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Biology

Øjenstilk ablation for at øge æggestokkens modning i mudderkrabber

Published: March 31, 2023 doi: 10.3791/65039
Automatic Translation
1, 1, 1,2,3, 2,4, 5, 1,2,3
1Higher Institution Centre of Excellence (HICoE), Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, 2Centre for Chemical Biology, Universiti Sains Malaysia, 3Department of Aquaculture, Faculty of Fisheries, Kasetsart University, 4School of Biological Sciences, Universiti Sains Malaysia, 5International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University

ERRATUM NOTICE

Summary

To øjenstilk ablationsprotokoller (dvs. cauterization og kirurgi tilgange) blev udført på bedøvede kvindelige krabber. Øjenstilkens ablation af mudderkrabber fremskyndede modningen af æggestokke uden at reducere overlevelsesraten.

Abstract

Mudderkrabber (Scylla spp.) er kommercielt vigtige krebsdyrarter, der kan findes i hele det indovestlige Stillehavsområde. Under kultur er induktion af ovariemodning vigtig for at imødekomme forbrugernes efterspørgsel efter modne mudderkrabber og fremskynde frøproduktionen. Øjenstængelablation er et effektivt værktøj til at forbedre modningen af æggestokkene i mudderkrabber. Der er dog ingen standardprotokol for øjenstilk-ablation af mudderkrabber. I denne undersøgelse beskrives to øjenstilk-ablationsteknikker: cauterization (brugen af varmt metal til at ablate øjenstilken på en bedøvet krabbe) og kirurgi (fjernelse af øjenstilken ved hjælp af kirurgisk saks). Før øjenstilk-ablation blev kønsmodne hunner (CW > 86 mm) bedøvet ved hjælp af en ispose (-20 °C) med havvand. Når vandtemperaturen nåede 4 °C, blev isposen taget ud af vandet. Strømmende havvand (omgivelsestemperatur: 28 °C) blev brugt til genopretning fra anæstesien umiddelbart efter ablation af øjenstilken. Dødelighed forekom ikke under eller efter processen med ablation af øjenstilk. Øjenstilkens ablationsprotokol, der præsenteres her, fremskyndede æggestokkenes modning af mudderkrabberne.

Introduction

Alle fire mudderkrabbearter, der tilhører slægten Skylla, er kommercielt vigtige krebsdyrarter i akvakultur 1,2. Væksten af krebsdyr, herunder mudderkrabber, og deres transformation fra den præmodne (sub-voksen eller pubertale) fase til den seksuelt modne (voksne) fase sker gennem en smelteproces, der involverer periodisk afgivelse af ældre og mindre exoskeletter. Carapace bredde (CW), chelipeds og abdominal flap morfologier bruges i vid udstrækning til at bestemme den seksuelle modenhed af Scylla spp. 3,4,5. Smelteprocessen reguleres af virkningen af forskellige hormoner og kræver en enorm mængde energi6. Ud over den normale smelteproces fremskynder tabet af lemmer, enten frivilligt eller fremkaldt af eksterne faktorer, smeltningen af krabber uden at påvirke deres overlevelsesrate 7,8,9. Derfor anvendes lemmernes autotomi almindeligvis til induktion af molt i softshell-mudderkrabbeopdrætsindustrien 7,9.

Unilateral eller bilateral øjenstilk ablation er mest populær i ferskvandsrejer og marine rejer til gonademodning og frøproduktion10,11,12,13. Almindelige øjenstilk-ablationsteknikker hos krebsdyr inkluderer følgende: (i) ligering ved bunden af øjenstilken ved hjælp af en streng14,15; ii) cauterization af øjenstilken ved hjælp af varm tang eller elektrokauteri anordninger16 iii) fjernelse eller direkte klemning af øjenstilken for at efterlade et åbent sår12 og (iv) fjernelse af øjenstilkens indhold gennem snit efter skæring af den distale del af øjet med en barbermaskine17. Øjenstilken X-organer er vigtige endokrine organer i krebsdyr, da de regulerer krebsdyr hyperglykæmiske hormoner (CHH), smeltehæmmende hormoner (MIH) og vitellogenese-hæmmende hormoner (VIH) 6,18,19,20,21,22. Eyestalk X-organer (eller sinuskirtelkomplekset) syntetiserer og frigiver gonadehæmmende hormoner (GIH), også kendt som vitellogenesehæmmende hormoner (VIH), der tilhører neuropeptidhormonfamilien6. Unilateral eller bilateral øjenstilk-ablation reducerer GIH-syntese, hvilket resulterer i dominans af stimulerende hormoner (dvs. gonadestimulerende hormoner, GSH) og accelerationen af ovariemodningsprocessen i krebsdyr23,24,25,26. Uden indflydelse fra GIH efter øjenstængelablation bruger kvindelige krebsdyr deres energi på udvikling af æggestokke27. Det har vist sig, at ensidig ablation af øjenstilken er tilstrækkelig til induktion af modning af æggestokkene hos krebsdyr11, og at den ablerede øjenstilk hos rejer og krabber kan regenerere efter flere smeltninger28. Der er registreret fire ovarieudviklingsstadier i Scylla spp.: i) umodne (stadie-1), ii) tidlig modning (stadie-2), iii) præmodning (stadie-3) og iv) fuldt modne (stadie-4)29,30. Det umodne ovariestadium findes hos umodne kvinder. Efter pubertetssmeltning og parring begynder den umodne æggestok at udvikle sig og modnes endelig (fase 4) før gytning31.

En øjenstilk ablationsprotokol er afgørende for udvikling af mudderkrabber og frøproduktion. På det globale fødevaremarked foretrækkes modne mudderkrabber med fuldt modne æggestokke (stadie-4) snarere end krabber med højere muskelindhold af forbrugerne og har således en højere kommerciel værdi, endnu højere end store hanner. Der er ingen komplet protokol for øjenstilk ablation af mudderkrabber. Øjenstilkens ablationsprotokol i dette arbejde minimerer stress ved at bruge fuldt bedøvede krabber og minimerer fysisk skade på personale fra krabbebid. Denne protokol er nem og omkostningseffektiv. Her præsenterer vi en protokol for øjenstilk-ablation af Scylla spp., der kan fremkalde modning af gonaden. To teknikker til øjenstilk ablation (cauterization og kirurgi) blev testet og deres effektivitet sammenlignet baseret på gonadal udviklingshastighed af kvindelige mudderkrabber.

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Protocol

Denne protokol følger den malaysiske adfærdskodeks for pleje og brug af dyr til videnskabelige formål skitseret af Laboratory Animal Science Association of Malaysia. Ofringen af forsøgsprøverne blev udført i henhold til National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH Publications No. 8023, revideret 1978). Seksuelt præmodne kvindelige mudderkrabber (orange mudderkrabbe S. olivacea) blev indsamlet fra det lokale marked (5°66′62′′N, 102°72′33′′E) ved Setiu vådområder i Malaysia. Mudderkrabbearten blev identificeret ud fra morfologiske egenskaber1.

1. Prøveopsamling og desinfektion

  1. Saml sunde, aktive og præmodne kvindelige mudderkrabber (figur 1).
    BEMÆRK: For modne hunkrabber har trekantede og lyse maveklapper sammen med et CW-område på 80-85 mm.
  2. Vask krabberne med kloreret ledningsvand (ferskvand) for at fjerne snavs og osmofile parasitter.
  3. Læg krabberne i blød i 150 ppm formaldehyd med 20 ppt saltholdighed i 30 min.
  4. Oprethold kontinuerlig og skånsom beluftning med luftsten under formaldehydbehandlingen. Luftningskilden kan være fra enten en central beluftningslinje eller en akvarieluftpumpe.
  5. Vask krabberne med strømmende havvand for at fjerne eventuel resterende formaldehyd.

Figure 1
Figur 1: Abdominal morfologi af kvindelige mudderkrabber, der bruges til at identificere de seksuelle modningsstadier. Klik her for at se en større version af denne figur.

2. Akklimatisering

  1. Overfør hver desinficeret hun til en separat 32 L cirkulær tank.
  2. Bag hunnerne i 3 dage i 20 ppt saltholdighed og fortsæt med at fodre to gange om dagen (morgen 09:00 og aften 20:00) med hakket havfisk på ca. 4% -5% af krabbens kropsvægt.
  3. Fjern overskydende og ufortyndet foder ved at sifonere før morgenfodring.
  4. Udskift 10% af krabbeopdræt havvand (20 ppt) dagligt.

3. Induceret smeltning for seksuel modenhed

  1. Skær alle benene undtagen svømmebenene ved hjælp af steriliseret saks.
    1. Fang krabben med et skovlenet, og hold krabben forsigtigt. Klip begge chelipeds først og derefter gangbenene ved det andet led ved hjælp af en saks. Krabben autotomiserer automatisk de beskadigede vedhæng. Anæstesi er ikke nødvendig for lemmer autotomi.
  2. Vask krabben i ferskvand umiddelbart efter lemmernes autotomi.
  3. Overfør de lem-autotomiserede krabber individuelt til perforerede plastkurve (28 cm L x 22 cm B x 7 cm H), og læg dem i en glasfibertank (305 cm L x 120 cm B x 60 cm H).
    BEMÆRK: To kurve kan bindes og klippes sammen. Den øverste kurv bruges som dæksel, så krabben ikke kan flygte fra kurven.
  4. Brug et recirkulerende akvakultursystem (RAS) med 20 ppt saltholdighed og en vanddybde på mindst 10 cm for at sikre, at hele plastkurven er nedsænket.
  5. Fortsæt med at fodre den lem-autotomiserede kvindelige krabbe med hakket havfisk to gange om dagen ved 5% -7% af krabbens kropsvægt.
  6. Bageste krabberne, indtil de er seksuelt modne gennem smeltning (35 dage).
    BEMÆRK: Induceret smeltning kan springes over til kommerciel modning af æggestokkene og frøproduktion med vilde modne kvindelige mudderkrabber. Høstede modne kvinder fra naturen skal akklimatiseres og direkte udsættes for koldchokbedøvelse og efterfølgende øjenstilk-ablation.

