natgillin/translations-raw huggingface dataset:
Sumpter, J.P. and S. Jobling, 1995, “Vitellogenesis As A Biomarker for Estrogenic Contamination of the Aquatic Environment”, Environmental Health Perspectives 103, pp. 173-178.
→ 45 sekund pri 56 °C
Hutchinson, S. Maddix, and H. Trip (1999), “An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin”, Environmental Toxicology and Chemistry 18, pp. 337-347.
Podaljševanje
Pedersen, and P. Bjerregaard (2001a), “Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio)”, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 130, pp. 119-131.
→ 1 minuta pri 68 °C
Andersen, L., P. Bjerregaard, and B. Korsgaard (2003), “Vitellogenin induction and brain aromatase activity in adult male and female zebrafish exposed to endocrine disrupters”, Fish Physiology and Biochemistry 28, pp. 319-321.
15 minut pri 68 °C
Petersen (2003), “Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone”, Aquatic Toxicology 65, pp. 397-411.
Program PCR za gena X in Y:
Tyler (2002), “Utility of a juvenile fathead minnow screening assay for detecting (anti-)estrogenic substances”, Environmental Toxicology and Chemistry 21, pp. 319-326.
v tem programu PCR za odkrivanje genov X in Y se uporabijo vzorci neokrnjene DNA.
Wang (2007), “Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults”, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, pp. 533-541.
Pri moški DNA morata biti vidni dve progi, pri ženski DNA pa ena proga (po barvanju in gelski elektroforezi).
LeBlanc, and C.V.Sullivan (1999), “Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds”, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 123, pp. 113-125.
V to izvajanje programa je treba vključiti eno pozitivno kontrolo za moške (vzorec XY) in eno za ženske (vzorec XX).
Porcher (2002), “Development and validation of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio)”, Environmental Toxicology and Chemistry 21, pp. 1699-1708.
‚PG 17,5‘ (zgornji/smerni) → CCG GGT GCC CAA GTG CTC CCG CTG
Hatano, H. Mizukami, M. Yamashita, R. Sakakibara, and E. Tamiya (2002), “Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin”, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 132, pp. 161-169.
‚PG 17,6‘ (spodnji/protismerni) → GAT CGT CCC TCC ACA GAG AAG AGA
Bengtsson (2004), “The juvenile three-spined stickleback (Gasterosteus aculeatus L.) as a model organism for endocrine disruption — II — kidney hypertrophy, vitellogenin and spiggin induction”, Aquatic Toxicology 70, pp. 311-326.
Cikel (40-krat):
Tatarazako, N., M. Koshio, H. Hori, M. Morita, and T. Iguchi (2004), “Validation of an enzyme-linked immunosorbent assay method for vitellogenin in the medaka”, Journal of Health Science 50, pp. 301-308.
→ 45 sekund pri 55 °C
Eidem, J.K., H. Kleivdal, K. Kroll, N. Denslow, R. van Aerle, C. Tyler, G. Panter, T. Hutchinson, and A. Goksoyr (2006), “Development and validation of a direct homologous quantitative sandwich ELISA for fathead minnow (Pimephales promelas) vitellogenin.
→ 1 minuta 30 sekund pri 68 °C
Ankley (2006), “Evaluation of a commercial kit for measuring vitellogenin in the fathead minnow (Pimephales promelas)”, Ecotoxicology and Environmental Safety 64, pp. 101-105.
ta program PCR preverja rezultate ‚programa PCR za gena X in Y‘.
Holbech, H., Petersen, G. I., Norman, A., Örn, S, Norrgren, L., and Bjerregaard, P (2001b), “Suitability of zebrafish as test organism for detection of endocrine disrupting chemicals.
Pri ‚moških vzorcih‘ mora biti vidna ena proga, pri ‚ženskih vzorcih‘ pa proge ne smejo biti vidne (po barvanju in gelski elektroforezi).
Comparison of vitellogenin in plasma and whole body homogenate from zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss)”, Nordic Council of Ministers, TemaNord 2001:597, pp. 48-51.
‚DMTYa (zgornji/smerni)‘ → GGC CGG GTC CCC GGG TG
Porcher, and A. Goksoyr (2004), “Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening”, Analytical and Bioanalytical Chemistry 378, pp. 621-633.
