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Practical Euthanasia Method for Common Sea Stars (Asterias rubens) That Allows for High-Quality RNA Sampling
Sea stars in research are often lethally sampled without available methodology to render them insensible prior to sampling due to concerns over sufficient sample quality for applied molecular techniques. The objectives of this study were to describe an inexpensive and effective two-step euthanasia m...
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Published in: | Animals (Basel) 2021-06, Vol.11 (7), p.1847 |
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description | Sea stars in research are often lethally sampled without available methodology to render them insensible prior to sampling due to concerns over sufficient sample quality for applied molecular techniques. The objectives of this study were to describe an inexpensive and effective two-step euthanasia method for adult common sea stars (Asterias rubens) and to demonstrate that high-quality RNA samples for further use in downstream molecular analyses can be obtained from pyloric ceca of MgCl2-immersed sea stars. Adult common sea stars (n = 15) were immersed in a 75 g/L magnesium chloride solution until they were no longer reactive to having their oral surface tapped with forceps (mean: 4 min, range 2–7 min), left immersed for an additional minute, and then sampled with sharp scissors. RNA from pyloric ceca (n = 10) was isolated using a liquid–liquid method, then samples were treated with DNase and analyzed for evaluation of RNA integrity number (RIN) for assessment of the quantity and purity of intact RNA. Aversive reactions to magnesium chloride solution were not observed and no sea stars regained spontaneous movement or reacted to sampling. The calculated RIN ranged from 7.3–9.8, demonstrating that the combination of animal welfare via the use of anesthesia and sampling for advanced molecular techniques is possible using this low-cost technique. |
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The objectives of this study were to describe an inexpensive and effective two-step euthanasia method for adult common sea stars (Asterias rubens) and to demonstrate that high-quality RNA samples for further use in downstream molecular analyses can be obtained from pyloric ceca of MgCl2-immersed sea stars. Adult common sea stars (n = 15) were immersed in a 75 g/L magnesium chloride solution until they were no longer reactive to having their oral surface tapped with forceps (mean: 4 min, range 2–7 min), left immersed for an additional minute, and then sampled with sharp scissors. RNA from pyloric ceca (n = 10) was isolated using a liquid–liquid method, then samples were treated with DNase and analyzed for evaluation of RNA integrity number (RIN) for assessment of the quantity and purity of intact RNA. Aversive reactions to magnesium chloride solution were not observed and no sea stars regained spontaneous movement or reacted to sampling. The calculated RIN ranged from 7.3–9.8, demonstrating that the combination of animal welfare via the use of anesthesia and sampling for advanced molecular techniques is possible using this low-cost technique.</description><identifier>ISSN: 2076-2615</identifier><identifier>EISSN: 2076-2615</identifier><identifier>DOI: 10.3390/ani11071847</identifier><identifier>PMID: 34206249</identifier><language>eng</language><publisher>Basel: MDPI AG</publisher><subject>Alkalinity ; Ammonia ; Anesthesia ; Animal behavior ; Animal tissues ; Animal welfare ; Aquariums ; Asterias rubens ; Biodiversity ; Cutting tools ; Deoxyribonuclease ; Digestive glands ; Euthanasia ; invertebrate ; Invertebrates ; killing ; Laboratory animals ; Magnesium ; Magnesium chloride ; Marine ecosystems ; Marine invertebrates ; Medical instruments ; Methods ; Salinity ; Sampling ; sea star ; starfish ; Water quality</subject><ispartof>Animals (Basel), 2021-06, Vol.11 (7), p.1847</ispartof><rights>2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). 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The objectives of this study were to describe an inexpensive and effective two-step euthanasia method for adult common sea stars (Asterias rubens) and to demonstrate that high-quality RNA samples for further use in downstream molecular analyses can be obtained from pyloric ceca of MgCl2-immersed sea stars. Adult common sea stars (n = 15) were immersed in a 75 g/L magnesium chloride solution until they were no longer reactive to having their oral surface tapped with forceps (mean: 4 min, range 2–7 min), left immersed for an additional minute, and then sampled with sharp scissors. RNA from pyloric ceca (n = 10) was isolated using a liquid–liquid method, then samples were treated with DNase and analyzed for evaluation of RNA integrity number (RIN) for assessment of the quantity and purity of intact RNA. Aversive reactions to magnesium chloride solution were not observed and no sea stars regained spontaneous movement or reacted to sampling. The calculated RIN ranged from 7.3–9.8, demonstrating that the combination of animal welfare via the use of anesthesia and sampling for advanced molecular techniques is possible using this low-cost technique.