4. Anæstesi

  1. Vælg seksuelt modne kvinder med en mørkfarvet ovalformet maveklap med en CW >86 mm (figur 1).
  2. Fang krabberne med et scoop net, og hold dem individuelt i små akvarier til anæstesi.
  3. Efter 5 minutters akklimatiseringsperiode tilsættes 2-phenoxyethanol (2-PE) ved 2 ml / L i hvert akvarium og tillades 15 minutters anæstesibehandling.
  4. Sørg for, at krabberne er fuldt bedøvet af manglen på spontan bevægelse.

5. Ablation af øjenstilk

  1. Cauterization teknik
    1. Udfør alle procedurer oven på et bord og i et åbent område.
    2. Tag en fladhovedet nikkelstålstang (f.eks. En skruetrækker) med et træ- eller plasthåndtag, og dæk håndtaget med et vådt bomuldshåndklæde.
    3. Steriliser to rustfri kirurgiske tang i en autoklave.
    4. Forbered 70% ethanol i en sprayflaske og hold den væk fra brandrelaterede kilder, såsom blæselampe og rødglødende skruetrækker. Hav silkepapir klar til brug.
      BEMÆRK: Ethanol er meget brandfarligt. Hold en sikker afstand fra brandkilder.
    5. Tilslut en blæselampe til en gasflaske (butan) sikkert.
      FORSIGTIG: Følg instruktionerne på blæselampen og gasflasken. Sørg for, at blæselampen er slukket, når du tilslutter til gasflasken. Læs og følg alle de brandsikkerhedsforanstaltninger, der er nævnt på gasflasken.
    6. Brug tykke bomuldshandsker for at undgå skader fra varme genstande.
    7. Udsæt spidsen af metalstangen for blæselampens ild, indtil metalstangen er lys rød.
    8. Dæk den bedøvede krabbe med et vådt bomuldshåndklæde.
      BEMÆRK: Dæk krabbens antenner for at undgå unødig skade.
    9. Hold et øje på krabben med steriliseret tang.
      BEMÆRK: Steriliser tangen i en autoklave til første gangs brug, og desinficer med 70% ethanol til efterfølgende brug på andre krabber.
    10. Hold den rødglødende metalflade spids på krabbens øje og tryk let i ca. 10-15 s, indtil øjenstilken får en orange eller rødlig-orange farve. Vær forsigtig, når du udfører dette trin for at undgå skader på tilstødende strukturer.
      BEMÆRK: To personer er nødvendige for at udføre øjenstilk-ablation efter cauterization-metoden: en til at holde krabben og en anden til at udføre ablationsproceduren.
    11. Desinficer tangen med 70% ethanolspray for at sikre, at der ikke er krydskontaminering mellem krabber.
      BEMÆRK: Udfør kun dette trin mindst 5 minutter efter øjenstilkens ablationsprocedure for at sikre, at tangen køles ned før desinfektion ved hjælp af 70% ethanol for at forhindre potentiel brandfare.
    12. Efter at have udført øjenstilkens ablation på alle krabber, dypp den varme nikkelstålmetalstang (skruetrækker) i ledningsvand.
    13. Desinficer håndklædet inden genbrug. Flere håndklæder kan bruges til at spare tid.
      BEMÆRK: Vask håndklædet med postevand, og dypp det i 30 ppm klorvand i 5 min. Derefter vaskes håndklædet med postevand igen, og dypp det i en 1 g / L natriumthiosulfatopløsning.
    14. Opbevar blæselampen et sikkert sted, når du har slukket den, og vent, indtil den vender tilbage til omgivelsestemperatur (ca. 30 min), før du afbryder forbindelsen.
  2. Kirurgi teknik
    1. Udfør proceduren i et godt ventileret område.
    2. Steriliser to kirurgiske saks og tang i en autoklave.
    3. Hæld 50 ml 70% ethanol i et 100 ml glasbægerglas.
    4. Brug tykke bomuldshandsker.
    5. Hold den bedøvede krabbe, og dæk den med et vådt bomuldshåndklæde.
    6. Hold et øje på krabben med steriliseret tang.
    7. Skær hurtigt øjenstilken af ved hjælp af steriliseret kirurgisk saks.
      BEMÆRK: Hemolymph kan gå tabt fra den sårede del af krabben.
    8. Dyp saksen og tangen i 70% ethanol efter hver brug, og tør dem med silkepapir inden genbrug.

6. Pleje efter anæstesi

  1. Forbered 20 ppt filtreret havvand, og opbevar i en overliggende tank med kontinuerlig beluftning.
  2. Tilslut et fleksibelt rør med overheadtanken for tyngdekraftsvandstrøm.
  3. Umiddelbart efter ablation af øjenstilken placeres krabben i kurven og udsættes krabben for strømmende havvand (omgivende vandtemperatur: 28 °C) fra overheadtanken.
  4. Hold havvandet flydende, og overvåg krabben, indtil den kan bevæge sig spontant, hvilket indikerer genopretning fra anæstesi.
    BEMÆRK: Havvand kan tilberedes i en jordtank, og en nedsænket vandpumpe kan bruges til vandstrømmen.
  5. Opbevar krabberne individuelt i 20 ppt havvand med beluftning i et akvarium i 30 minutter til yderligere observation.
    BEMÆRK: De genvundne krabber vil blive dyrket individuelt i den efterfølgende yngeldyrkningsproces.

7. Observation af modning af æggestokkene

  1. Opdræt af gydekvæg
    1. Overfør de modne krabber til individuelle 32 L cirkulære tanke.
    2. Fortsæt fodring med hakkede havfisk (frosset ved -20 ° C) to gange om dagen (morgen 09:00 og aften 20:00), og fjern ufortyndet foder før morgenfodring.
    3. Bageste yngelstammen individuelt i 30 dage i 20 ppt saltholdighed.
    4. Fjern afføring, og udskift 10% af havvandet (20 ppt) dagligt.
  2. Dissektion
    1. Rengør en dissekeringsbakke, saks og tang med 70% ethanol.
    2. Bedøv hunnerne individuelt med 2-PE nedsænkningsanæstesimetoden.
    3. Vælg tilfældigt nyligt modne kvinder (efter smeltning af præmature kvinder), der ikke har gennemgået øjenstilk-ablation for at bekræfte deres gonadale stadier.
    4. Ofre alle øjenstilk-ablerede eksperimentelle hunner individuelt og identificere gonademodningsstadierne. Ødelæg krabbens thoraxganglier ved hjælp af en skarp steril syl. Fjern først den øverste rygskjold og derefter hepatopancreas for at gøre æggestokken synlig. Overhold æggestokkens farve, og identificer æggestokkens modningsstadium (figur 2).
  3. Identifikation af ovariemodningsstadier
    1. Overhold æggestokkens farve med det blotte øje eller under et stereomikroskop.
    2. Identificer æggestokkenes modningsstadier baseret på farve30: den umodne (fase-1) viser en gennemskinnelig eller cremet hvid farve; den tidlige modning (trin-2) viser en lys til lysegul farve; iii) Formodningen (trin 3) har en gul til lys orange farve og (iv) den fuldt modne (trin-4) viser en mørk orange til rødlig farve.

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Representative Results

Gonad modning
Cremet hvidt ovarievæv (umodne æggestokke, stadium-1) blev fundet hos 100% af de dissekerede kvinder (n = 6), før de udførte øjenstilkens ablation (figur 2). Gonademodningshastigheden for øjenstilk-ablerede hunkrabber (n = 63; 31 hunner med kauteriseringsteknikken og 32 hunner med operationsteknikken) var højere sammenlignet med hunkrabber, der ikke blev udsat for øjenstilk-ablation (n = 31) efter 30 dages individuel opdræt (figur 3). De højeste procentdele af præmodne æggestokke (stadie-3) blev fundet i øjenstilk-ablerede hunkrabber (figur 3; både cauterization og kirurgiske teknikker), og en envejsanalyse af varians (ANOVA) viste signifikante forskelle (p < 0,05) blandt de eksperimentelle hunkrabbers modningsstadier i æggestokkene (tabel 1). Kontrolgruppen havde en højere forekomst af umodne kvindelige krabber sammenlignet med cauterization og kirurgi behandlingsgrupper (Tukey's HSD test, p < 0,001). Cauterization og kirurgi behandlinger viste ingen signifikante forskelle med hensyn til procentdelen af kvindelige krabber på tværs af alle modningsstadier (Tukeys HSD-test, alle p > 0,1). Både cauterization (Tukeys HSD-test, p = 0,004) og kirurgi (Tukeys HSD-test, p = 0,006) behandlinger havde signifikant højere procentdele af præ-modne fase 3 kvindelige krabber end kontrolbehandlingen, og kun cauterization og kirurgi behandlinger var i stand til at producere fase 4 kvindelige krabber fra et umodent stadium inden for 30 dage efter behandlingen (tabel 2).

Figure 2
Figur 2: Fire modningsstadier af æggestokkene hos hunkrabber. Forskelle i farve og volumen af æggestokkene mellem stadierne påpeges tydeligt af de sorte pile. Klik her for at se en større version af denne figur.

