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 the back stand pipe of each tank and flows into a sump that empties into a sanitary drain. In this experiment, a water exchange rate of one gallon (US) per hour through a drip emitter was used.</li>
	<li>Maintain the temperature of each tank with an independent heating element that is used to manually alter the temperature during the priming process.</li>
	<li>Set up individual rows in a way to enable separate photoperiods in each. Install doors on each row that can be closed to prevent light from entering or escaping. 
	NOTE: The automated controller can enable manipulation of all photoperiods with the least disturbance to the fish.</li>
	<li>Equip the rack with a red work light and blackout curtains for access during dark hours.</li>
</ol>
2. Adjusting photoperiod and priming fish for gamete collection
<ol>
	<li>Remove the desired fish (Figure 2a) from general system racks and place in breeding racks to allow for adjustment of the photoperiod 14 days prior to priming. 
	NOTE: This allows the fish to acclimatize to a new environment.</li>
	<li>Keep the fish at 22.8 &#176;C (73 &#176;F) during this period using the installed aquatic heating system. The normal photoperiod is from 6 a.m. to 8 p.m. light and 8 p.m. to 6 a.m. dark. For the light cycle rack, shift the photoperiod to 10 p.m. to 12 p.m. light and 12 p.m. to 10 p.m. dark by adjusting the timer that powers the light within the rack. 
	NOTE: Males and females are housed in the same tank to allow for natural priming behaviors to take place. While spawning may take place in the tank, fish can still be used for in vitro fertilization since gametes are released in stages11.</li>
	<li>Once the fish are acclimatized, start priming the animals for spawning5 as described in steps 2.3.1 to 2.3.5. 
	NOTE: This procedure takes six days in total. Over this time, change the temperature to prime the ova production using an installed aquatic heating system. Using the 50W Aquatic Heaters (see Table of Materials), set the heater directly to the temperature (scale on the heater is in Fahrenheit) given in the protocol at each step. Depending on the size of the tank and flow-through rate of the water, time of temperature adjustment may differ. In this experiment, the temperature was adjusted at noon and temperature equilibration took over the next 18 h.
	<ol>
		<li>On day 1, raise the temperature from 22.8 &#176;C (73 &#176;F) to 24.4 &#176;C (76 &#176;F).</li>
		<li>On day 2, raise the temperature from 24.4 &#176;C (76 &#176;F) to 26.1 &#176;C (79 &#176;F).</li>
		<li>On day 3 and 4, keep the temperature at 26.1 &#176;C (79 &#176;F). Fish will be ready to spawn during the day and IVF can be performed. 
		NOTE: Depending on the individual fish, females can spawn on day 3 and/or day 4. We recommend trying to obtain ova on day 3 and/or day 4 depending on the success of the ova collection.</li>
		<li>On day 5, lower the temperature from 26.1 &#176;C (79 &#176;F) to 24.4 &#176;C (76 &#176;F).</li>
		<li>On day 6, lower the temperature from 24.4 &#176;C (76 &#176;F) to 22.8 &#176;C (73 &#176;F). 
		NOTE: Provide a 7-day gap before repeating this temperature cycle. It is recommended to continue keeping the fish in this photoperiod since this will reduce the overall time needed by the fish to adjust to this shifted light cycle.</li>
	</ol>
	</li>
</ol>
3. Female gamete collection
<ol>
	<li>Begin by inserting a moistened tissue wipe into a Petri dish lid and closing the dish to create a humidified chamber and prevent the ova from drying out during the collection process.</li>
	<li>Next choose a female for collection. Gravid fish with large, protruding abdomens will likely be the best choice for this procedure (Figure 2a). 
	NOTE: To differentiate between males and females of adult A. mexicanus, the cotton ball method was used12.</li>
	<li>Immobilize a female using chilled water and place her in the supine position in a moistened sponge animal holder. Do so by placing the fish in 4 &#176;C system water for at least 30 s or until the fish is immobilized (i.e., loss of gill movement, see Ross and Ross13 for details). 