‚DMTYd (spodnji/protismerni)‘ → TTT GGG TGA ACT CAC ATG G
Orn, S., S. Yamani, and L. Norrgren (2006), “Comparison of vitellogenin induction, sex ratio, and gonad morphology between zebrafish and Japanese medaka after exposure to 17 alpha-ethinylestradiol and 17 beta-trenbolone”, Archives of Environmental Contamination and Toxicology 51, pp. 237-243.
Barvanje vzorcev PCR
Scholz, S. and N. Kluver (2009), “Effects of Endocrine Disrupters on Sexual, Gonadal Development in Fish, Sexual Development 3”, pp. 136-151.
Raztopine za barvanje
Fenske, M., G. Maack, C. Schafers, and H. Segner (2005), “An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio”, Environmental Toxicology and Chemistry 24, pp. 1088-1098.
dstotni glicerol
Nagahama (2004), “Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes”, Developmental Dynamics 231, pp. 518-526.
0,25-odstotno bromofenolno modro
Shinomiya, A., H. Otake, K. Togashi, S. Hamaguchi, and M. Sakaizumi (2004), “Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations”, Zoological Science 21, pp. 613-619.
0,25-odstotni ksilenksianol
Flick (2007), “Collapse of a fish population after exposure to a synthetic estrogen”, Proceedings of the National Academy of Sciences of the United States of America 104, pp. 8897-8901.
V vsako epruveto se s pipeto doda 1 μl raztopine za barvanje.
Kidd (2009), “Interspecies differences in biochemical, histopathological, and population responses in four wild fish species exposed to ethynylestradiol added to a whole lake”, Canadian Journal of Fisheries and Aquatic Sciences 66, pp. 1920-1935.
Začetek gelske elektroforeze:
Tyler (2006), “Development of chronic tests for endocrine active chemicals — Part 1.
pripravljeni 1-odstotni agarozni gel se prenese v komoro za gelsko elektroforezo, napolnjeno s pufrom 1 × TAE;
An extended fish early-life stage test for oestrogenic active chemicals in the fathead minnow (Pimephales promelas)”, Aquatic Toxicology 77, pp. 279-290.
v utor na agaroznem gelu se s pipeto vnese 10–15 μl obarvanega vzorca PCR;
Petersen, P. Jackson, K. Hylland, L. Norrgren, and P. Bjerregaard (2006), “Detection of endocrine disrupters: Evaluation of a Fish Sexual Development Test (FSDT)”, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 144, pp. 57-66.
v ločen utor se s pipeto vnese tudi 5–15 μl Invitrogena (lestvica 1 kb);
Andersen, L., K. Kinnberg, H. Holbech, B. Korsgaard, and P. Bjerregaard (2004), “Evaluation of a 40 day assay for testing endocrine disrupters: Effects of an anti-estrogen and an aromatase inhibitor on sex ratio and vitellogenin concentrations in juvenile zebrafish (Danio rerio)”, Fish Physiology and Biochemistry 30, pp. 257-266.
elektroforezo je treba začeti z 200 V;
Morthorst, J.E., H. Holbech, and P. Bjerregaard (2010), “Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations”, Aquatic Toxicology 98, pp. 336-343.
končati jo je treba po 30–45 minutah.
Metcalf (2003), “Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes)”, Aquatic Toxicology 63, pp. 391-403.
Določanje prog:
Tyler (2004), “Successful detection of (anti-) androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development”, Aquatic Toxicology 70, pp. 11-21.
agarozni gel je treba očistiti v destilirani H2O;
Petersen, and P. Bjerregaard (2007), “Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio)”, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, pp. 165-170.
nato je treba agarozni gel za 15–30 minut prenesti v etidijev bromid;
Chapter C.14 of this Annex, Fish Juvenile Growth Test.
po tem je treba v UV-svetlobni škatli posneti sliko agaroznega gela;
Chapter C.4 of this Annex, Ready Biodegradability.
na koncu se vzorci analizirajo glede na pozitivno kontrolno progo (ali proge) in lestvico.
Chapter C.37 of this Annex, 21-Day Fish Assay:
Dodatek 10
A Short Term Screening for Oestrogenic and Androgenic Activity, and Aromatase Inhibition.
Smernica za vzorčenje tkiva za določitev genetskega spola z metodo PCR pri zetu
Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., Segner, H (2007), “Concentration- and time-dependent effects of the synthetic estrogen, 17 alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio” Journal of Toxicology and Environmental Health-Part A, 70, 9-10 pp 768-779.
