DETERMINATION OF PLOIDY FROM EYED ATLANTIC SALMON EGGS USING FLOW CYTOMETRY AND VALIDATION OF A PROCEDURE FOR THE PRODUCTION OF TRIPLOID AQUADVANTAGE SALMON.

Debbie A. Plouffe*, Charmaine A. Milligan, Dawn Runighan, Jonathan R. Veinot, and John T. Buchanan.

Aqua Bounty Canada Inc.
Prince Edward Island, Canada
dplouffe@aquabounty.com
The AquAdvantage Salmon product is defined as: A triploid Atlantic salmon (Salmo salar) bearing a single copy of the stably integrated -form of the opAFP-GHc2 gene construct at the -locus in the EO-1line. Ploidy of the salmon is routinely determined by flow cytometric analysis of propidium iodide-stained erythrocytes. For the large-scale production of triploid salmon it would be advantageous to have the ability to assess ploidy as early in development as possible. To this end, a series of studies were conducted to develop a procedure for flow cytometric determination of ploidy from fertilized egg samples, and to employ the procedure to validate the conditions used to generate triploid AquAdvantage Salmon.

To evaluate ploidy at the earliest possible stage of development, a method was developed in which homogenates from fertilized eggs could be used for analysis by flow cytometry. Fertilized eggs (diploid and triploid full siblings) collected from three separate crosses were homogenized and the cell suspensions stained using propidium iodide at 150, 250, and 350 degree days post-fertilization. In addition, individual fertilized eggs (degree days) were homogenized and the resulting cell suspensions were pooled in groups of ten (nine triploid and one diploid egg) in order to validate a high-throughput method for analysis of pooled samples of mixed ploidy. It was established that for conclusive analysis of ploidy from Atlantic salmon eggs, the eggs should be well-eyed, approximately 300-350 degree days post-fertilization. At this stage a single diploid eyed-egg can be reliably detected in a group of nine triploid eyed-eggs.

Following the development of the method for the evaluation of ploidy from eyed-eggs, a study was performed to validate the conditions for the induction of triploidy by hydrostatic pressure treatment. To this end, one-to-one crosses were established with eggs from non-transgenic female Atlantic salmon and milt from AquAdvantage salmon males hemizygous for EO-1a. The fertilized eggs from each cross were apportioned volumetrically into five replicate groups: one diploid control group that was not submitted to pressure treatment; and, four treated replicates that were subjected to hydrostatic pressure shock (9500 psi for five minutes at 300 deg-min post-fertilization). Ploidy was determined for a sub-sample of 350 eyed-eggs collected from each of the treated replicates from five different crosses in order to estimate the efficiency of triploid induction from a total of twenty pressure-shocked groups.

The results of this study, and of an additional study performed to validate the use of a commercial-scale pressure chamber, demonstrate that the conditions used for the induction of triploidy result in production of batches of AquAdvantage salmon eyed-eggs that are >99% triploid.