Figure 3
Figur 3: Ovariemodningsstadier hos hunkrabber, der udsættes for øjenstilk-ablation (kirurgi og kauterisering) og kontrol efter 30 dages opdrætsperiode (n = 94). Fejllinjen repræsenterer standardafvigelsen. De hævede bogstaver angiver signifikante forskelle mellem behandlingerne på hvert modningsstadium ved p < 0,05. Klik her for at se en større version af denne figur.

Modning fase Summen af kvadrater Df Gennemsnitlig firkant F P
Umoden (fase-1) Mellem grupper 3755.556 2 1877.778 169 <0,001
Inden for grupper 66.667 6 11.111
Total 3822.222 8
Tidlig modning (etape-2) Mellem grupper 1355.556 2 677.778 8.714 0.017
Inden for grupper 466.667 6 77.778
Total 1822.222 8
Præ-modning (fase-3) Mellem grupper 4688.889 2 2344.444 17.58 0.003
Inden for grupper 800 6 133.333
Total 5488.889 8
Fuldt modnet (fase-4) Mellem grupper 822.222 2 411.111 9.25 0.015
Inden for grupper 266.667 6 44.444
Total 1088.889 8
Bemærk: Den gennemsnitlige forskel er signifikant på p = 0,05 niveau.

Tabel 1: Sammenligning af øjenstilk-ableret (cauterization og kirurgi) og kontrol kvindelige krabbers gonadale modningsstadier efter en envejs ANOVA test. Den gennemsnitlige forskel var signifikant ved p = 0,05.

Modning fase Behandling Sammenligning behandling P
Umoden (fase-1) Kauterisering Kirurgi 1
Kauterisering Kontrol <0,001
Kirurgi Kontrol <0,001
Tidlig modning (etape-2) Kauterisering Kirurgi 0.129
Kauterisering Kontrol 0.014
Kirurgi Kontrol 0.232
Præ-modning (fase-3) Kauterisering Kirurgi 0.934
Kauterisering Kontrol 0.004
Kirurgi Kontrol 0.006
Fuldt modnet (fase-4) Kauterisering Kirurgi 0.109
Kauterisering Kontrol 0.012
Kirurgi Kontrol 0.237
Bemærk: Den gennemsnitlige forskel er signifikant på p = 0,05 niveau.

Tabel 2: Post-hoc Tukeys HSD-test af forskellene mellem øjenstilk-ableret (cauterization og kirurgisk) og kontrol af kvindelige krabbers gonadale modningsstadier. Den gennemsnitlige forskel var signifikant ved p = 0,05.

Overlevelsesraten
Den gennemsnitlige overlevelsesrate for øjenstilke-ablerede hunkrabber var 95,45% ± 4,98% (gennemsnitlig ± standardafvigelse) i opdrætsperioden på 30 dage. Der forekom ingen mortalitet inden for de første 7 dage efter ablation og håndtering af øjenstilken. I løbet af de 30 dages opdræt efter øjenstilk-ablation var dødeligheden ikke signifikant forskellig (Kruskal-Wallis-test, p = 0,67) mellem behandlingerne. Succesraten for smeltning af de lemautotomiserede hunkrabber var 80% ± 2,86% (n = 115).

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Discussion

Denne protokol blev udviklet til øjenstilk-ablation af mudderkrabben, Scylla spp., Og kan anvendes som en effektiv metode til at inducere gonademodning. Denne protokol kan let replikeres til kommerciel modning af æggestokke af mudderkrabber og kan implementeres for at reducere den latente periode (tid fra en gydning til en anden) i mudderkrabbefrøproduktion.

Øjenstilkens ablation af krebsdyr (dvs. ferskvandsrejer, marine rejer) udføres typisk for at fremkalde gonademodning og gydning uden for sæsonen11,12,13. Øjenstilk ablation i brachyuran krabber er også blevet gjort for at studere smeltning 25,32,33, hormonel regulering 18, gonad modning34, og induceret avl og reproduktionsevne35,36,37,38,39. Anæstesi via nedsænkning i 2-phenoxyethanol blev anvendt, da det er sammenligneligt med brugen af tricainmethansulfonat (MS-222) i arthopoder, men billigere og ikke kræver brug af yderligere buffer40. Ensidig eller bilateral øjenstilk ablation påvirker krebsdyrets fysiologi. Ablation af øjenstilk efter protokollen angivet i denne undersøgelse påvirker også modningshastigheden for æggestokkene hos mudderkrabber. I kontrolbehandlingen (uden øjenstilkablation) havde 43,33% ± 5,77% af hunkrabberne en umoden æggestok (fase 1). I samme opdrætsperiode (30 dage) havde øjenstilk-ablerede hunkrabber imidlertid præmodne æggestokke (stadium 3; 56,67% ± 11,55% og 53,33% ± 15,28% med henholdsvis cauterization og kirurgi teknikker), hvilket viser, at øjenstilk ablation kan øge gonademodningen af mudderkrabber. Tidligere undersøgelser har også rapporteret, at ovarieudviklingen af intakte krabber (uden øjenstilk-ablation) er langsommere end for øjenstilk-ablerede krabber25,31. På grund af den langsommere gonadale udvikling hos intakte krebsdyr udføres øjenstilk-ablation i vid udstrækning i kommercielle reje- og rejerugerier. I denne protokol opnåede de øjenstilk-ablerede hunkrabber højere procentdele af ovariemodning sammenlignet med hunkrabber uden øjenstilk-ablationsbehandling (figur 3).

Mudderkrabbens gonademodning reguleres af hormoner21,41,42. Øjenstammen indeholder vigtige endokrine kirtler (dvs. X-organ-sinuskirtelkomplekset), der spiller vitale roller i den gonadale modningsproces af mudderkrabber18,21. Ensidig øjenstilk-ablation, enten ved cauterization eller kirurgi, beskadiger en af de store endokrine kirtler, der er involveret i syntese og frigivelse af hæmmende hormoner (fx VIH), hvilket resulterer i et højere niveau af gonadestimulerende hormoner (dvs. VSH).

Ovariemodningsstadierne af Scylla spp. kan differentieres ved at observere ovarievævsfarvningen med det blotte øje 29,30,43. Gennemsigtigt eller cremet hvidt ovarievæv er indikationer på umodne æggestokke 29,30,43,44. I denne undersøgelse blev umodne æggestokke (fase 1) stadig fundet i gruppen af kvindelige krabber uden øjenstilk-ablation på grund af den langsommere modningsproces i æggestokkene. Imidlertid viste krabberne i øjenstilk-ablerede grupper (både ved cauterization og kirurgiske teknikker) for det meste præ-modne æggestokke (stadium-3), hvor nogle individer udviser fuldt modne æggestokke (stadium-4). Derfor kan protokollen for øjenstilk-ablation beskrevet her bruges til at øge æggestokkenes modning hos kvindelige mudderkrabber. Denne protokol kan også anvendes direkte på vildtindsamlede modne kvindelige mudderkrabber for at fremskynde deres frøproduktion. For at evaluere effektiviteten af cauterization og kirurgiske metoder på mudderkrabbe gonad modning og for at sikre den nøjagtige estimering af smeltevarighed blev seksuelt præmodne krabber brugt. Efter (induceret) smeltning af seksuelt præmature hunkrabber bemærkede vi, at deres æggestokke stadig var i de umodne eller tidlige udviklingsstadier 29,45. Efter 30 dages opdræt af de nyligt modne hunkrabber (enten øjenstilk-ableret eller uden øjenstilk-ablation) blev ovarieudviklingsstadierne (trin 1 til trin 4) bestemt af farven på æggestokkene. Denne protokol tilskynder til brug af cauterization teknik til at udføre øjenstilk ablation i mudderkrabber for at undgå hæmolympisk tab og forhindre infektion på de ablerede steder. Cauterization forsegler straks såret, mens operationsteknikken tager tid for såret at hele, og dette ville give mulighed for risiko for infektion. Til kommercielle formål bør større modne krabber, helst på et senere stadium af æggestokkenes modning, vælges til øjenstilk-ablation for at forkorte tiden til at nå det fuldt modne ovariestadium til efterfølgende handel eller yngelkultur. Ud over ablation af øjenstilke kan individuel opdræt med sandsubstrat og tilstrækkelig fodring, helst med levende foder, øge kønsmodningshastigheden for mudderkrabber i fangenskab 30,35,46,47.

Krebsdyrblod kaldes hæmolymp og kan gå tabt under øjenstilk ablation. Et overdreven tab af hæmolymp kan føre til død af øjenstilk-ablerede krabber, især når der udføres kirurgi for at fjerne øjenstilken. Hæmolympen kan koagulere i den sårede del for at forhindre tab. I sammenligning med operationsteknikken forsegler cauteriseringsteknikken imidlertid straks den sårede del og derved forhindrer tab af hæmoolymp og mulig infektion.

Mudderkrabbedødelighed efter ensidig øjenstilk-ablation med enten cauterization eller kirurgi blev ikke fundet inden for de første 7 dage. Således kan øjenstilk ablation udføres med en højere overlevelsesrate. Ensidig øjenstilk ablation hæmmer ikke krabbens overlevelsesrate33.