	NOTE: Work quickly and try to avoid warming the fish until the procedure is completed. This may include periodically dipping the gloved tips of the fingers into cold water or offering supplemental anesthesia. Other anesthesia methods (e.g., MS-22213) may be used as well. Under the IACUC guidelines of the Stowers Institute for Medical Research, manual collection of ova is considered a non-invasive procedure, which does not require complete anesthesia (e.g., through MS-222).</li>
	<li>Once positioned, blot the ventral side of the fish with a delicate tissue wipe as contact with water will cause the ova to activate.</li>
	<li>Hold the female between the thumb and index finger. Gently squeeze against the lateral sides of the coelomic cavity in the direction of the urogenital opening while rolling the fingers slightly. Collect the expressed ova using a disposable spatula.</li>
	<li>Transfer these ova to the humidified Petri dish. 
	NOTE: Several clutches of ova may be combined in the same dish if specific parentage data is not needed. The ova can be stored at 24 &#176;C and are best when used for IVF within 30-60 min after collection.</li>
	<li>After collection, gently return the fish to a recovery tank filled with system water. 
	NOTE: Place the fish back into dark cabinet tank for future ova collection when necessary.</li>
</ol>
4. Male gamete collection
<ol>
	<li>Choose a male for collection. 
	NOTE: There are no outwardly visible signs of male gamete quality. However, fish should appear healthy in appearance before use in this procedure. To differentiate between males and females of adult A. mexicanus, the cotton ball method was used12.</li>
	<li>Immobilize a male using chilled water and place him in the supine position in a moistened sponge animal holder. Immobilize by placing the fish in 4 &#176;C system water for at least 30 seconds or until the fish is immobilized (i.e., loss of gill movement, see Ross and Ross13 for details). 
	NOTE: Work quickly and try to avoid warming the fish until the procedure is completed. This may include periodically dipping the gloved tips of the fingers into cold water or offering supplemental anesthesia. Other anesthesia methods (e.g., MS-22213) may be used here as well. Under the IACUC guidelines of the Stowers Institute for Medical Research, manual collection of sperm is considered a non-invasive procedure, which does not require complete anesthesia (e.g., through MS-222).</li>
	<li>Blot the ventral side of the fish with a delicate tissue wipe as contact with water will activate the milt.</li>
	<li>Gently place the end of a capillary tube at the urogenital opening.</li>
	<li>Expel milt by applying gentle pressure on the sides of the fish with the thumb and forefinger. Start distal to the gills, moving towards the urogenital opening.</li>
	<li>Collect the milt in the end of a capillary tube. Gentle suction may be necessary by use of an aspirator tube. Avoid any feces that may be expelled with the milt.</li>
	<li>Dispense the milt into an empty 1.5 mL centrifuge tube and dilute with twice the volume of Sperm Extender E400 (see Table of Materials). Keep on ice. 
	NOTE: Milt from multiple males may be pooled together if specific parentage data is not needed. This step can be used to extend the working time of the milt for several hours, but it is not required for immediate fertilization.</li>
	<li>After collection, gently return the fish to a recovery tank filled with system water. 
	NOTE: Place fish back into dark cabinet tank for future sperm collection when necessary.</li>
</ol>
5. In vitro fertilization
<ol>
	<li>Using a new pipette for each stock, mix the sperm by pipetting and/or agitating the side of the tube before fertilizing as sperm in milt can settle in the E400 solution over time.</li>
	<li>Dispense the milt or extended milt solution into the freshly collected ova.</li>
	<li>Quickly add 1 mL of system water to the clutch to activate the sperm and eggs for fertilization. Avoid mixing or agitating the dish contents and allow 2 min for fertilization to occur. 
	NOTE: Mixing and agitating greatly reduces the fertilization rates and should, therefore, be avoided14.</li>
	<li>Add E2 Embryo Media to fill the dish 2/3rd full. 
	NOTE: Depending on the subsequent procedure, embryos can be either used right away (e.g., for injection of genetic constructs as described before15) or embryos can be incubated in E2 Embryo Media at 23 &#176;C until they reach 5 dpf. At this point transfer embryos to the main recirculating housing system using system water.</li>
</ol>