Vzorčenje tkiva in ekstrakcija DNA
Abbreviations and definitions
DNA se lahko ekstrahira z različnimi komercialno dostopnimi reagenti ter ročnimi in samodejnimi ekstrakcijskimi sistemi.
Apical endpoint
V nadaljevanju je opisan protokol, ki se uporablja v laboratoriju Cefas Weymouth, kjer je potrebno, pa so bili dodani alternativni pristopi.
Causing effect at population level
Z vsake posamezne ribe se z natančnimi škarjami odstrani majhen kos tkiva (10–20 mg) z dorzolateralnega območja (ko se odstranita glava in rep za analizo VTG).
Air saturation value
Tkivo se vnese v epruveto in postavi neposredno na tekoči dušik (za shranjevanje pri – 80 °C) ali napolni s 70-odstotnim etanolom (za prenos in naknadno shranjevanje pri 4 °C).
A substance or a mixture.
Alikvot (200 μl) produkta razkroja se prenese v 96-mestni S-blok (Qiagen), DNA pa se ekstrahira v 96-mestnem formatu s sistemom Qiagen Universal BioRobot in priborom QIamp Investigator BioRobot.
Days post hatch
DNA se eluira v 50 μl proste vode DNaze in RNaze.
Y-specific DM-domain gene required for male development in the medaka fish
Če se za ekstrakcijo DNA uporabljajo trda tkiva (kot je bodica ali prsna plavut), bo morda treba vzorec homogenizirati v liznem pufru s sistemom za lizo tkiva FastPrep® ali enakovrednim sistemom za razkroj tkiva.
Enzyme-Linked Immunosorbent Assay
Ali pa:
Fish weight
tkivo se čez noč razkraja s proteinazo K v 400 μl liznega pufra G2 (Qiagen), DNA pa se ekstrahira iz 200 μl produkta razkroja z enostavnim priborom EZ-1 DNA Tissue Kit in EZ-1 BioRobot ali enostavnim priborom DNA Mini Kit.
Fish wet weight (blotted dry)
DNA se eluira v 50 μl zmesi;
FSDT
tkiva se obdelajo z reagentom DNAzol.
Fish Sexual Development Test
Vzorci tkiv se v 1,5-mililitrski mikrocentrifugirki 10 minut razkrajajo v 1 ml DNAzola in nato 5 minut centrifugirajo pri 13000 o/min, da se odstranijo vsi delci.
Hypothalamic-pituitary-gonadal axis
Vzorec, ki je prestal lizo, se prenese v novo 1,5-mililitrsko mikrocentrifugirko s 500 μl 100-odstotnega molekularno čistega etanola in nato 10 minut centrifugira pri 13000 o/min, da se DNA obori.
Intersex fish
Etanol se odstrani in nadomesti s 400 μl 70-odstotnega etanola molekulske stopnje, 5 minut centrifugira pri 13000 o/min, kosi DNA pa se raztapljajo v 50 μl proste vode molekulske DNaze in RNaze.
Fish with more than one oocyte in testis per 6 sections analysed or spermatogenetic cells in ovaries (yes/no)
Če se uporabljajo trda tkiva (prsna plavut), bo morda treba vzorec pred ekstrakcijo DNA homogenizirati v liznem pufru s sistemom za lizo tkiva FastPrep® ali enakovrednim sistemom za razkroj tkiva.
Wet weight of fish per volume of water
DNA se shranjuje pri – 20 °C, dokler se ne potrebuje.
Mode of action
med postopki je treba nositi rokavice.
Reverse Transcriptase Polymerase Chain-Reaction
Analiza verižne reakcije s polimerazo (PCR)
Undifferentiated fish
Amplifikacije so se izvedle z 2,5 μl ekstrakta DNA v 50 μl reakcijske zmesi z začetnimi oligonukleotidi lokusov (kot je opisano v Peichel idr., 2004.
Experimental conditions for the FSDT (freshwater species)
protismerni začetni oligonukleotid
Japanese medaka (Oryzias latipes)
Reagente PCR dobavljajo številni dobavitelji.
Zebrafish (Danio rerio)
V nadaljevanju opisana metoda se trenutno uporablja v laboratoriju Cefas Weymouth.
Three-spined Stickleback (Gasterostreus aculeatus)
Priprava ‚reakcijske zmesi‘ (50 μl na vzorec):
Flow-through or semi-static
Izhodiščna zmes se pripravi, kot sledi.
Fluorescent bulbs (wide spectrum
Pripravi se lahko vnaprej in se do uporabe shrani zamrznjena pri – 20 °C.
