We have developed an "all fish" growth hormone (GH) chimeric gene construct by using an antifreeze protein gene (AFP) promoter from ocean pout linked to a chinook salmon GH cDNA clone. After microinjection into fertilized, nonactivated Atlantic salmon eggs via the micropyle, transgenic Atlantic salmon were generated. The presence of the transgene was detected by polymerase chain reaction (PCR) using specific oligonucleotide primers. A number of these transgenic fish showed dramatic increases in their growth rate. At one year old, the average increase of the transgenic fish was 2 to 6 fold and the largest transgenic fish was 13 times that of the average non-transgenic control.

Over the past 20 years we have generated stable lines of transgenic Atlantic salmon possessing either antifreeze protein (AFP) genes or a salmon growth hormone (GH) gene construct. The AFP gene transfer studies were initiated in 1982. The AFP transgene integrated into salmon genomic DNA and AFP has been found in the blood of all 5 generations to date. However, AFP levels are low and a means to raise these levels needs to be developed. Our GH gene transfer studies were initiated in 1989. Evidence to date indicates that a single copy of the GH transgene integrated into chromosomal DNA and has been passed down in Mendelian fashion, along with its rapid growth phenotype, over 6 generations. Laboratory studies indicate that our GH transgene enhances growth rates with Atlantic salmon reaching market size (4–6 kg) a year earlier than non-transgenics cultured commercially in Atlantic Canada. This GH gene transfer technology was patented and licensed to Aqua Bounty Farms Inc., and the transgenic salmon are currently under review by various government regulatory authorities in the USA and Canada for use in commercial aquaculture ventures. Our experience with the regulatory authorities, the industry and the press indicates that the successful introduction of transgenic salmon into the aquaculture industry involves issues concerning not only science but also food safety, environmental safety, animal welfare and consumer acceptance. This communication centres on our experience with Atlantic salmon and outlines our plans and progress towards demonstrating the safety of transgenic fish to the consumer and to the environment.

The influence of water temperature and photoperiod on the timing of the annual cycle of plasma antifreeze glycoproteins (AFGP) was examined in Atlantic cod. Long day lengths (18 h) or continuous light had no effect on the time of appearance or disappearance of AFGP from the plasma. Cold water (0 °C) advanced the time of AFGP appearance by as much as 100 days. Long day lengths had no effect on this early induction of AFGP production. AFGP was not detectable in the plasma of fish exposed to water temperatures greater than 1 °C. Although small amounts of AFGP did appear in the plasma of cod exposed to 1 °C, it immediately began to disappear while plasma levels in normal and 0 °C acclimated cod continued to rise. The biological half time of AFGP activity was very sensitive to temperature, ranging from 15.6 days at 5 °C to 99.4 days at 0 °C. The results of this study suggest that the appearance of AFGP in cod during the winter months is dependent on the cod's exposure to water temperatures at least as low as 1 °C. Although 1 °C appears to be capable of initiating production of AFGP, it is not low enough to allow normal protective levels to be built up in the plasma.

Atlantic salmon (Salmo salar) freeze to death if they come into contact with ice at water temperatures below −0.7 °C. Consequently, sea-pen culture of this species in cold water is severely limited. Winter flounder (Pseudopleuronectes americanus) survive in ice-laden seawater by producing a set of antifreeze polypeptides (AFP). We are attempting to make the Atlantic salmon more freeze resistant by transferring antifreeze protein genes from the winter flounder to the genome of the salmon. Salmon eggs were microinjected with linearized DNA after fertilization. Individual fingerlings (1–2 g) were analyzed for flounder AFP genes by genomic Southern blotting. DNA from 2 out of 30 fingerlings showed hybridization to the flounder DNA probe. Hybridization bands following cleavage by restriction enzymes Sst l and Bam HI were identical to those of the injected DNA. Hybridization following Hind III digestion indicated that the flounder AFP gene was linked to the salmon genome. These hybridization signals were absent in the DNA from control fish. The intensity of the hybridization signals indicated that there was on average at least one copy of the AFP gene present per cell.

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