity and spectral quality in affecting the BPG axis and the initiation/postponement of puberty Feb 1) Sea bass - continuous light exposure has an inhibitory effect on percentage of precocious males (<5%) and gonadal growth - No effects on growth performance under a feed regime based on apparent Dicentrarchus labrax satiety conditions Atlantic Salmon - continuous light has an ambiguous effect: it inhibits or accelerates male puberty. Plasma testosterone levels can be used as an early marker for delayed puberty in LL treated animal Plasma testosterone levels as a predictive marker for puberty outcome Atlantic salmon NL males recruited successively into maturation LL treatment either blocks or accelerates testis maturation Low testis weight associated with low 11-KT levels Very low 11-KT levels and inactive testis in LL low fish Elevated 11-KT levels often precede elevations in GSI GSI 1.5 Feb 18 Mar 19 Apr Jun /9 4/1 3/12. * 2/8 1/8 4/9 /12 4/9 * NL / low NL / high LL / low LL / high TREATMENT / KT group - significantly different from the respective NL/low or LL/low group * # - significantly different from the respective NL/high group * * * # # * 4

5 Improve knowledge on the underlying mechanisms of the delayed puberty by a continuous light treatment: identify target genes involved in the brain-pituitary pituitary-gonad (BPG) axis activation Photoperiod Diet Growth Adiposity Genetics Brain GnRHs New candidates genes ERα, ERβ1, ERβ2 sea bass and trout Gene characterization and expression analysis mrna levels Localization assays Plasma levels Steroids 11 KT, E2 Gonads ARs Pituitary FSH FSH-R GnRH-Rs GTH-α FSH-β LH-β germ cells LH-R LH somatic cells GnRHRs GnRHR1 sea bass and trout Purified Salmon Gthα Gonadotropins receptors Salmon and trout Two ARs in Salmon GnRHR2 in sea bass and trout mrna levels protein levels Cellular Localization Gonadotropins RIA mrna levels protein levels Cellular Localization Hormone plasma levels Morphology Proliferation Apoptosis Search for seabass and salmonid orthologous genes From gene prediction to gene charaterization Consensus sequence model determined from known genes (fish and mammals) In silico analysis of fish models with fully sequenced genome ESTs databases Takifugu rubripes Tetraodon nigroviridis Orysias latipes Molecular phylogeny Degenerate primers (RT-PCR, genome walking) Danio rerio eel GnRH-R2 Identification of new family members relevant for puberty onset - spatial and temporal gene expression analysis - regulation over the reproductive cycle - pharmacology zfgnrh-r2 zfgnrh-r4 fggnrh-r3 Amberjack GnRH-R Striped bass GnRH-R Sea bass GnRH-R1 fggnrh-r1 mkgnrh-r1 fggnrh-r2 fggnrh-r5 eel GnRH-R1 rtgnrh-r2 cfgnrh-r2 zfgnrh-r1 gfgnrh-rb cfgnrh-r1 zfgnrh-r3 gfgnrh-ra Astatotilapia GnRH-R1 mkgnrh-r2 fggnrh-r4 Human GnRH-R2 Type II Type I JAS PHASE 15 et 16 mars 25 5

6 Evidence for a sea bass GnRH-Rs Rs subfunctionalization dlgnrh-r2a R2A as a marker of the BPG axis activation Several GnRH-Rs are expressed in sea bass: dlgnrhr2a and dlgnrhr1b Only dlgnrhr2a is preferentially expressed in the pituitary during puberty onset and is activated by the endogenous hypophysiotrope form of GnRH (SbGnRH) In situ hybridization Sea bass pituitary sbgnrh-rs mrna/28s fold induction Male pituitary Meiosis onset Gonadal stages dlgnrhr-2a dlgnrhr-1b dlgnrhr1b dlgnrhr2a % relative to cgnrh -II cgnrh-ii sgnrh sbgnrh % relative to cgnrh -II cgnrh-ii sgnrh sbgnrh antisense sense antisense peptide concentration peptide concentration dlgnrhr2a dlgnrhr1b Future directions Towards a global gene expression profiling approach Characterization of key candidate genes (sexual steroids and peptide hormones and cognate receptors) involved in puberty onset Use plasma testosterone levels to predict the puberty outcome. Analyse and compare the relevant candidate genes in animals predicted to have a normal versus delayed puberty caused by a continuous light treatment To compare animal with normal or delayed puberty: use the relevant candidate genes as landmarks to cross check and validate relevance of the future global gene expression analysis using arrays - arrays with salmon cdna (from SGP, Norway/UK) - arrays with trout cdna arrays (from AGENAE, France) under progress 6

7 PUBERTIMING Coord.: G. L. Tarranger University of Stirling H. Migaud University of Bergen G.L. Tarranger University of Utrecht R. Schulz, J. Bogerd INRA and Rennes University J.J. Lareyre O. Kah CSIC, Torre de la Sal M. Carrillo S. Zanuy Alicia Felip Edo J.M. Cerdá Reverter Thanks for your attention! 7