Optimization of transfer conditions in rainbow trout recipient embryo by zona radiata removal and determination of embryonic stage effect
Optimization of the donor embryonic stage and environmental condition for successful transplantation.

Embryonic stem cells were successfully transplanted in mammals resulting in the production of interspecies chimeras. If this technique becomes successful in fish, it will open vast opportunities for gene transfer methods and recovery of entire genomes from extinct species, strains, if embryonic cells are cryopreserved. The improvement of the efficiency of embryonic cell transfer can accelerate progress in transgenic research with aquatic animals. Transgenic methods can be incorporated as a way to increase growth, fertility, or diseases resistance in fish of industrial importance.

This study was conducted on germplasm (spermatozoa, oocytes, fertilized eggs) from rainbow trout. Two color morphs, golden and regular genotypes were used in order to evaluate mosaicism in successful chimeras. The fertilized eggs at different developmental stages were dechorionated (zona radiata removal) chemically (Collodi et al. 1992) or manually (Nilsson and Cloud, 1993). Embryonic cells were dissected from the yolk and dissociated (Varadi and Horvath, 1997). Two methods were investigated to transfer the donor cell(s) into recipient embryo: microinjection and aggregation of the donor cells into dechorionated embryo.

Facilities: Principal investigator (PI) has extensive knowledge in controlled fish reproduction and gametes quality in salmonids. The PI already took training course (July 1999) in Woods Hole, M.B.L. "Microinjection Techniques in Cell Biology" and consequently is familiar with equipment required and pitfalls encountered with using transplantation methods. The laboratory of the PI has basic equipment for fish reproduction and gamete storage, however, instruments needed for micromanipulation and microinjection are missing. Therefore, budget request includes supply expenses for microinjectors M-16 ($1,250) for precise injections and IM-50B ($1050) for holding oocyte on a holding pipette. Additionally coarse manipulator M-152 ($745) is requested. All instruments are produced by Narashige International USA, Inc., East Meadow, NY.

We developed relevant procedures for: (1) chilled storage of salmonid gametes and embryos at the earliest developmental stages; and (2) isolation and transplantation of entire blastulas in salmonids.

(1) Chilled storage of salmonid gametes and embryos at the earliest developmental stages. Embryonic cells at the blastula stage are the most suitable for transplantation experiments, as they are small, pluripotent, and the blastula can be easily separated from a periblast. Cells at the earlier developmental stages are large (50 micrometer), thus are sensitive to manipulations, while late blastula-stage cells are no longer pluripotent. In rainbow trout, embryos at developmental stage 6-7 (Ballard 1973) are the most suitable for transplantation experiments (Nilsson and Cloud 1992). Depending on incubation temperature, embryos at this developmental stage are available for up to 1 day. Therefore, we developed procedures for chilled storage of gametes and fertilized eggs of salmonids to prolong the availability of cells at the blastula stage. We found that 4-day storage of the gametes insignificantly affected embryo viability. Chilled storage of fertilized eggs at 2°C, followed by a transfer to the hatchery water (10-14°C), prolonged the availability of the blastula stage embryos up to 7 days. The transfer of embryos from lower to higher incubation temperature did not cause any visible malformations in hatched embryos and swim-up larvae. We found that the chilled storage of fertilized eggs is a simple, useful technique for obtaining the prolonged access to blastula-stage embryos in salmonids.

(2) Isolation and transplantation of entire blastulas in salmonids. The existing methods for embryonic cell transplantations in fish consist of isolation and dispersion of donor cells and microinjection of those cells into recipient embryo (Nilsson and Cloud 1992; Joly et al. 1999). No method was reported for transfer of an entire blastula into the host egg. The reason is that because the size of the blastula is too large (in salmonids it has a diameter of more than 1 mm) to use the microinjection technique. To overcome this problem, we developed a novel method of isolation and transplantation of entire blastulas. We introduced a surgical method to cut out a large opening in the salmonid chorion enabling direct access to the entire blastula. Survival of embryos having such openings in their chorions was comparably high as in controls at hatching (60-90%). Entire blastulas were easily separated from periblast and transplanted into recipient eggs. We replaced the entire blastulas of rainbow trout and cutthroat trout. They could integrate into the egg of other species and develop until the stage of one-third of epiboly. This is a very promising indication that the transplantation of entire blastulas is possible.

This research also was supported by Fulbright Scholar Program, U.S. Department of State, Washington, DC.