Stress under krabbehåndtering og øjenstilk ablation kan bidrage til krabbedødelighed. Korrekt anæstesi er nødvendig for at minimere håndtering af stress under øjenstilk ablation. I ablation af krebsdyr øjenstilk anvendes kemiske anæstetika (dvs. xylocain, lidokain) i bunden af øjenstilken før øjenstilk-ablation14,15,17,48. På grund af mudderkrabbernes aggressive natur og store størrelse er brugen af anæstesi kun i bunden af øjenstilken imidlertid ikke tilstrækkelig og kan resultere i yderligere stress for dyrene under injektionen. På den anden side er anæstesi ved at udsætte dem for en lavere vandtemperatur mere økonomisk og sikrere. Brugen af koldt vand til anæstesi i mudderkrabber er almindelig og er blevet brugt i andre undersøgelser på grund af dets effektivitet, enkelhed og minimale indvirkning på genopretning og overlevelse 37,49,50. Derudover anbefales fremtidig forskning i smertevurdering efter øjenstilk-ablation på mudderkrabber for at fremhæve ændringen i adfærd forbundet med smerte og stress, som det fremgår af ferskvandsrejer Macrobrachium americanum51.

Selvom øjenstilk ablation ved hjælp af både cauterization og kirurgi metoder har en minimal effekt på krabbe overlevelse og forbedrer ovarie modning, udfører øjenstilk ablation kræver professionel beherskelse af teknikkerne. Timingen mellem trinene er kritisk, da enhver forsinkelse mellem protokoller tilføjer yderligere stress for krabberne. I modsætning til operationsteknikken er cauteriseringsteknikken farlig, fordi den involverer brug af brandfarligt udstyr (dvs. en blæselampe og butangas). Således er ekstra forsigtighed nødvendig, når du udfører cauterization teknikken.

Krabber er kannibalistiske i naturen, og de er kendt for at bytte andre, der lige har afsluttet deres smeltning og stadig er i deres soft-shell betingelser 7,52,53. Således kan opdræt af krabberne individuelt undgå unødvendig dødelighed på grund af kannibalisme. Individuel opdræt i mudderkrabbekultur praktiseres almindeligvis, både i højdensitetskultur og damkultur, til opfedning og dyrkning af blødskalskrabber 8,53. Denne protokol brugte også individuel opdræt og vedligeholdelse. Under transporten af krabberne til opdræt eller handel bindes krabberne sikkert (eller endda autotomiseres) for at forhindre kamp, unødvendig skade og tab af lemmer34.

Den beskrevne protokol for øjenstilk ablation skal udføres med flere personer. Efter afslutning af øjenstilkens ablation skal ikke-engangsudstyr (f.eks. Akvarium, bakke, håndklæde osv.) desinficeres med 30 ppm klor. Krabberne skal overvåges mindst to gange om dagen. Døde krabber, ufortyndet foder, ablerede lemmer eller smeltede krabbeskaller skal bortskaffes hurtigt (dvs. begravet i jord med blegepulver) for at forhindre ethvert potentiale for sygdomsspredning.

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Disclosures

Ingen af forfatterne har nogen interessekonflikter.

Acknowledgments

Denne undersøgelse blev støttet af Undervisningsministeriet, Malaysia, under Higher Institution Centre of Excellence (HICoE) -programmet, Malaysia, akkrediteret til Institut for Tropisk Akvakultur og Fiskeri, Universiti Malaysia Terengganu (Vot nr. 63933 &; Vot nr. 56048). Vi anerkender støtten fra Universiti Malaysia Terengganu og Sayap Jaya Sdn. Bhd. via Private Partnership Research Grant (Vot. nr. 55377). En adjungeret akademisk stipendiat fra Universiti Sains Malaysia til Khor Waiho og Hanafiah Fazhan anerkendes også.

Materials

Name Company Catalog Number Comments
Aeration tube  Ming Yu Three N/A aquarium and pet shop
Airstone Ming Yu Three N/A aquarium and pet shop
Autoclave machine HIRAYAMA MANUFACTURING CORPORATION N/A MADE IN JAPAN
Bleaching powder (Hi-Chlon 70%) Nippon Soda Co.Ltd,Japan N/A N/A
Blow torch  MR D.I.Y. Group Berhad N/A N/A
Circular tank (32L) BEST PLASTIC INDUSTRY SDN. BHD.  N/A N/A
Cotton hand gloves (thick)  MR D.I.Y. Group Berhad N/A N/A
Cotton towel MR D.I.Y. Group Berhad N/A N/A
Digital thermometer Hanna Instrument HI9814 Hanna Instruments GroLine Hydroponics Waterproof pH / EC / TDS / Temp. Portable Meter HI9814
Digital Vernier Caliper INSIZE Co., Ltd. N/A
Dissecting tray Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu
Dropper bottle/Plastic Pipettes Dropper Shopee Malaysia N/A N/A
Ethanol 70% Thermo Scientific Chemicals 033361.M1 Diluted to 70% using double distilled water
Fiberglass tank (1 ton) Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu
Fine sand N/A N/A collected from Sea beach of Universiti Malaysia Terengganu
First Aid Kits Watsons Malaysia N/A N/A
Flat head nickel steel metal rod (Screw driver) MR D.I.Y. Group Berhad N/A N/A
Formaldehyde Thermo Scientific Chemicals 119690010
Gas cylinder (butane gas) for blow  torch MR D.I.Y. Group Berhad N/A N/A
Gas lighter gun (long head) MR D.I.Y. Group Berhad N/A N/A
Glass beaker (100 mL)) Corning Life Sciences 1000-100
Ice bag  Watsons Malaysia N/A N/A
Perforated plastic baskets  Eco-Shop Marketing Sdn. Bhd. N/A N/A
PVC pipe 15mm Bina Plastic Industries Sdn Bhd (HQ) N/A N/A
Refractometer ATAGO CO.,LTD.
Refrigerator Sharp Corporation Japan N/A Chest Freezer SHARP 110L - SJC 118
Scoop net MR D.I.Y. Group Berhad N/A
Seawater Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu
Siphoning pipe MR D.I.Y. Group Berhad N/A N/A
Spray bottle Mr. DIY Sdn Bhd N/A N/A
Stainless surgical forceps  N/A N/A N/A
Stainless surgical scissors  N/A N/A N/A
Submersible water pump  AS N/A model: Astro 4000
Tincture of iodine solution  (Povidone Iodine) Farmasi Fajr Sdn Bhd N/A N/A
Tissue paper  N/A N/A
Transparent plastic aquarium Ming Yu Three N/A aquarium and pet shop
Waterproof table Hatcheri AKUATROP  N/A Research Center of Universiti Malaysia Terengganu

DOWNLOAD MATERIALS LIST

References

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Erratum

Formal Correction: Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs
Posted by JoVE Editors on 05/26/2023. Citeable Link.

An erratum was issued for: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs. The Introduction, Protocol, Discussion and References were updated.

The forth sentence in the third paragraph of the Introduction has been updated from:

The eyestalk ablation protocol in this work minimizes stress by using fully sedated crabs and minimizes physical injury to personnel from crab bites. 

to:

The eyestalk ablation protocol in this work minimizes stress by using fully anesthetized crabs and minimizes physical injury to personnel from crab bites. 

The start of the Protocol has been updated from:

This protocol follows the Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes outlined by the Laboratory Animal Science Association of Malaysia. The sacrifice of the experimental samples was done according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Sexually pre-mature female mud crabs (orange mud crab S. olivacea) were collected from the local market (5°66′62′′N, 102°72′33′′E) at the Setiu Wetlands in Malaysia. The mud crab species was identified based on morphological characteristics1.

to:

This protocol follows the Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes outlined by the Laboratory Animal Science Association of Malaysia and was approved by the Universiti Malaysia Terengganu's Research Ethics Committee (Animal ethics approval number: UMT/JKEPHMK/2023/96). The sacrifice of the experimental samples was done according to the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. Sexually pre-mature female mud crabs (orange mud crab Scylla olivacea) were collected from the local market (5°66′62′′N, 102°72′33′′E) at the Setiu Wetlands in Malaysia. The mud crab species was identified based on morphological characteristics1.

Section 4 of the Protocol has been updated from:

4. Cold-shock anesthesia

  1. Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
  2. Catch the crabs with a scoop net, and keep them individually in small aquariums for cold shock anesthesia.
  3. Prepare 2 L of 4 °C to 1 °C seawater (20 ppt) in a transparent plastic aquarium. Maintain the temperature using (−20 °C) ice bags for cold shock anesthesia.
    NOTE: Check the temperature with a digital thermometer.
  4. Immerse the crab in the 4 °C seawater until sedated (about 3−5 min).
  5. Ensure the crabs are fully anesthetized by the lack of spontaneous movement. The legs and chelipeds joints will still show minor movements when touched with forceps.

to:

4. Anesthesia

  1. Select sexually mature females with a dark-colored oval-shaped abdominal flap with a CW >86 mm (Figure 1).
  2. Catch the crabs with a scoop net, and keep them individually in small aquariums for anesthesia.
  3. After 5 min of acclimatization period, add 2-phenoxyethanol (2-PE) at 2 mL/L into each aquarium and allow 15 min of anesthesia treatment.
  4. Ensure the crabs are fully anesthetized by the lack of spontaneous movement.

