Name: gamete collection and in vitro fertilization of astyanax mexicanus
Uploaded: 2019-05-25T13:00:00
Description: Watch this Scientific Journal Video about Gamete Collection and In Vitro Fertilization of Astyanax mexicanus at JoVE.com

The authors would like to thank Philippe Noguera and Kimberly Bland for their support on the video production. The authors would also like to acknowledge the entire Aquatics Team of the Stowers Institute for animal husbandry. This work was supported by institutional funding to DPB and NR. NR was supported by the Edward Mallinckrodt Foundation and JDRF. RP was supported by a grant from the Deutsche Forschungsgemeinschaft (PE 2807/1-1).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Stowers Institute for Medical Research.
1. Light cycle manipulation
<ol>
	<li>Set up fish tanks within an opaque, fully enclosed (light protection), flow-through aquaculture system containing multiple rows of tanks (Figure 1). 
	NOTE: The flow-through system as shown in Figure 1 uses system water to flush waste through th...

<ol>
	<li>Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regressive+evolution+in+Astyanax+cavefish.">Regressive evolution in Astyanax cavefish.</a> Annual Review Genetics. 43, 25-47 (2009).</li><li>Gross, J. B., Borowsky, R., Tabin, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+Mc1r+in+the+parallel+evolution+of+depigmentation+in+independent+populations+of+the+cavefish+Astyanax+mexicanus.">A novel role for Mc1r in the parallel evolution of depigmentation in independent populations of the cavefish Astyanax mexicanus.</a> PLoS Genetics. 5, e1000326 (2009).</li><li>Riddle, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+in+cavefish+as+an+adaptation+to+a+nutrient-limited+environment.">Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.</a> Nature. 555, 647-651 (2018).</li><li>Xiong, S., Krishnan, J., Peu&#223;, R., Rohner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+adipogenesis+contributes+to+excess+fat+accumulation+in+cave+populations+of+Astyanax+mexicanus.">Early adipogenesis contributes to excess fat accumulation in cave populations of Astyanax mexicanus.</a> Developmental Biology. 441 (2), 297-304 (2018).</li><li>Borowsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+Astyanax+mexicanus+through+Natural+Spawning.">Breeding Astyanax mexicanus through Natural Spawning.</a> COLD SPRING HARBOR Protocols. , (2008).</li><li>Borowsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Fertilization+of+Astyanax+mexicanus.">In Vitro Fertilization of Astyanax mexicanus.</a> COLD SPRING HARBOR Protocols. , (2008).</li><li>Beale, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythms+in+Mexican+blind+cavefish+Astyanax+mexicanus+in+the+lab+and+in+the+field.">Circadian rhythms in Mexican blind cavefish Astyanax mexicanus in the lab and in the field.</a> Nature Communications. 4, 2769 (2013).</li><li>Sato, Y., Sampaio, E. V., Fenerich-Verani, N., Verani, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+biology+and+induced+breeding+of+two+Characidae+species+(Osteichthyes,+Characiformes)+from+the+S&#227;o+Francisco+River+basin,+Minas+Gerais,+Brazil.">Reproductive biology and induced breeding of two Characidae species (Osteichthyes, Characiformes) from the S&#227;o Francisco River basin, Minas Gerais, Brazil.</a> Revista Brasileira Zoology. 23 (1), 267-273 (2006).</li><li>Yasui, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+gamete+quality+and+its+short-term+storage:+an+approach+for+biotechnology+in+laboratory+fish.">Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish.</a> Animal. 9 (3), 464-470 (2015).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Simon, V., Hyacinthe, C., Retaux, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+behavior+in+the+blind+Mexican+cavefish+and+its+river-dwelling+conspecific.">Breeding behavior in the blind Mexican cavefish and its river-dwelling conspecific.</a> PLoS One. 14 (2), e0212591 (2019).</li><li>Borowsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+Sex+of+Adult+Astyanax+mexicanus.">Determining the Sex of Adult Astyanax mexicanus.</a> COLD SPRING HARBOR Protocols. , (2008).</li><li>Ross, L. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transgenesis+in+Astyanax+mexicanus+using+the+Tol2+transposase+system.">Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system.</a> Developmental Dynamics. , 1-9 (2019).</li><li>Elipot, Y., Legendre, L., Pere, S., Sohm, F., Retaux, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astyanax+transgenesis+and+husbandry:+how+cavefish+enters+the+laboratory.">Astyanax transgenesis and husbandry: how cavefish enters the laboratory.</a> Zebrafish. 11, 291-299 (2014).</li><li>Gross, J. B., Borowsky, R., Tabin, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+Mc1r+in+the+parallel+evolution+of+depigmentation+in+independent+populations+of+the+cavefish+Astyanax+mexicanus.">A novel role for Mc1r in the parallel evolution of depigmentation in independent populations of the cavefish Astyanax mexicanus.</a> PLoS Genetics. 5 (1), e1000326 (2009).</li><li>Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8.+Evolution+and+development+in+the+cavefish+Astyanax.">Chapter 8. Evolution and development in the cavefish Astyanax.</a> Current Topics in Developmental Biology. 86, 191-221 (2009).</li><li>Protas, M., Conrad, M., Gross, J. B., Tabin, C., Borowsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regressive+evolution+in+the+Mexican+cave+tetra,+Astyanax+mexicanus.">Regressive evolution in the Mexican cave tetra, Astyanax mexicanus.</a> Current Biology. 17 (5), 452-454 (2007).</li><li>Hinaux, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+staging+table+for+Astyanax+mexicanus+surface+fish+and+Pachon+cavefish.">A developmental staging table for Astyanax mexicanus surface fish and Pachon cavefish.</a> Zebrafish. 8, 155-165 (2011).</li><li>Draper, B. W., Moens, C. 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While IVF is a standardized method for many different model organisms such as zebrafish, existing protocols for A. mexicanus do not take into account that this species naturally spawns during night hours6. Given that cavefish and surface fish differ quite drastically in their circadian rhythms, the maturation cycle of the ova also differs between the cave and surface morphotypes. While the staging temperatures and times for surface A. mexicanus are well studied1...