<ol><li>Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regressive+evolution+in+Astyanax+cavefish%5BTitle%5D)+AND+%22Annual+Review+Genetics%22%5BJournal%5D)">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/pubmed?term=((A+novel+role+for+Mc1r+in+the+parallel+evolution+of+depigmentation+in+independent+populations+of+the+cavefish+Astyanax+mexicanus%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">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/pubmed?term=((Insulin+resistance+in+cavefish+as+an+adaptation+to+a+nutrient-limited+environment%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.</a> Nature. 555, 647-651 (2018).</li>
<li>Xiong, S., Krishnan, J., Peuß, R., Rohner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+adipogenesis+contributes+to+excess+fat+accumulation+in+cave+populations+of+Astyanax+mexicanus%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((Breeding+Astyanax+mexicanus+through+Natural+Spawning%5BTitle%5D)+AND+%22COLD+SPRING+HARBOR+Protocols%22%5BJournal%5D)">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/pubmed?term=((In+Vitro+Fertilization+of+Astyanax+mexicanus%5BTitle%5D)+AND+%22COLD+SPRING+HARBOR+Protocols%22%5BJournal%5D)">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/pubmed?term=((Circadian+rhythms+in+Mexican+blind+cavefish+Astyanax+mexicanus+in+the+lab+and+in+the+field%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Reproductive+biology+and+induced+breeding+of+two+Characidae+species+%28Osteichthyes%2C+Characiformes%29+from+the+S%C3%A3o+Francisco+River+basin%2C+Minas+Gerais%2C+Brazil%5BTitle%5D)+AND+%22Revista+Brasileira+Zoology%22%5BJournal%5D)">Reproductive biology and induced breeding of two Characidae species (Osteichthyes, Characiformes) from the São 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/pubmed?term=((Improvement+of+gamete+quality+and+its+short-term+storage%3A+an+approach+for+biotechnology+in+laboratory+fish%5BTitle%5D)+AND+%22Animal%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWesterfield%2C+M+%22The+zebrafish+book+%3A+a+guide+for+the+laboratory+use+of+zebrafish+%28Danio+rerio%29%22">The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio)</a>. , University of Oregon Press. (2000).</li>
<li>Simon, V., Hyacinthe, C., Retaux, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Breeding+behavior+in+the+blind+Mexican+cavefish+and+its+river-dwelling+conspecific%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Determining+the+Sex+of+Adult+Astyanax+mexicanus%5BTitle%5D)+AND+%22COLD+SPRING+HARBOR+Protocols%22%5BJournal%5D)">Determining the Sex of Adult Astyanax mexicanus.</a> COLD SPRING HARBOR Protocols. , (2008).</li>
<li>Ross, L. G., Ross, B. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRoss%2C+LG+%22Anaesthetic+and+Sedative+Techniques+for+Aquatic+Animals%22">Anaesthetic and Sedative Techniques for Aquatic Animals</a>. , 3rd edn, Wiley-Blackwell. (2008).</li>
<li>Matthews, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+to+Extender%2C+Cryoprotective+Medium%2C+and+In+Vitro+Fertilization+Improve+Zebrafish+Sperm+Cryopreservation%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation.</a> Zebrafish. 15 (3), 279-290 (2018).</li>
<li>Stahl, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stable+transgenesis+in+Astyanax+mexicanus+using+the+Tol2+transposase+system%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">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/pubmed?term=((Astyanax+transgenesis+and+husbandry%3A+how+cavefish+enters+the+laboratory%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">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/pubmed?term=((A+novel+role+for+Mc1r+in+the+parallel+evolution+of+depigmentation+in+independent+populations+of+the+cavefish+Astyanax+mexicanus%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">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/pubmed?term=((Chapter+8.+Evolution+and+development+in+the+cavefish+Astyanax%5BTitle%5D)+AND+%22Current+Topics+in+Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((Regressive+evolution+in+the+Mexican+cave+tetra%2C+Astyanax+mexicanus%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">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/pubmed?term=((A+developmental+staging+table+for+Astyanax+mexicanus+surface+fish+and+Pachon+cavefish%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+method+for+zebrafish+sperm+cryopreservation+and+in+vitro+fertilization%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiment%22%5BJournal%5D)">A high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization.</a> Journal of Visualized Experiment. (29), (2009).</li>
</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>1.5 mL Centrifuge Tube</td><td>Eppendorf</td><td>#22364111</td><td></td></tr><tr><td>100 mm Petri Dishes</td><td>VWR International</td><td>#25384-302</td><td></td></tr><tr><td>Aspirator Tube</td><td>Drummond&amp;nbsp;</td><td>#2-000-000</td><td></td></tr><tr><td>Calibrated 1-5 &amp;micro;L Capillary Tubes</td><td>Drummond</td><td>#2-000-001</td><td></td></tr><tr><td>Dispolable Spatulas</td><td>VWR International</td><td>#80081-188</td><td></td></tr><tr><td>HMA-50S &amp;nbsp;50W Aquatic Heaters</td><td>Finnex</td><td>HMA-50S</td><td></td></tr><tr><td>P1000 Pipette</td><td>Eppendorf</td><td>#3123000063</td><td></td></tr><tr><td>P1000 Pipette Tips</td><td>Thermo Scientific</td><td>#2079E</td><td></td></tr><tr><td>Sanyo MIR-554 incubator&amp;nbsp;</td><td>Panasonic Health Care</td><td>MIR-554-PA</td><td></td></tr><tr><td>Sperm Extender E400</td><td></td><td></td><td>130 mM KCl, 50 mM NaCl, 2 mM CaCl&lt;sub&gt;2&lt;/sub&gt; (2H&lt;sub&gt;2&lt;/sub&gt;O), 1 mM MgSO&lt;sub&gt;4&lt;/sub&gt; (7H&lt;sub&gt;2&lt;/sub&gt;O), 10 mM D (+)-Glucose, 30 mM HEPES&lt;br/&gt; Adjust to pH 7.9 with&amp;nbsp; 5M KOH and filter sterilize. Solution can be stored at 4 ?C for up to 6 months.</td></tr><tr><td>Sponge Animal Holder</td><td></td><td></td><td>Made from scrap foam</td></tr><tr><td>System Water</td><td></td><td></td><td>Deionized water supplemented with Instant Ocean Sea Salt [Blacksburg, VA] to reach a specific conductance of 800 &amp;micro;S/cm.&amp;nbsp; 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 &amp;deg;C; pH, 7.65; dissolved oxygen 100 %)</td></tr><tr><td>Tissue Wipes</td><td>Kimberly-Clark Professional</td><td>#21905-026</td><td></td></tr><tr><td>ZIRC E2 Embryo Media</td><td></td><td></td><td>15 mM NaCl, 0.5 mM KCl, 1.0 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 150 &amp;micro;M KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 50 &amp;micro;M Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;,&lt;br/&gt; 1.0 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.7 mM NaHCO&lt;sub&gt;3. &lt;/sub&gt;Adjust pH to 7.2 to 7.4 using 2 N hydrochloric acid. Filter sterilize. Stored at room temperature for a maximum of two weeks.</td></tr></tbody></table>