Section 5 of the Protocol has been updated from:

5. Eyestalk ablation

  1. Cauterization technique
    1. Perform all procedures on top of a table and in an open area.
    2. Take a flat head nickel-steel metal rod (e.g., a screwdriver) with a wooden or plastic handle, and cover the handle with a wet cotton towel.
    3. Sterilize two stainless surgical forceps in an autoclave.
    4. Prepare 70% ethanol in a spray bottle. Have tissue paper ready for use.
      NOTE: Ethanol is highly flammable. Maintain a safe distance from fire sources.
    5. Connect a blowtorch to a gas cylinder (butane) securely.
      CAUTION: Follow the instructions on the blowtorch and gas cylinder. Make sure that the blowtorch is switched off when connecting with the gas cylinder. Read and follow all the fire safety precautions mentioned on the gas cylinder.
    6. Wear thick cotton gloves to avoid injury from hot objects.
    7. Subject the tip of the metal rod to the fire of the blowtorch until the metal rod is bright red.
    8. Cover the anesthetized (sedated) crab with a wet cotton towel.
      NOTE: Cover all the tentacles of the crab to avoid unnecessary damage.
    9. Hold one eye of the crab with sterilized forceps.
      NOTE: Sterilize the forceps in an autoclave for first-time use, and disinfect using 70% ethanol for subsequent use on other crabs.
    10. Hold the red-hot metal flat tip onto the eye of the crab and press slightly for about 10−15 s until the eyestalk turns an orange or reddish-orange color.
      NOTE: Two people are needed to execute eyestalk ablation following the cauterization method: one to hold the crab and another to perform the ablation procedure.
    11. Disinfect the forceps with 70% ethanol spray to ensure no cross-contamination between crabs.
    12. After performing the eyestalk ablation on all crabs, dip the hot nickel steel metal rod (screwdriver) into tap water.
    13. Disinfect the towel before reuse. Multiple towels can be used to save time.
      NOTE: Wash the towel with tap water, and dip it into 30 ppm chlorinated water for 5 min. Then, wash the towel with tap water again, and dip it in a 1 g/L sodium thiosulphate solution.
    14. Keep the blowtorch in a safe place after turning it off, and wait until it returns to environmental temperature (about 30 min) before disconnecting.
  2. Surgery technique
    1. Perform the procedure in a well-ventilated area.
    2. Sterilize two surgical scissors and forceps in an autoclave.
    3. Pour 50 mL of 70% ethanol into a 100 mL glass beaker.
    4. Prepare the tincture of iodine solution in a dropper bottle.
      NOTE: Tincture of iodine (iodine tincture or weak iodine solution) is made up of 2%-7% elemental iodine and potassium iodide, or sodium iodide, dissolved in ethanol and water.
    5. Wear thick cotton gloves.
    6. Hold the sedated crab, and cover it with a wet cotton towel.
    7. Hold one eye of the crab with sterilized forceps.
    8. Swiftly cut off the eyestalk using sterilized surgical scissors.
      NOTE: Hemolymph may be lost from the wounded part of the crab.
    9. Dip the scissors and forceps in 70% ethanol after every use, and dry them using tissue paper before reuse.
    10. Apply two to three drops of iodine tincture to the wounded part of the eyestalk immediately after cutting it off.
      NOTE: Tincture of iodine is used for healing and to prevent infection.

to:

5. Eyestalk ablation

  1. Cauterization technique
    1. Perform all procedures on top of a table and in an open area.
    2. Take a flat head nickel-steel metal rod (e.g., a screwdriver) with a wooden or plastic handle, and cover the handle with a wet cotton towel.
    3. Sterilize two stainless surgical forceps in an autoclave.
    4. Prepare 70% ethanol in a spray bottle and keep it away from any fire-related sources, such as blow torch and red hot screwdriver. Have tissue paper ready for use.
      NOTE: Ethanol is highly flammable. Maintain a safe distance from fire sources.
    5. Connect a blowtorch to a gas cylinder (butane) securely.
      CAUTION: Follow the instructions on the blowtorch and gas cylinder. Make sure that the blowtorch is switched off when connecting with the gas cylinder. Read and follow all the fire safety precautions mentioned on the gas cylinder.
    6. Wear thick cotton gloves to avoid injury from hot objects.
    7. Subject the tip of the metal rod to the fire of the blowtorch until the metal rod is bright red.
    8. Cover the anesthetized crab with a wet cotton towel.
      NOTE: Cover the antennae of the crab to avoid unnecessary damage.
    9. Hold one eye of the crab with sterilized forceps.
      NOTE: Sterilize the forceps in an autoclave for first-time use, and disinfect using 70% ethanol for subsequent use on other crabs.
    10. Hold the red-hot metal flat tip onto the eye of the crab and press slightly for about 10−15 s until the eyestalk turns an orange or reddish-orange color. Be careful when conducting this step to avoid damage to adjacent structures. 
      NOTE: Two people are needed to execute eyestalk ablation following the cauterization method: one to hold the crab and another to perform the ablation procedure.
    11. Disinfect the forceps with 70% ethanol spray to ensure no cross-contamination between crabs.
      NOTE: Only perform this step at least waiting for 5 min after the eyestalk ablation procedure to ensure the forceps are cooled down before disinfection using 70% ethanol to prevent potential fire hazards.
    12. After performing the eyestalk ablation on all crabs, dip the hot nickel steel metal rod (screwdriver) into tap water.
    13. Disinfect the towel before reuse. Multiple towels can be used to save time.
      NOTE: Wash the towel with tap water, and dip it into 30 ppm chlorinated water for 5 min. Then, wash the towel with tap water again, and dip it in a 1 g/L sodium thiosulphate solution.
    14. Keep the blowtorch in a safe place after turning it off, and wait until it returns to environmental temperature (about 30 min) before disconnecting.
  2. Surgery technique
    1. Perform the procedure in a well-ventilated area.
    2. Sterilize two surgical scissors and forceps in an autoclave.
    3. Pour 50 mL of 70% ethanol into a 100 mL glass beaker.
    4. Wear thick cotton gloves.
    5. Hold the anesthetized crab, and cover it with a wet cotton towel.
    6. Hold one eye of the crab with sterilized forceps.
    7. Swiftly cut off the eyestalk using sterilized surgical scissors.
      NOTE: Hemolymph may be lost from the wounded part of the crab.
    8. Dip the scissors and forceps in 70% ethanol after every use, and dry them using tissue paper before reuse.

Step 7.2.2 of the Protocol has been updated from:

Sedate the females individually with the cold shock anesthesia method.

to:

Anesthetize the females individually with the 2-PE immersion anesthesia method.

The Discussion has been updated from:

This protocol was developed for the eyestalk ablation of the mud crab, Scylla spp., and can be applied as an efficient method to induce gonad maturation. This protocol can be easily replicated for the commercial ovary maturation of mud crabs and can be implemented to reduce the latent period (time from one spawning to another) in mud crab seed production.

The eyestalk ablation of crustaceans (i.e., freshwater prawn, marine shrimp) is typically done to induce gonad maturation and out-of-season spawning11,12,13. Eyestalk ablation in brachyuran crabs has also been done to study molting25,32,33, hormonal regulation18, gonad maturation34, and induced breeding and reproductive performance35,36,37,38,39. Unilateral or bilateral eyestalk ablation influences the physiology of the crustacean. Eyestalk ablation following the protocol stated in this study also influences the ovarian maturation rate of mud crabs. In the control treatment (without eyestalk ablation), 43.33% ± 5.77% of female crabs had an immature ovary (stage-1). However, in the same rearing period (30 days), eyestalk-ablated female crabs had pre-maturing ovaries (stage-3; 56.67% ± 11.55% and 53.33% ± 15.28% with the cauterization and surgery techniques, respectively), which shows that eyestalk ablation can increase the gonad maturation of mud crabs. Previous studies have also reported that the ovarian development of intact crabs (without eyestalk ablation) is slower than that of eyestalk-ablated crabs25,31. Due to the slower gonadal development in intact crustaceans, eyestalk ablation is widely done in commercial prawn and shrimp hatcheries. In this protocol, the eyestalk-ablated female crabs achieved higher percentages of ovarian maturation compared to the female crabs without the eyestalk ablation treatment (Figure 3).

The gonad maturation of the mud crab is regulated by hormones21,40,41. The eyestalk contains important endocrine glands (i.e., the X-organ-sinus gland complex) that play vital roles in the gonadal maturation process of mud crabs18,21. Unilateral eyestalk ablation, either by cauterization or surgery, damages one of the major endocrine glands that is involved in the synthesis and release of inhibiting hormones (e.g., VIH), thereby resulting in a higher level of gonad-stimulating hormones (i.e., VSH).

The ovarian maturation stages of Scylla spp. can be differentiated by observing the ovarian tissue coloration with the naked eye29,30,42. Translucent or creamy white ovarian tissues are indications of immature ovaries29,30,42,43. In this study, immature ovaries (stage-1) were still found in the group of female crabs without eyestalk ablation due to the slower ovarian maturation process. However, the crabs in the eyestalk-ablated groups (both by the cauterization and surgery techniques) mostly showed pre-maturing ovaries (stage-3), with some individuals exhibiting fully matured ovaries (stage-4). Therefore, the protocol of eyestalk ablation described here can be used to increase ovarian maturation in female mud crabs. This protocol can also be applied directly to wild-collected mature female mud crabs to hasten their seed production. To evaluate the effectiveness of cauterization and surgery methods on mud crab gonad maturation and to ensure the accurate estimation of molting duration, sexually pre-mature crabs were used. After the (induced) molting of sexually pre-mature female crabs, we noticed that their ovaries were still in the immature or early developing stages29,44. After 30 days of rearing the newly mature female crabs (either eyestalk-ablated or without eyestalk ablation), the ovarian development stages (stage-1 to stage-4) were determined by the color of the ovarian tissues. This protocol encourages the use of the cauterization technique to perform eyestalk ablation in mud crabs to avoid any hemolymph loss and prevent infection at the ablated sites. Cauterization immediately seals the wound, whereas the surgery technique requires an additional step of disinfection using iodine. For commercial purposes, larger mature crabs, preferably at a later stage of ovarian maturation, should be selected for eyestalk ablation to shorten the time to reach the fully matured ovary stage for subsequent commerce or brood stock culture. In addition to eyestalk ablation, individual rearing with sand substrate and sufficient feeding, preferably with live feed, can increase the gonad maturation rate of mud crabs in captivity30,35,45,46.