In recent years, Astyanax mexicanus has become a model organism in different fields such as developmental biology, evolutionary biology, behavioral biology, and physiology1,2,3,4. The uniqueness of this system comes from this species having several morphotypes that have adapted to very different environments. The surface dwelling morphotype lives in rivers where there is high biodiversity and plenty of food sources for the fish. In contrast, the cave morphotypes of A. mexicanus, the cavefish, live in caves where biodiversity, food sources, and oxygen are drastically diminished1. Cavefish differ from the surface fish in a variety of phenotypes such as the absence of eyes and pigmentation, insulin resistance, and the ability to store fat2,3,4. However, surface fish and cavefish still belong to the same species and are, therefore, interfertile.
For both morphotypes, a set of conditions has been defined to allow routine maintenance and breeding under laboratory conditions5,6. However, genetic modifications, proper embryonic developmental studies, and creation of hybrids are still challenging for several reasons. A. mexicanus primarily spawn during night hours&#160;which is inconvenient for subsequent experiments on early embryonic stages&#160;such as injection of genetic constructs or monitoring of early embryonic developmental processes. In addition, generation of surface and cave hybrids is challenging using natural spawning, since the cave morphotypes have an altered circadian rhythm7 that ultimately affects the production of viable ova. Successful, yet invasive, IVF procedures have been described for other Astyanax species, where gamete production and spawning behavior was primed using hormonal injections8,9. Less invasive IVF procedures (i.e., obtaining gametes from manual spawning without the injection of hormonal preparations) have been described but do not consider the differences in the spawning cycle between cave and surface morphotypes of A. mexicanus6.
Other fish model organisms, such as the zebrafish, can easily be genetically modified and studied at an embryonic level because the obstacles stated above have been successfully resolved. Implementation of standardized breeding techniques, in vitro fertilization, and sperm cryopreservation have all pushed zebrafish forward and solidified the model&#39;s use in the biological sciences10. Therefore, extending these techniques to A. mexicanus will further strengthen it as a model system.
Here, we present a detailed protocol for IVF that will help to make A. mexicanus more accessible. We will present a breeding setup that enables shifting the light-cycles of the fish from daytime to nighttime so that viable ova can be obtained during day hours without injection of hormonal preparations. We then provide a detailed description of how to obtain the ova and milt used for IVF. This method will enable the production of embryos during normal working hours and make further downstream applications more feasible compared to using embryos from natural spawning.

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			<td style="width:175px;">130 mM KCl, 50 mM NaCl, 2 mM CaCl2 (2H2O), 1 mM MgSO4 (7H2O), 10 mM D (+)-Glucose, 30 mM HEPES 
			Adjust to pH 7.9 with&#160; 5M KOH and filter sterilize. Solution can be stored at 4 &#730;C for up to 6 months.</td>
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			<td style="width:175px;">Deionized water supplemented with Instant Ocean Sea Salt [Blacksburg, VA] to reach a specific conductance of 800 &#181;S/cm.&#160; Water quality parameters are maintained within safe limits (Upper limit of total ammonia nitrogen range, 1 mg/L; upper limit of nitrite range, 0.5 mg/L; upper limit of nitrate range, 60 mg/L; temperature, 22 &#176;C; pH, 7.65; dissolved oxygen 100 %)</td>
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gamete collection and in vitro fertilization of astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

The protocol presented here is mainly based on a previously published protocol6. However, since A. mexicanus spawns during night hours, we designed a housing rack for fish breeding that can change the photoperiod independent of working hours (Figure 1). The fish light cycle is altered within a fully enclosed, flow-through aquaculture system containing three rows of tanks (Figure 1). Each tank cont...

Watch this Scientific Journal Video about Gamete Collection and In Vitro Fertilization of Astyanax mexicanus at JoVE.com

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus (Video) | JoVE

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

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