Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus, also known as the blind Mexican cave fish, is emerging as a model organism for a variety of research fields in biological science, specifically in understanding the evolution of adaptive genetic traits. This species consists of a river-dwelling surface morph and a cave-dwelling morph allowing us to analyze how two populations of the same species diverged over time. However, in order for this research to take place we must be able to obtain healthy time-matched embryos and hybrid outcrosses.

While this species is easily maintained and bred in the lab the fish primarily spawn during night hours making it challenging to obtain freshly laid embryos during the daytime. In this protocol we show how by acclimatizing Astyanax mexicanus to altered light cycles and temperature changes we are able to shift breeding cycles to a more convenient time of day. Next, we show how to identify suitable parent fish and collect gametes from anesthetized males and females.

Finally, we demonstrate how to produce viable offspring using in vitro fertilization and discuss signs of successful fertilization. We expect this protocol to enable procedures such as injection of genetic constructs and developmental analysis to be easily conducted during normal working hours. In addition this technique can be useful for creating hybrid outcrosses between all cave and surface-dwelling populations.

Set up fish tanks within an opaque, fully enclosed flow-through aquaculture system containing multiple rows of tanks. Maintain the temperature of each tank with an independent heating element that is used to manually alter the temperature during the priming process. 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. The automated controller can enable manipulation of all photoperiods with the least disturbance to the fish. Equip the rack with a red work light and blackout curtains for access during dark hours.

Remove desired fish from general system racks and place in breeding racks to allow for adjustment of the photoperiod 14 days prior to priming. This allows the fish to acclimatize to a new environment. Keep the fish at 22.8 degrees Celsius during this period using the installed aquatic heating system.

The normal photoperiod is six a.m. to eight p. m light, and eight p.m.

to six a.m.dark. For 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.

Once the fish are acclimatized start priming the animals for spawning. This is a procedure that takes six days in total. Over this time change the temperature to prime the ova production using an installed aquatic heating system.

On day one raise the temperature from 22.8 degrees Celsius to 24.4 degrees Celsius. On day two raise the temperature from 24.4 degrees Celsius to 26.1 degrees Celsius. On day three and four keep the temperature at 26.1 degrees Celsius.

Fish will be ready to spawn during the day and IVF can be performed. On day five lower the temperature from 26.1 degrees Celsius to 24.4 degrees Celsius. On day six lower the temperature from 24.4 degrees Celsius to 22.8 degrees Celsius.

Provide a seven-day gap before repeating this temperature cycle. We recommend continuing to keep the fish in this photoperiod since this will reduce the overall time needed by the fish to adjust to the shifted light cycle. 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.

Next choose a female for collection. Gravid fish with large protruding abdomens will likely be the best choice for this procedure. Immobilize a female using chilled water and place her in supine position in a moistened sponge animal holder.

Once positioned blot the ventral side of the fish with a delicate tissue wipe as contact with water will cause the ova to activate. Hold the female between thumb and index finger. Gently squeeze against the lateral sides of the coelomic cavity in the direction of the urogenital opening while rolling the finger slightly.

Collect the expressed ova using a disposable spatula. Transfer these over to the humidified Petri dish. Several clutches of ova may be combined in the same dish if specific parentage data is not needed.

After collection gently return the fish to a recovery tank filled with system water. Choose a male for collection. There are no outwardly visible signs of male gamete quality.

However, fish should appear healthy in appearance before use in this procedure. Immobilize a male using chilled water and place him in supine position in a moistened sponge animal holder. Blot the ventral side of the fish with a delicate tissue wipe as contact with water will activate the milt.

Gently place the end of a capillary tube at the urogenital opening. 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.

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.

Dispense the milt into an empty 1.5-milliliter centrifuge tube and dilute with twice the volume of sperm extender E400. Keep on ice. 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 is not required for immediate fertilization. After collection gently return the fish to a recovery tank filled with system water. Using a new pipette for each stock mix the sperm by pipetting and/or agitating the sides of the tube before fertilizing as sperm and milt and settle in E400 solution over time.

Dispense the milt or extended milt solution into the freshly collected ova. Quickly add one milliliter of system water to the clutch to activate the sperm and eggs for fertilization. Avoid mixing or agitating the dish contents and allow two minutes for fertilization to occur.

Add E2 embryo media to fill the dish 2/3 full. Depending on the subsequent procedure embryos can either be used right away for downstream applications or they can be incubated in E2 embryo media at 23 degrees Celsius until they reach five days post-fertilization. At this point we transfer embryos to the main recirculating housing system using system water.

The major strength of this method is the reliable production of cave and surface hybrids of Astyanax mexicanus. Conventional methods of hybrid production are very challenging due to the loss of circadian rhythm in the cave morphotype of Astyanax mexicanus, which results in altered spawning times of these fish. For a successful IVF procedure in Astyanax mexicanus the quality of the collected ova is of major importance.

Gravid female fish with large protruding abdomens are most likely to release viable ova, which appear clear and even in appearance. Adding the collected milt to such ova results in the development of fertilized embryos usually within 20 to 30 minutes. Viable embryos will become slightly more translucent before entering the one cell stage of the developmental cycle.

Unfertilized ova will appear more uneven and opaque. Using IVF to generate hybrids of tenaja cavefish and surface fish one can examine a broad range of morphological phenotypes. For example, surface/cave hybrids have eyes indicating the presence of eyes is a dominant trait.

In surface cave F2 hybrids however, we obtain a broad range of eye sizes indicating that there are multiple loci that control eye size in Astyanax mexicanus making it a quantitative trait. Another example is pigmentation. Observing the F1 hybrid of surface and cavefish, it can be concluded that body pigmentation is a dominant trait as the fish are fully pigmented.

In the F2 generation the variation in body pigmentation again points towards a quantitative trait. Combination of this phenotypic data with sequencing data can reveal underlying genetic loci responsible for these phenotypes. In this protocol we learned how to shift the breeding cycles of Astyanax mexicanus to more convenient hours of the day, and to obtain healthy viable embryos using in vitro fertilization.

In addition to allowing work with early-stage embryos it also enables the future exploration of sperm cryopreservation. Standardization of this technique strengthens Astyanax mexicanus as a model system for early developmental and genetic studies.