Crustacean blood is called hemolymph and can be lost during eyestalk ablation. An excessive loss of hemolymph may lead to the death of eyestalk-ablated crabs, especially when performing surgery to remove the eyestalk. The hemolymph can coagulate in the wounded part to prevent loss. The application of a tincture of iodine can prevent infection of the wounded part. However, in comparison to the surgery technique, the cauterization technique seals the wounded part immediately, thereby preventing the loss of hemolymph and possible infection.

Mud crab mortality after unilateral eyestalk ablation with either cauterization or surgery was not found within the first 7 days. Thus, eyestalk ablation can be done with a higher survival rate. Unilateral eyestalk ablation does not hamper the survival rate of the crab33.

Stress during crab handling and eyestalk ablation may contribute to crab mortality. Proper anesthesia is needed to minimize handling stress during eyestalk ablation. In crustacean eyestalk ablation, chemical anesthetics (i.e., xylocaine, lidocaine) are used at the base of the eyestalk before eyestalk ablation14,15,17,47. However, due to the aggressive nature and large size of mud crabs, the use of anesthesia only at the base of the eyestalk is not sufficient and might result in additional stress to the animals during the injection. On the other hand, anesthesia by subjecting them to a lower water temperature is more economical and safer. The use of cold water for anesthesia in mud crabs is common and has been used in other studies due to its efficiency, simplicity, and minimal impact on recovery and survival37,48,49.

Although eyestalk ablation using both cauterization and surgery methods has a minimal effect on crab survival and enhances ovarian maturation, performing eyestalk ablation requires professional mastery of the techniques. The timing between the steps is critical as any delay between protocols adds additional stress for the crabs. Unlike the surgery technique, the cauterization technique is dangerous because it involves the use of flammable equipment (i.e., a blow torch and butane gas). Thus, extra caution is needed when performing the cauterization technique.

Crabs are cannibalistic in nature, and they are known to prey on others that have just completed their molt and are still in their soft-shell conditions7,50,51. Thus, rearing the crabs individually can avoid unnecessary mortality due to cannibalism. The use of individual rearing in mud crab culture is commonly practiced, both in high-density culture and pond culture, for fattening and soft-shell crab farming purposes8,52. This protocol also utilized individual rearing and maintenance. During the transportation of the crabs for rearing or commerce, the crab chelipeds are tied up securely (or even autotomized) to prevent fighting, unnecessary injury, and limb loss34.

The described protocol for eyestalk ablation should be performed with multiple persons. After completing the eyestalk ablation, non-disposable equipment (e.g., the aquarium, tray, towel, etc.) should be disinfected with 30 ppm chlorine. The crabs must be monitored at least twice per day. Any dead crabs, uneaten feed, ablated limbs, or molted crab shells should be swiftly disposed of (i.e., buried in soil with bleaching powder) to prevent any potential for disease spread.

to:

This protocol was developed for the eyestalk ablation of the mud crab, Scylla spp., and can be applied as an efficient method to induce gonad maturation. This protocol can be easily replicated for the commercial ovary maturation of mud crabs and can be implemented to reduce the latent period (time from one spawning to another) in mud crab seed production.

The eyestalk ablation of crustaceans (i.e., freshwater prawn, marine shrimp) is typically done to induce gonad maturation and out-of-season spawning11,12,13. Eyestalk ablation in brachyuran crabs has also been done to study molting25,32,33, hormonal regulation18, gonad maturation34, and induced breeding and reproductive performance35,36,37,38,39. Anesthesia via immersion in 2-phenoxyethanol was used as it is comparable to the use of tricaine methanesulfonate (MS-222) in arthopods but cheaper and does not require the use of additional buffer40. Unilateral or bilateral eyestalk ablation influences the physiology of the crustacean. Eyestalk ablation following the protocol stated in this study also influences the ovarian maturation rate of mud crabs. In the control treatment (without eyestalk ablation), 43.33% ± 5.77% of female crabs had an immature ovary (stage-1). However, in the same rearing period (30 days), eyestalk-ablated female crabs had pre-maturing ovaries (stage-3; 56.67% ± 11.55% and 53.33% ± 15.28% with the cauterization and surgery techniques, respectively), which shows that eyestalk ablation can increase the gonad maturation of mud crabs. Previous studies have also reported that the ovarian development of intact crabs (without eyestalk ablation) is slower than that of eyestalk-ablated crabs25,31. Due to the slower gonadal development in intact crustaceans, eyestalk ablation is widely done in commercial prawn and shrimp hatcheries. In this protocol, the eyestalk-ablated female crabs achieved higher percentages of ovarian maturation compared to the female crabs without the eyestalk ablation treatment (Figure 3).

The gonad maturation of the mud crab is regulated by hormones21,41,42. The eyestalk contains important endocrine glands (i.e., the X-organ-sinus gland complex) that play vital roles in the gonadal maturation process of mud crabs18,21. Unilateral eyestalk ablation, either by cauterization or surgery, damages one of the major endocrine glands that is involved in the synthesis and release of inhibiting hormones (e.g., VIH), thereby resulting in a higher level of gonad-stimulating hormones (i.e., VSH).

The ovarian maturation stages of Scylla spp. can be differentiated by observing the ovarian tissue coloration with the naked eye29,30,43. Translucent or creamy white ovarian tissues are indications of immature ovaries29,30,43,44. In this study, immature ovaries (stage-1) were still found in the group of female crabs without eyestalk ablation due to the slower ovarian maturation process. However, the crabs in the eyestalk-ablated groups (both by the cauterization and surgery techniques) mostly showed pre-maturing ovaries (stage-3), with some individuals exhibiting fully matured ovaries (stage-4). Therefore, the protocol of eyestalk ablation described here can be used to increase ovarian maturation in female mud crabs. This protocol can also be applied directly to wild-collected mature female mud crabs to hasten their seed production. To evaluate the effectiveness of cauterization and surgery methods on mud crab gonad maturation and to ensure the accurate estimation of molting duration, sexually pre-mature crabs were used. After the (induced) molting of sexually pre-mature female crabs, we noticed that their ovaries were still in the immature or early developing stages29,45. After 30 days of rearing the newly mature female crabs (either eyestalk-ablated or without eyestalk ablation), the ovarian development stages (stage-1 to stage-4) were determined by the color of the ovarian tissues. This protocol encourages the use of the cauterization technique to perform eyestalk ablation in mud crabs to avoid any hemolymph loss and prevent infection at the ablated sites. Cauterization immediately seals the wound, whereas the surgery technique takes time for the wound to heal and this would allow for chance of infection. For commercial purposes, larger mature crabs, preferably at a later stage of ovarian maturation, should be selected for eyestalk ablation to shorten the time to reach the fully matured ovary stage for subsequent commerce or brood stock culture. In addition to eyestalk ablation, individual rearing with sand substrate and sufficient feeding, preferably with live feed, can increase the gonad maturation rate of mud crabs in captivity30,35,46,47.

Crustacean blood is called hemolymph and can be lost during eyestalk ablation. An excessive loss of hemolymph may lead to the death of eyestalk-ablated crabs, especially when performing surgery to remove the eyestalk. The hemolymph can coagulate in the wounded part to prevent loss. However, in comparison to the surgery technique, the cauterization technique seals the wounded part immediately, thereby preventing the loss of hemolymph and possible infection.

Mud crab mortality after unilateral eyestalk ablation with either cauterization or surgery was not found within the first 7 days. Thus, eyestalk ablation can be done with a higher survival rate. Unilateral eyestalk ablation does not hamper the survival rate of the crab33.

Stress during crab handling and eyestalk ablation may contribute to crab mortality. Proper anesthesia is needed to minimize handling stress during eyestalk ablation. In crustacean eyestalk ablation, chemical anesthetics (i.e., xylocaine, lidocaine) are used at the base of the eyestalk before eyestalk ablation14,15,17,48. However, due to the aggressive nature and large size of mud crabs, the use of anesthesia only at the base of the eyestalk is not sufficient and might result in additional stress to the animals during the injection. On the other hand, anesthesia by subjecting them to a lower water temperature is more economical and safer. The use of cold water for anesthesia in mud crabs is common and has been used in other studies due to its efficiency, simplicity, and minimal impact on recovery and survival37,49,50. In addition, future research on pain assessment following eyestalk ablation on mud crabs is recommended to highlight the change in behaviours associated with pain and stress, as evident in freshwater prawn Macrobrachium americanum51.

Although eyestalk ablation using both cauterization and surgery methods has a minimal effect on crab survival and enhances ovarian maturation, performing eyestalk ablation requires professional mastery of the techniques. The timing between the steps is critical as any delay between protocols adds additional stress for the crabs. Unlike the surgery technique, the cauterization technique is dangerous because it involves the use of flammable equipment (i.e., a blow torch and butane gas). Thus, extra caution is needed when performing the cauterization technique.

Crabs are cannibalistic in nature, and they are known to prey on others that have just completed their molt and are still in their soft-shell conditions7,52,53. Thus, rearing the crabs individually can avoid unnecessary mortality due to cannibalism. The use of individual rearing in mud crab culture is commonly practiced, both in high-density culture and pond culture, for fattening and soft-shell crab farming purposes8,53. This protocol also utilized individual rearing and maintenance. During the transportation of the crabs for rearing or commerce, the crab chelipeds are tied up securely (or even autotomized) to prevent fighting, unnecessary injury, and limb loss34.

