The Zebrafish Science Monitor Vol 3(5)
DELAYED IN VITRO FERTILIZATION OF ZEBRAFISH EGGS USING COHO SALMON (ONCHORHYNCHUS KISUTCH) OVARIAN FLUIDBy G.E. Corley-Smith, C.J. Lim, and B.P. Brandhorst, Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., V5A 1S6, CANADA
Many experiments that involve in vitro fertilization can be facilitated by being able to delay fertilization of zebrafish eggs for periods up to a few hours after they are extruded from the female. For example, when injecting 1-4 cell embryos, the injections could be done in batches that were fertilized at progressively later times. Delayed fertilization is also required if eggs must be manipulated prior to fertilization; e.g. for microinjection, nuclear transplantation, or irradiation to produce haploid or diploid androgenotes.
We have held zebrafish eggs for over 6 hours after extrusion from the female; when subsequently fertilized, they developed into apparently normal zebrafish. Recently we held 76 zebrafish eggs for 50 minutes between the time the eggs were extruded from the female and when milt was added to fertilize the eggs. In this group, 72% were fertilized and developed as normal looking embryos which hatched. The remaining 28%, observed at 24 hours, showed no embryonic development and appeared unfertilized. The technique does not appear to lead to an increased incidence of abnormal development or decreased fertility of adults. Fish derived from eggs that were held for times exceeding 1.5 hours, have matured and been used for breeding in our lab.
The technique uses the natural body fluid that surrounds mature coho salmon eggs, ovarian fluid. We have successfully used coho salmon and rainbow trout (O. mykiss) ovarian fluid. Our batches of coho ovarian fluid collected in 1991 are more effective than our batches of rainbow trout ovarian fluid. However, Pat Gibbs (Gibbs et al., 1994) has used rainbow trout ovarian fluid successfully. We suspect that ovarian fluid from any fish species that holds eggs in a mature state for long periods is likely to be effective. Thus, ovarian fluid from any salmonid species is likely to work, although we expect the most effective ovarian fluid to be from species that undergo prolonged upstream migrations to their spawning streams and consequently probably hold their eggs longer.
When coho ovarian fluid containing zebrafish eggs is diluted with water, the chorions of the eggs rise, and cytoplasmic streaming that leads to formation of the blastodisks is initiated. Although the blastodisk forms, no cell divisions take place. Thus, egg activation without insemination is initiated, and the egg can no longer be fertilized. Very little dilution of coho ovarian fluid is required for initiation of activation. Increasing the proportion of water added, up to a plateau, increases the rate of chorion elevation.
Quality of Ovarian Fluid
Care should be exercised in collecting ovarian fluid. For salmon, if a few eggs are ruptured in a batch of eggs, the subsequent fertilization rate of the non-ruptured eggs is substantially reduced. Contamination of salmon eggs and ovarian fluid with blood reduces their subsequent fertilization rate. Furthermore, dilution of ovarian fluid with water leads to activation of eggs. Thus, we believe the ovarian fluid must be collected in such a manner as to reduce contamination by broken salmon eggs, blood and water.
The female fish from which ovarian fluid is collected should be mature and ready to deliver eggs but should not be overly ripe with broken eggs and watery ovarian fluid. Ovarian fluid from non-ripe females may be useable, but the volume of ovarian fluid will be less.
We normally collect ovarian fluid at a salmon hatchery. We separate some ovarian fluid from the salmon eggs and turn the salmon eggs over to the hatchery staff for fertilization, which is not impaired. Approximately 50 mls of ovarian fluid can be collected per coho salmon. One good batch of 50 mls is sufficient for holding the eggs from over 1,000 zebrafish females.
As batches of ovarian fluid (ovarian fluid from one fish) vary in their effectiveness for holding zebrafish eggs, we test individual batches of ovarian fluid. When a batch is identified that allows for holding zebrafish eggs for 1.5 hours with high subsequent fertilization rates, we then aliquot this batch into 1.5 ml screw cap microcentrifuge tubes and freeze at -20C or -80C. Coho salmon ovarian fluid is robust and can undergo repeated freezing and thawing and can be left for several days at room temperature and still function. However, for optimal fertilization rates we suggest more careful storage.
Collection of Ovarian Fluid
Coho salmon die shortly after spawning; thus, eggs are normally collected at hatcheries immediately following lethal cranial trauma. The euthanized female coho salmon is immediately hung by its tail and all the gills on both sides of the had are slit to drain blood. Pushing a wad of paper towels under the operculum will help soak up blood and impede coagulation. After 5 minutes, dry the fish with a towel so that no water can drip into eggs and ovarian fluid when they are collected. Have an assistant hold the fish by its head and tail, with its belly downward over a clean dry bowl, and slit the fish from anus to from of the body cavity. Eggs and ovarian fluid will fall into the bowl. Release eggs from skein (ovarian connective tissue) and remove pieces of skein from bowl containing free eggs and ovarian fluid. Remove 75% of ovarian fluid to 50 ml conical plastic tube. A non-abrasive kitchen strainer or colander, stainless steel or plastic, is useful to separate eggs from ovarian fluid. Handle eggs gently at all times to prevent breakage. Give the salmonid eggs to hatchery staff and put the ovarian fluid on ice. Back at the lab, centrifuge the tubes of ovarian fluid at 5500 g for 5 minutes at 4C to sediment cellular debris. Remove the supernatant and freeze it.
Use of Ovarian Fluid for In Vitro Fertilization
This technique is similar to normal in vitro fertilization as outlined by Charline Walker and George Streisinger in Eugene's The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). We collect eggs with silanized capillary tubes (Kimax-51, Kimble Products Art. No. 34502, ID 0.8-1.1mm, length 100mm) that we pre-wet with ovarian fluid. When eggs are collected, they are placed into 100ul of salmonid ovarian fluid in a small petri dish. The actual volume of ovarian fluid is adjusted to the number of eggs. All eggs must be covered with ovarian fluid. We get better subsequent fertilization rates when we hold the eggs at room temperature (18-22C) than when we hold them chilled at approximately 4C. Immediately before fertilization, we remove most of the ovarian fluid from the zebrafish eggs with a Pipetman, centrifuge it at 5500g for 5 min at 4C to sediment cellular debris, and then remove the supernatant which can be frozen for reuse. Following removal of ovarian fluid, eggs are fertilized as normally done for in vitro fertilization.
A step by step protocol for using coho ovarian fluid for delayed in vitro fertilization is expected to be included in an update to The Zebrafish Book which is scheduled to be issued later this year.
Acknowledgements
We gratefully acknowledge Pat Gibbs and Charline Walker. This technique was refined based on friendly advice received from both. Also, they were both instrumental in infecting us with their enthusiasm for zebrafish research, and teaching us the basics of running a zebrafish research facility.
Zebrafish Science Monitor Vol 3(5)
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