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Biology

Øjenstilk ablation for at øge æggestokkens modning i mudderkrabber

Published: March 31, 2023
doi:
10.3791/65039
, , , , ,
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

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.

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 vådo…

Representative Results

Gonad modningCremet 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 (fig…

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 gyd…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

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
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Tags

Keywords: Eyestalk AblationMud CrabOvarian MaturationStress MinimizationAnesthesiaCauterizationSurgical AblationOvarian Maturation Stages
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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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