The described protocol for eyestalk ablation should be performed with multiple persons. After completing the eyestalk ablation, non-disposable equipment (e.g., the aquarium, tray, towel, etc.) should be disinfected with 30 ppm chlorine. The crabs must be monitored at least twice per day. Any dead crabs, uneaten feed, ablated limbs, or molted crab shells should be swiftly disposed of (i.e., buried in soil with bleaching powder) to prevent any potential for disease spread.

The References have been updated from:

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  2. Fazhan, H. et al. Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp. PeerJ. 8, e8066 (2020).
  3. Ikhwanuddin, M., Bachok, Z., Hilmi, M. G., Azmie, G., Zakaria, M. Z. Species diversity, carapace width-body weight relationship, size distribution and sex ratio of mud crab, genus Scylla from Setiu Wetlands of Terengganu coastal waters, Malaysia. Journal of Sustainability Science and Management. 5 (2), 97-109 (2010).
  4. Ikhwanuddin, M., Bachok, Z., Mohd Faizal, W. W. Y., Azmie, G., Abol-Munafi, A. B. Size of maturity of mud crab Scylla olivacea (Herbst, 1796) from mangrove areas of Terengganu coastal waters. Journal of Sustainability Science and Management. 5 (2), 134-147 (2010).
  5. Waiho, K. et al. On types of sexual maturity in brachyurans, with special reference to size at the onset of sexual maturity. Journal of Shellfish Research. 36 (3), 807-839 (2017).
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  10. Okumura, T. et al. Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 143 (2), 246-253 (2006).
  11. Pervaiz, P. A., Jhon, S. M., Sikdar-bar, M. Studies on the effect of unilateral eyestalk ablation in maturation of gonads of a freshwater prawn Macrobrachium dayanum. World Journal of Zoology. 6 (2), 159-163 (2011).
  12. Primavera, J. H. Induced maturation and spawning in five-month-old Penaeus monodon Fabricius by eyestalk ablation. Aquaculture. 13 (4), 355-359 (1978).
  13. Shyne Anand, P. S. et al. Reproductive performance of wild brooders of Indian white shrimp, Penaeus indicus: Potential and challenges for selective breeding program. Journal of Coastal Research. 86 (sp1), 65 (2019).
  14. Diarte-Plata, G. et al. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012).
  15. Vargas-Téllez, I. et al. Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: Behavioral evaluation. Latin American Journal of Aquatic Research. 49 (4), 576-594 (2021).
  16. Chu, K. H., Chow, W. K. Effects of unilateral versus bilateral eyestalk ablation on molting and growth of the shrimp, Penaeus chinensis (Osbeck, 1765) (Decapoda, Penaeidea). Crustaceana. 62 (3), 225-233 (1992).
  17. Taylor, J. Minimizing the effects of stress during eyestalk ablation of Litopenaeus vannamei females with topical anesthetic and a coagulating agent. Aquaculture. 233 (1-4), 173-179 (2004).
  18. Wang, M., Ye, H., Miao, L., Li, X. Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain. Aquaculture. 560, 738493 (2022).
  19. Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
  20. Kornthong, N. et al. Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression. General and Comparative Endocrinology. 185, 28-36 (2013).
  21. Kornthong, N. et al. Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration. Animal Reproduction Science. 208, 106122 (2019).
  22. Liu, C. et al. VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1. General and Comparative Endocrinology. 255, 1-11 (2018).
  23. Chen, H.-Y., Kang, B. J., Sultana, Z., Wilder, M. N. Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 237 (300), 110552 (2019).
  24. Rotllant, G., Nguyen, T. V., Aizen, J., Suwansa-ard, S., Ventura, T. Toward the identification of female gonad-stimulating factors in crustaceans. Hydrobiologia. 825 (1), 91-119 (2018).
  25. Supriya, N. T., Sudha, K., Krishnakumar, V., Anilkumar, G. Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda). Chinese Journal of Oceanology and Limnology. 35 (3), 645-657 (2017).
  26. Wilder, M. N. Advances in the science of crustacean reproductive physiology and potential applications to new seed production technology. Journal of Coastal Research. 86 (sp1), 6-10 (2019).
  27. Arcos, G. F., Ibarra, A. M., Vazquez-Boucard, C., Palacios, E., Racotta, I. S. Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone. Aquaculture Research. 34 (9), 749-755 (2003).
  28. Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
  29. Wu, Q. et al. Growth performance and biochemical composition dynamics of ovary, hepatopancreas and muscle tissues at different ovarian maturation stages of female mud crab, Scylla paramamosain. Aquaculture. 515, 734560 (2020).
  30. Ghazali, A., Azra, M. N., Noordin, N. M., Abol-Munafi, A. B., Ikhwanuddin, M. Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) fed with various diets. Aquaculture. 468 (Part 1), 45-52 (2017).
  31. Farhadi, A. et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8 (2021).
  32. Sukardi, P., Prayogo, N. A., Harisam, T., Sudaryono, A. Effect of eyestalk-ablation and differences salinity in rearing pond on molting speed of Scylla serrata. AIP Conference Proceedings. 2094, 020029 (2019).
  33. Stella, V. S., López Greco, L. S., Rodríguez, E. M. Effects of eyestalk ablation at different times of the year on molting and reproduction of the estuarine grapsid crab Chasmagnathus granulata (Decapoda, Brachyura). Journal of Crustacean Biology. 20 (2), 239-244 (2000).
  34. Jang, I. K. et al. The effects of manipulating water temperature, photoperiod, and eyestalk ablation on gonad maturation of the swimming crab, Portunus trituberculatus.Crustaceana. 83 (2), 129-141 (2010).
  35. Millamena, O. M., Quinitio, E. The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata. Aquaculture. 181 (1-2), 81-90 (2000).
  36. Zeng, C. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development. Aquaculture Research. 38 (14), 1478-1485 (2007).
  37. Rana, S. Eye stalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding. International Journal of Pure and Applied Zoology. 6 (3), 30-34 (2018).
  38. Yi, S.-K., Lee, S.-G., Lee, J.-M. Preliminary study of seed production of the Micronesian mud crab Scylla serrata (Crustacea: Portunidae) in Korea. Ocean and Polar Research. 31 (3), 257-264 (2009).
  39. Azra, M. N., Abol-Munafi, A. B., Ikhwanuddin, M. A review of broodstock improvement to brachyuran crab: Reproductive performance. International Journal of Aquaculture. 5 (38), 1-10 (2016).
  40. Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
  41. Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
  42. Ghazali, A., Mat Noordin, N., Abol-Munafi, A. B., Azra, M. N., Ikhwanuddin, M. Ovarian maturation stages of wild and captive mud crab, Scylla olivacea fed with two diets. Sains Malaysiana. 46 (12), 2273-2280 (2017).
  43. Aaqillah-Amr, M. A., Hidir, A., Noordiyana, M. N., Ikhwanuddin, M. Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development. Animal Reproduction Science. 195, 274-283 (2018).
  44. Amin-Safwan, A., Muhd-Farouk, H., Mardhiyyah, M. P., Nadirah, M., Ikhwanuddin, M. Does water salinity affect the level of 17β-estradiol and ovarian physiology of orange mud crab, Scylla olivacea (Herbst, 1796) in captivity? Journal of King Saud University - Science. 31 (4), 827-835 (2019).
  45. Wu, X. et al. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock. Aquaculture. 273 (4), 602-613 (2007).
  46. Azra, M. N., Ikhwanuddin, M. A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective. Saudi Journal of Biological Sciences. 23 (2), 257-267 (2016).
  47. Maschio Rodrigues, M., López Greco, L. S., de Almeida, L. C. F., Bertini, G. Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation. Acta Zoologica. 103 (3), 326-334 (2022).
  48. Zhang, C. et al. Changes in bud morphology, growth-related genes and nutritional status during cheliped regeneration in the Chinese mitten crab, Eriocheir sinensis. PLoS One. 13 (12), e0209617 (2018).
  49. Zhang, C. et al. Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation. Fish & Shellfish Immunology. 81, 266-275 (2018).
  50. Mirera, D. O., Moksnes, P. O. Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: Effect of shelter, crab size and stocking density. Aquaculture International. 23 (1), 155-173 (2015).
  51. Ut, V. N., Le Vay, L., Nghia, T. T., Hong Hanh, T. T. Development of nursery cultures for the mud crab Scylla paramamosain (Estampador). Aquaculture Research. 38 (14), 1563-1568 (2007).
  52. Fazhan, H. et al. Limb loss and feeding ability in the juvenile mud crab Scylla olivacea: Implications of limb autotomy for aquaculture practice. Applied Animal Behaviour Science. 247, 105553 (2022).

to:

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  4. Ikhwanuddin, M., Bachok, Z., Mohd Faizal, W. W. Y., Azmie, G., Abol-Munafi, A. B. Size of maturity of mud crab Scylla olivacea (Herbst, 1796) from mangrove areas of Terengganu coastal waters. Journal of Sustainability Science and Management. 5 (2), 134-147 (2010).
  5. Waiho, K. et al. On types of sexual maturity in brachyurans, with special reference to size at the onset of sexual maturity. Journal of Shellfish Research. 36 (3), 807-839 (2017).
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  10. Okumura, T. et al. Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 143 (2), 246-253 (2006).
  11. Pervaiz, P. A., Jhon, S. M., Sikdar-bar, M. Studies on the effect of unilateral eyestalk ablation in maturation of gonads of a freshwater prawn Macrobrachium dayanum. World Journal of Zoology. 6 (2), 159-163 (2011).
  12. Primavera, J. H. Induced maturation and spawning in five-month-old Penaeus monodon Fabricius by eyestalk ablation. Aquaculture. 13 (4), 355-359 (1978).
  13. Shyne Anand, P. S. et al. Reproductive performance of wild brooders of Indian white shrimp, Penaeus indicus: Potential and challenges for selective breeding program. Journal of Coastal Research. 86 (sp1), 65 (2019).
  14. Diarte-Plata, G. et al. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012).
  15. Vargas-Téllez, I. et al. Impact of unilateral eyestalk ablation on Callinectes arcuatus (Ordway, 1863) under laboratory conditions: Behavioral evaluation. Latin American Journal of Aquatic Research. 49 (4), 576-594 (2021).
  16. Chu, K. H., Chow, W. K. Effects of unilateral versus bilateral eyestalk ablation on molting and growth of the shrimp, Penaeus chinensis (Osbeck, 1765) (Decapoda, Penaeidea). Crustaceana. 62 (3), 225-233 (1992).
  17. Taylor, J. Minimizing the effects of stress during eyestalk ablation of Litopenaeus vannamei females with topical anesthetic and a coagulating agent. Aquaculture. 233 (1-4), 173-179 (2004).
  18. Wang, M., Ye, H., Miao, L., Li, X. Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain. Aquaculture. 560, 738493 (2022).
  19. Nagaraju, G. P. C. Reproductive regulators in decapod crustaceans: an overview. Journal of Experimental Biology. 214 (1), 3-16 (2011).
  20. Kornthong, N. et al. Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression. General and Comparative Endocrinology. 185, 28-36 (2013).
  21. Kornthong, N. et al. Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration. Animal Reproduction Science. 208, 106122 (2019).
  22. Liu, C. et al. VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1. General and Comparative Endocrinology. 255, 1-11 (2018).
  23. Chen, H.-Y., Kang, B. J., Sultana, Z., Wilder, M. N. Variation of protein kinase C-α expression in eyestalk removal-activated ovaries in whiteleg shrimp, Litopenaeus vannamei. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 237 (300), 110552 (2019).
  24. Rotllant, G., Nguyen, T. V., Aizen, J., Suwansa-ard, S., Ventura, T. Toward the identification of female gonad-stimulating factors in crustaceans. Hydrobiologia. 825 (1), 91-119 (2018).
  25. Supriya, N. T., Sudha, K., Krishnakumar, V., Anilkumar, G. Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura: Decapoda). Chinese Journal of Oceanology and Limnology. 35 (3), 645-657 (2017).
  26. Wilder, M. N. Advances in the science of crustacean reproductive physiology and potential applications to new seed production technology. Journal of Coastal Research. 86 (sp1), 6-10 (2019).
  27. Arcos, G. F., Ibarra, A. M., Vazquez-Boucard, C., Palacios, E., Racotta, I. S. Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone. Aquaculture Research. 34 (9), 749-755 (2003).
  28. Desai, U. M., Achuthankutty, C. T. Complete regeneration of ablated eyestalk in penaeid prawn, Penaeus monodon. Current Science. 79 (11), 1602-1603 (2000).
  29. Wu, Q. et al. Growth performance and biochemical composition dynamics of ovary, hepatopancreas and muscle tissues at different ovarian maturation stages of female mud crab, Scylla paramamosain. Aquaculture. 515, 734560 (2020).
  30. Ghazali, A., Azra, M. N., Noordin, N. M., Abol-Munafi, A. B., Ikhwanuddin, M. Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) fed with various diets. Aquaculture. 468 (Part 1), 45-52 (2017).
  31. Farhadi, A. et al. The regulatory mechanism of sexual development in decapod crustaceans. Frontiers in Marine Science. 8 (2021).
  32. Sukardi, P., Prayogo, N. A., Harisam, T., Sudaryono, A. Effect of eyestalk-ablation and differences salinity in rearing pond on molting speed of Scylla serrata. AIP Conference Proceedings. 2094, 020029 (2019).
  33. Stella, V. S., López Greco, L. S., Rodríguez, E. M. Effects of eyestalk ablation at different times of the year on molting and reproduction of the estuarine grapsid crab Chasmagnathus granulata (Decapoda, Brachyura). Journal of Crustacean Biology. 20 (2), 239-244 (2000).
  34. Jang, I. K. et al. The effects of manipulating water temperature, photoperiod, and eyestalk ablation on gonad maturation of the swimming crab, Portunus trituberculatus.Crustaceana. 83 (2), 129-141 (2010).
  35. Millamena, O. M., Quinitio, E. The effects of diets on reproductive performance of eyestalk ablated and intact mud crab Scylla serrata. Aquaculture. 181 (1-2), 81-90 (2000).
  36. Zeng, C. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development. Aquaculture Research. 38 (14), 1478-1485 (2007).
  37. Rana, S. Eye stalk ablation of freshwater crab, Barytelphusa lugubris: An alternative approach of hormonal induced breeding. International Journal of Pure and Applied Zoology. 6 (3), 30-34 (2018).
  38. Yi, S.-K., Lee, S.-G., Lee, J.-M. Preliminary study of seed production of the Micronesian mud crab Scylla serrata (Crustacea: Portunidae) in Korea. Ocean and Polar Research. 31 (3), 257-264 (2009).
  39. Azra, M. N., Abol-Munafi, A. B., Ikhwanuddin, M. A review of broodstock improvement to brachyuran crab: Reproductive performance. International Journal of Aquaculture. 5 (38), 1-10 (2016).
  40.  Archibald, K. E., Scott, G. N., Bailey, K. M., Harms, C. A. 2-phenoxyethanol (2-PE) and tricaine methanesulfonate (MS-222) immersion anesthesia of American horseshoe crabs (Limulus polyphemus). Journal of Zoo and Wildlife Medicine. 50 (1), 96-106 (2019).
  41. Muhd-Farouk, H., Abol-Munafi, A. B., Jasmani, S., Ikhwanuddin, M. Effect of steroid hormones 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on ovary external morphology of orange mud crab, Scylla olivacea. Asian Journal of Cell Biology. 9 (1), 23-28 (2013).
  42. Muhd-Farouk, H., Jasmani, S., Ikhwanuddin, M. Effect of vertebrate steroid hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 1796). Aquaculture. 451, 78-86 (2016).
  43. Ghazali, A., Mat Noordin, N., Abol-Munafi, A. B., Azra, M. N., Ikhwanuddin, M. Ovarian maturation stages of wild and captive mud crab, Scylla olivacea fed with two diets. Sains Malaysiana. 46 (12), 2273-2280 (2017).
  44. Aaqillah-Amr, M. A., Hidir, A., Noordiyana, M. N., Ikhwanuddin, M. Morphological, biochemical and histological analysis of mud crab ovary and hepatopancreas at different stages of development. Animal Reproduction Science. 195, 274-283 (2018).
  45. Amin-Safwan, A., Muhd-Farouk, H., Mardhiyyah, M. P., Nadirah, M., Ikhwanuddin, M. Does water salinity affect the level of 17β-estradiol and ovarian physiology of orange mud crab, Scylla olivacea (Herbst, 1796) in captivity? Journal of King Saud University - Science. 31 (4), 827-835 (2019).
  46. Wu, X. et al. Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock. Aquaculture. 273 (4), 602-613 (2007).
  47. Azra, M. N., Ikhwanuddin, M. A review of maturation diets for mud crab genus Scylla broodstock: Present research, problems and future perspective. Saudi Journal of Biological Sciences. 23 (2), 257-267 (2016).
  48. Maschio Rodrigues, M., López Greco, L. S., de Almeida, L. C. F., Bertini, G. Reproductive performance of Macrobrachium acanthurus (Crustacea, Palaemonidae) females subjected to unilateral eyestalk ablation. Acta Zoologica. 103 (3), 326-334 (2022).
  49. Zhang, C. et al. Changes in bud morphology, growth-related genes and nutritional status during cheliped regeneration in the Chinese mitten crab, Eriocheir sinensis. PLoS One. 13 (12), e0209617 (2018).
  50. Zhang, C. et al. Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation. Fish & Shellfish Immunology. 81, 266-275 (2018).
  51. Diarte-Plata, G., Sainz-Hernandez, J. C., Aguiñaga-Cruz, J. A., Fierro-Coronado, J. A., Polanco-Torres, A., Puente-Palazuelos, C. Eyestalk ablation procedures to minimize pain in the freshwater prawn Macrobrachium americanum. Applied Animal Behaviour Science. 140 (3-4), 172-178 (2012). 
  52. Mirera, D. O., Moksnes, P. O. Comparative performance of wild juvenile mud crab (Scylla serrata) in different culture systems in East Africa: Effect of shelter, crab size and stocking density. Aquaculture International. 23 (1), 155-173 (2015).
  53. Ut, V. N., Le Vay, L., Nghia, T. T., Hong Hanh, T. T. Development of nursery cultures for the mud crab Scylla paramamosain (Estampador). Aquaculture Research. 38 (14), 1563-1568 (2007).
  54. Fazhan, H. et al. Limb loss and feeding ability in the juvenile mud crab Scylla olivacea: Implications of limb autotomy for aquaculture practice. Applied Animal Behaviour Science. 247, 105553 (2022).
Øjenstilk ablation for at øge æggestokkens modning i mudderkrabber
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Asmat-Ullah, M., Rozaimi, R.,… More

Asmat-Ullah, M., Rozaimi, R., Fazhan, H., Shu-Chien, A. C., Wang, Y., Waiho, K. Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs. J. Vis. Exp. (193), e65039, doi:10.3791/65039 (2023).

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