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

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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9.2K Views.  Stowers Institute for Medical Research. 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. 

Video: Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Genetics

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Developmental Biology

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.

This goal of this protocol is to produce chimeric axolotls with haploid forelimbs derived from Cas9-mutagenized donor tissue using embryonic tissue grafting techniques.

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) and blind cave (cave fish) morphs in this species presents an opportunity to interrogate the mechanisms underlying morphological and behavioral evolution. Cave fish have evolved novel constructive and regressive traits. The constructive changes include increases in taste buds and jaws, lateral line sensory organs, and body fat. The regressive changes include loss or reduction of eyes. melanin pigmentation, schooling behavior, aggression, and sleep. To experimentally interrogate these changes, it is crucial to obtain large numbers of spawned embryos. Since the original A. mexicanus surface fish and cave fish were collected in Texas and Mexico in the 1990s, their descendants have been routinely stimulated to breed and spawn large numbers of embryos bimonthly in the Jeffery laboratory. Although breeding is controlled by food abundance and quality, light-dark cycles, and temperature, we have found that incremental temperature changes play a key role in stimulating maximal spawning. The gradual increase of temperature from 72 &#176;F to 78 &#176;F in the first three days of a breeding week provides two-three consecutive spawning days with maximal numbers of high-quality embryos, which is then followed by a gradual decrease of temperature from 78 &#176;F to 72 &#176;F during the last three days of the spawning week. The procedures shown in this video outline the workflow before and during a laboratory breeding week for incremental temperature stimulated spawning.

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

This article outlines the basic laboratory conditions and protocols for an incremental temperature regime to stimulate maximal spawning in the Mexican tetra Astyanax mexicanus, which is an emerging model for developmental and evolutionary studies.

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Summary

Explore More Videos

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

Manipulation of Gene Function in Mexican Cavefish

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Seawater Sampling and Collection

Wholemount In Situ Hybridization for Astyanax Embryos

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)