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/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. G., Ross, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anaesthetic and Sedative Techniques for Aquatic Animals. , (2008).</li><li>Matthews, J. L., 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=Changes+to+Extender,+Cryoprotective+Medium,+and+In+Vitro+Fertilization+Improve+Zebrafish+Sperm+Cryopreservation.">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/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. 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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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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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:366px;">0.6 mL Centrifuge Tube</td>
			<td style="width:179px;">Eppendorf</td>
			<td style="width:110px;">#22364111</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:366px;">100 mm Petri Dishes</td>
			<td style="width:179px;">VWR International</td>
			<td>#25384-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:366px;">Aspirator Tube</td>
			<td style="width:179px;">Drummond&#160;</td>
			<td style="width:110px;">#2-000-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calibrated 1-5 &#181;L Capillary Tubes</td>
			<td style="width:179px;">Drummond</td>
			<td style="width:110px;">#2-000-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:366px;">Dispolable Spatulas</td>
			<td style="width:179px;">VWR International</td>
			<td>#80081-188</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HMA-50S &#160;50W Aquatic Heaters</td>
			<td style="width:179px;">Finnex</td>
			<td style="width:110px;">HMA-50S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:366px;">P1000 Pipette</td>
			<td style="width:179px;">Eppendorf</td>
			<td>#3123000063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:366px;">P1000 Pipette Tips</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:110px;">#2079E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sanyo MIR-554 incubator&#160;</td>
			<td style="width:179px;">Panasonic Health Care</td>
			<td style="width:110px;">MIR-554-PA</td>
			<td></td>
		</tr>
		<tr height="218">
			<td height="218" style="height:218px;width:366px;">Sperm Extender E400</td>
			<td style="width:179px;"></td>
			<td style="width:110px;"></td>
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:366px;">Sponge Animal Holder</td>
			<td style="width:179px;"></td>
			<td style="width:110px;"></td>
			<td>Made from scrap foam</td>
		</tr>
		<tr height="374">
			<td height="374" style="height:375px;width:366px;">System Water</td>
			<td style="width:179px;"></td>
			<td style="width:110px;"></td>
			<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>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:366px;">Tissue Wipes</td>
			<td style="width:179px;">Kimberly-Clark Professional</td>
			<td>#21905-026</td>
			<td></td>
		</tr>
		<tr height="245">
			<td height="245" style="height:245px;width:366px;">ZIRC E2 Embryo Media</td>
			<td style="width:179px;"></td>
			<td style="width:110px;"></td>
			<td style="width:175px;">15 mM NaCl, 0.5 mM KCl, 1.0 mM MgSO4, 150 &#181;M KH2PO4, 50 &#181;M Na2HPO4, 
			1.0 mM CaCl2, 0.7 mM NaHCO3. 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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8.7K 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

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 ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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 ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

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 ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...