Light intensity
Pripraviti je treba zadostno izhodiščno zmes za negativno kontrolo (samo voda stopnje molekularne biologije).
10-20 μE/m2/s, 540-1080 lux, or 50-100 ft-c (ambient laboratory levels)
Prostornina (založna koncentracija)/vzorec
12-16 h light, 8-12 h dark
Reakcijski pufer 5xGoTaq®
Minimum chamber size
Nukleotidi (dATP, dCTP, dGTP, dTTP)
Individual chambers should contain a minimum of 7 l water volume
0,5 μl (25 mM vsak)
Age of test organisms at start of exposure
250 μM vsak
Newly fertilised eggs (Early blastula stage)
Smerni začetni oligonukleotid
Newly fertilised eggs
0,5 μl (0,1 nmol/μl)
No. of eggs per treatment
Protismerni začetni oligonukleotid
Minimum 120
2,0 μM
Minimum 3 (plus appropriate controls)
Voda stopnje molekularne biologije
No. replicates per treatment
Polimeraza GoTaq
Minimum 4 (unless square root allocation to controls)
V označeno 0,5-mililitrsko epruveto za PCR s tankimi stenami se odmeri 47,5 μl.
Live Artemia, frozen adult brine shrimp, flake food, etc.
V ustrezno označeno epruveto se doda 2,5 μl čiste DNA.
It is recommended to feed twice daily
Postopek se ponovi za vse vzorce in negativno kontrolo.
Special fry food, live Artemia, frozen adult brine shrimp, flake food, etc.
Prekrije se z dvema kapljicama mineralnega olja.
It is recommended to feed twice daily
Namesto tega se lahko uporabi ciklični termostat z ogrevanim pokrovom.
None unless DO concentration falls below 60 % saturation
Pokrovi se zaprejo.
None unless DO concentration falls below 70 % saturation
Vzorci so se 5 minut denaturirali v cikličnem termostatu PTC-225 pri 94 ± 2 °C, temu pa je sledilo 39 enominutnih ciklov pri 94 ± 2 °C, 55 ± 2 °C in 72 ± 2 °C ter na koncu desetminutno podaljševanje pri 72 ± 2 °C.
Clean surface, well or reconstituted water
Priprava agaroznega gela (2 %):
Test chemical exposure duration
Proizvodi PCR se običajno razgradijo na 20-odstotnem agaroznem gelu, ki vsebuje etidijev bromid.
Hatching success > 80 %
Dodata se 2 kapljici končne koncentracije etidijevega bromida 0,5 μg/ml.
Post hatch survival ≥ 70 %
Vroča raztopina se prenese v opremo za ulitje gela.
growth (Fish wet weight, blotted dry) > 150 mg
Gel naj se strdi.
growth (Fish wet weight, blotted dry) > 75 mg
Gelska elektroforeza:
growth (Fish wet weight, blotted dry) > 120 mg
Agarozni gel se prenese v opremo za elektroforezo in potopi v pufer 1 × TAE.
Length (standard length) > 20mm
V ločene vdolbinice se vnese 20 μl vsakega vzorca, pri čemer se v prosto vdolbinico doda molekulski velikostni standard (lestvica DNA 100 bp, Promega).
Length (standard length) > 14 mm
Elektroforeza se 30–45 minut izvaja pri 120 V.
Length (standard length) > 20 mm
Vizualizacija proizvodov amplifikacije
Sex ratio (% males or females) 30 %-70 %
Če je bil v agarozni gel vključen etidijev bromid, kot je opisano zgoraj, se proizvodi DNA vizualizirajo pod virom UV.
Chemical characteristics of an acceptable dilution water
V nasprotnem primeru se agarozni gel obarva tako, da se gel pred vizualizacijo za 30 minut prekrije z razredčeno raztopino etidijevega bromida (0,5 μg/ml v vodi).
CONCENTRATION
Dodatek 11
Particular matter
Smernica za postopek umetne oploditve pri zetu
From test method C.14/Guidance on test concentrations
Namen tega razdelka je opisati postopke za pridobitev oplojenih iker zeta za uporabo v preskusu spolnega razvoja rib.
Column (Number of concentrations between 100 and 10, or between 10 and 1) [1]
Postopki
A series of three (or more) successive concentrations may be chosen from a column.
Pridobivanje semenčic samcev
Mid-points between concentrations in column (x) are found in column (2x + 1).