</description><subject>Alkalinity</subject><subject>Ammonia</subject><subject>Anesthesia</subject><subject>Animal behavior</subject><subject>Animal tissues</subject><subject>Animal welfare</subject><subject>Aquariums</subject><subject>Asterias rubens</subject><subject>Biodiversity</subject><subject>Cutting tools</subject><subject>Deoxyribonuclease</subject><subject>Digestive glands</subject><subject>Euthanasia</subject><subject>invertebrate</subject><subject>Invertebrates</subject><subject>killing</subject><subject>Laboratory animals</subject><subject>Magnesium</subject><subject>Magnesium chloride</subject><subject>Marine ecosystems</subject><subject>Marine invertebrates</subject><subject>Medical instruments</subject><subject>Methods</subject><subject>Salinity</subject><subject>Sampling</subject><subject>sea star</subject><subject>starfish</subject><subject>Water quality</subject><issn>2076-2615</issn><issn>2076-2615</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>PIMPY</sourceid><sourceid>DOA</sourceid><recordid>eNpdkktr3DAQgE1paUKaU_-AoJeU4lYP6-FLYVnSJpA-Nz2LsSSvtcjWRpJb8u_rZENJqsuImU8fM2Kq6jXB7xlr8QeYPCFYEtXIZ9UxxVLUVBD-_NH9qDrNeYeXIzkjnLysjlhDsaBNe1yF7wlM8QYCOp_LABNkD-iLK0O0qI8JreM4xgltHKBNgZTR2SoXlzxklObOTfktuh6goFUI8U--f3Lht0P9Y4bgyy36-XWFNjDug5-2r6oXPYTsTh_iSfXr0_n1-qK--vb5cr26qk3Daak7Sh2jUrW8aRTjlhgraMe4WHK96ClRbcNs1zln2pa02Bojcd8JyxV3wICdVJcHr42w0_vkR0i3OoLX94mYthrSMnRwWjrloCfYYawaYgVgJYiVrLOkV9apxfXx4NrP3eiscVNJEJ5In1YmP-ht_K0Vw5i1chGcPQhSvJldLnr02bgQYHJxzpryZchWSHaHvvkP3cU5TctXLRRvmCCU8YV6d6BMijkn1_9rhmB9txT60VKwv4ztp3E</recordid><startdate>20210622</startdate><enddate>20210622</enddate><creator>Wahltinez, Sarah J.</creator><creator>Kroll, Kevin J.</creator><creator>Nunamaker, Elizabeth A.</creator><creator>Denslow, Nancy D.</creator><creator>Stacy, Nicole I.</creator><general>MDPI AG</general><general>MDPI</general><scope>AAYXX</scope><scope>CITATION</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0003-4258-4414</orcidid><orcidid>https://orcid.org/0000-0002-4232-4420</orcidid><orcidid>https://orcid.org/0000-0001-7639-4454</orcidid></search><sort><creationdate>20210622</creationdate><title>Practical Euthanasia Method for Common Sea Stars (Asterias rubens) That Allows for High-Quality RNA Sampling</title><author>Wahltinez, Sarah J. ; Kroll, Kevin J. ; Nunamaker, Elizabeth A. ; Denslow, Nancy D. ; Stacy, Nicole I.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c452t-b22e32789544835d1cd62b356278f6f218943dbbeec99190dcc70fb6d585ea3a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Alkalinity</topic><topic>Ammonia</topic><topic>Anesthesia</topic><topic>Animal behavior</topic><topic>Animal tissues</topic><topic>Animal welfare</topic><topic>Aquariums</topic><topic>Asterias rubens</topic><topic>Biodiversity</topic><topic>Cutting tools</topic><topic>Deoxyribonuclease</topic><topic>Digestive glands</topic><topic>Euthanasia</topic><topic>invertebrate</topic><topic>Invertebrates</topic><topic>killing</topic><topic>Laboratory animals</topic><topic>Magnesium</topic><topic>Magnesium chloride</topic><topic>Marine ecosystems</topic><topic>Marine invertebrates</topic><topic>Medical instruments</topic><topic>Methods</topic><topic>Salinity</topic><topic>Sampling</topic><topic>sea star</topic><topic>starfish</topic><topic>Water quality</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wahltinez, Sarah J.</creatorcontrib><creatorcontrib>Kroll, Kevin J.</creatorcontrib><creatorcontrib>Nunamaker, Elizabeth A.</creatorcontrib><creatorcontrib>Denslow, Nancy D.</creatorcontrib><creatorcontrib>Stacy, Nicole I.</creatorcontrib><collection>CrossRef</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest Central</collection><collection>ProQuest Central Essentials</collection><collection>AUTh Library subscriptions: ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Publicly Available Content Database (Proquest) (PQ_SDU_P3)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>Directory of Open Access Journals</collection><jtitle>Animals (Basel)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wahltinez, Sarah J.</au><au>Kroll, Kevin J.</au><au>Nunamaker, Elizabeth A.</au><au>Denslow, Nancy D.</au><au>Stacy, Nicole I.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Practical Euthanasia Method for Common Sea Stars (Asterias rubens) That Allows for High-Quality RNA Sampling</atitle><jtitle>Animals (Basel)</jtitle><date>2021-06-22</date><risdate>2021</risdate><volume>11</volume><issue>7</issue><spage>1847</spage><pages>1847-</pages><issn>2076-2615</issn><eissn>2076-2615</eissn><abstract>Sea stars in research are often lethally sampled without available methodology to render them insensible prior to sampling due to concerns over sufficient sample quality for applied molecular techniques. The objectives of this study were to describe an inexpensive and effective two-step euthanasia method for adult common sea stars (Asterias rubens) and to demonstrate that high-quality RNA samples for further use in downstream molecular analyses can be obtained from pyloric ceca of MgCl2-immersed sea stars. Adult common sea stars (n = 15) were immersed in a 75 g/L magnesium chloride solution until they were no longer reactive to having their oral surface tapped with forceps (mean: 4 min, range 2–7 min), left immersed for an additional minute, and then sampled with sharp scissors. RNA from pyloric ceca (n = 10) was isolated using a liquid–liquid method, then samples were treated with DNase and analyzed for evaluation of RNA integrity number (RIN) for assessment of the quantity and purity of intact RNA. Aversive reactions to magnesium chloride solution were not observed and no sea stars regained spontaneous movement or reacted to sampling. 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subjects | Alkalinity Ammonia Anesthesia Animal behavior Animal tissues Animal welfare Aquariums Asterias rubens Biodiversity Cutting tools Deoxyribonuclease Digestive glands Euthanasia invertebrate Invertebrates killing Laboratory animals Magnesium Magnesium chloride Marine ecosystems Marine invertebrates Medical instruments Methods Salinity Sampling sea star starfish Water quality |
title | Practical Euthanasia Method for Common Sea Stars (Asterias rubens) That Allows for High-Quality RNA Sampling |
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