Evtanazira se dobro obarvan samec želene populacije.
The values listed can represent concentrations expressed as percentage per volume or weight (mg/l or μg/l).
Moda se odrežejo z obeh strani ribe.
Values can be multiplied or divided by any power of 10 as appropriate.
Moda so običajno močno pigmentirane paličaste strukture, ki so že vidne ob lateralni sredinski črti telesa.
Column 1 might be used if there was considerable uncertainty on the toxicity level.
Uporabi se lahko katera koli od naslednjih metod.
Guidance for homogenisation of head & tail from juvenile zebrafish, fathead minnow, three spined stickleback and Japanese medaka
Z natančnimi škarjami se naredi 1–1,5-centimetrski rez z enim prerezom pod kotom približno 45 stopinj, ki se začne pri kloaki.
The purpose of this section is to describe the procedures that occur prior to the quantification of the VTG concentration.
S skalpelom se naredi majhen rez na boku ribe nekoliko posteriorno na medenico in ventralno na lateralne ploščice.
Other procedures that result in comparable VTG quantification can be used.
Moda se odstranijo s finimi kleščami in vstavijo v petrijevko.
It is an option to determine the VTG concentration in blood plasma or liver instead of head/tail homogenate.
Vsako modo se prekrije s 100 μl sveže narejene Hankove končne raztopine.
The head and tail are cut of the fish in accordance with the test description.
Moda se fino narežejo na kocke z britvico ali skalpelom.
All dissection instruments, and the cutting board should be rinsed and cleaned properly (e.g. with 96 % ethanol) between handling of each single fish to prevent “VTG pollution” from females or induced males to un-induced males.
Pri tem se sprostijo semenčice, ki dajo Hankovi raztopini mlečen videz.
When a homogenous mass is achieved an amount of 4-10 time the tissue weight of ice-cold homogenisation buffer is added (note the dilution).
Tekočina s semenčicami se vnese v epruveto, pri čemer se pazi, da se s pipetiranjem vanjo ne vnesejo deli mod.
The samples are placed on ice until centrifugation at 4 °C at 50000 g for 30 min.
V epruveto se doda 800 μl Hankove končne raztopine in dobro premeša.
Use a pipette to dispense portions of 20 to 50 μl (note the amount) supernatant into at least two tubes by dipping the tip of the pipette below the fat layer on the surface and carefully sucking up the supernatant without fat- or pellet fractions.
Po potrebi se lahko samec konzervira s fiksiranjem v 100-odstotnem etanolu ali drugem želenem fiksativu.
Place on ice during use
To je zlasti pomembno, če se v okviru študije določa izvor potomstva glede na starše.
Homogenisation buffer:50 mM Tris-HCl pH 7,4;
čeprav se lahko večina potrebnih založnih raztopin pripravi vnaprej, je treba zalogo 5 in zato končno raztopino pripraviti svežo na dan uporabe.
1 % Protease inhibitor cocktail (Sigma):
Zaloga 1
12 ml Tris-HCl pH 7,4 + 120 μl Protease inhibitor cocktail (or equivalent protease inhibitor cocktails).TRIS:
8,00 g
TRIS-ULTRA PURE (ICN)Protease inhibitor cocktail:
0,40 g
From Sigma (for mammalian tissue) Product number P 8340.
Destilirana voda
Guidance for quantification of head & tail homogenate vitellogenin in zebrafish (Danio rerio) (modified from Holbech et al., 2001). other procedures using homologous antibodies and standards can be used
100 ml
Purified zebrafish vitellogenin standard [1] is serially diluted to 0,2, 0,5, 1, 2, 5, 10 and 20 ng/ml in dilution buffer (**) and samples are diluted at least 200 times (to prevent matrix effect) in dilution buffer and applied to the plates.
HPO4 (brezvodni)
An assay control is applied in duplicate.
0,358 g
150 μl are applied to each well.
0,60 g
Standards are applied in duplicate and samples in triplicate.
Zaloga 3
Incubate over night at 4 °C on a shaker.
0,72 g
The plates are washed 5 times with washing buffer (*)
50 ml
HRP coupled to a dextran chain (e.g. AMDEX A/S, Denmark) and conjugated antibodies are diluted in washing buffer;
Zaloga 4
Actual dilution differs by batch and age.
MgSO4 7H2O
When the standard curve is fully developed the enzyme activity is stopped by adding 150 μl 0,2 M H2SO4 to each well.
če že imate nekatere od navedenih soli, vendar z drugačno vsebnostjo vode (tj. 2H2O namesto brezvodne), jih lahko uporabite, vendar najprej prilagodite maso na podlagi molekulske mase.
The absorbance is measured at 450 nm (e.g. on a Molecular Devices Thermomax plate reader).
Za Hankovo končno raztopino se po naslednjem vrstnem redu združijo:
Data are analysed on the associated software (e.g. Softmax).
zaloga 1
Washing buffer:
1,0 ml
PBS-stock (****)
zaloga 2
Adjust pH to 7,3 and fill to 5 l with millipore H2O.
0,1 ml
Store at 4 °C.
zaloga 3
Dilution buffer:
8,6 ml
PBS-Stock (****)
zaloga 4
TMB plus is a “ready-to-use” substrate produced by KemEnTec (Denmark).
Pred uporabo dobro premešajte.
It is sensitive to light.
Oploditev
PBS stock
V želeni populaciji se določijo velike in gravidne samice;
HPO4 · 2H2O
samice so pripravljene na stiskanje šele, ko se vidijo ikre, ki izstopajo iz kloake.
Adjust pH to 6,8 and fill with millipore H2O to 2 l.
Za pripravljene samice je značilna ‚pokončna‘ drža.
Store at room temperature.
S prstom oz. palcem se nežno premika po boku ribe do repa, da se spodbudi izločitev vrečke z ikrami v svežo petrijevko.
Battelle AP4.6.04 (1,18 mg/ml (AAA)), purified according to:
Postopek se ponovi na drugi strani in riba vrne v posodo.
Guidance for the preparation of tissue sections for sex determination and staging of gonads
Ikre se lahko razporedijo (tvorijo enoslojno plast) s finim čopičem.
The purpose of this section is to describe the procedures that occur prior to the evaluation of histological sections.
Pomembno je, da se poskusi čim več iker izpostaviti semenčicam, k čemur pripomore čim večja površina iker.
Other procedures that result in similar sex determination and gonadal staging can be used.
ikre morajo biti vlažne, zato se obložijo z vlažnim robcem (pomembno je, da se ikre ne dotikajo vode neposredno, saj bi lahko horij, ki preprečuje oploditev, prehitro otrdel).
With a few exceptions, these procedures are similar for Japanese medaka (JMD) and zebrafish (ZF).
Samice se zelo razlikujejo po številu iker, ki ga lahko proizvedejo, vendar bi moralo biti v povprečju iz ene gravidne samice enostavno pridobiti približno 150 iker.
Euthanasia, Necropsy, and Tissue Fixation
Po celotni površini iker se s čopičem enakomerno porazdeli 25μl semenčic v Hankovi zmesi.
Objectives:
Ko se oplojevanje začne, bodo ikre hitro otrdele in spremenile barvo (v minuti).
Provide for the humane sacrifice of fish.
Če se oceni, da je iker več kot 150, se postopek ponovi.
Obtain necessary body weights and measurements.
Če ikre ne otrdijo v minuti, se doda nekaj semenčic.
Evaluate secondary sex characteristics.
z dodajanjem semenčic se stopnja oplojenosti morda ne bo izboljšala.
Dissect tissues for VTG analysis.
Ikre in raztopina s semenčicami morajo biti v stiku vsaj 15 minut, oplojene ikre pa je treba vstaviti v akvarij za izpostavljanje v uri in pol po oploditvi.
Fixation of the gonads.
Postopek se ponovi z drugo samico, dokler ni zbrano želeno število iker.
Fish should be sacrificed immediately prior to necropsy.
Štetje in razporeditev iker v preskusnem akvariju
Therefore, unless multiple prosectors are available, multiple fish should not be sacrificed simultaneously.
Ikre je treba enakomerno porazdeliti med vsako stopnjo tretiranja, da se prepreči genetska nagnjenost.
Using the small dip net, a fish is removed from the experimental chamber and transported to the necropsy area in the transport container.
Vsako serijo oplojenih iker je treba razporediti v enako velike skupine (toliko, kot je stopenj tretiranja) s topim instrumentom (npr. s širokimi entomološkimi kleščami ali inokulacijsko zanko).
The fish is removed from the solution when there is cessation of respiration and the fish is unresponsive to external stimuli.
priporočljivo je dodati dodatnih 20 % (tj. 96 iker na stopnjo tretiranja), dokler se ne zagotovi 100-odstotna oploditev.