Sterility conference
Scientists from around the world share research findings
PRESSURE to farm fish that are reproductively incompetent comes from both within and out-with the aquaculture industry but for rather different reasons in each case.
Fish farmers are well aware that maturation and gonadal development are always achieved at the expense of somatic growth and, caviar producers excepted, this represents a bottom line cost to the business.
Anglers have similar concerns but for different reasons, reproductive competence in farmed stocks being perceived as a threat to wild ones should the former escape and gain access to them.
And so for over 30 years in the salmonid farming sector, the use of stocks which have been rendered sterile, usually through triploidisation, has been an accepted and well understood practice, mainly in trout but also in some salmon farming operations.
In the case of the latter, the propensity to produce and farm triploids came into, and out of, fashion in the early ‘90s due largely to some spectacularly poor farming outcomes.
While the use of triploid salmon finished at this time, the requirement for sterile fish didn’t. Because of this, research into sterile production in general, and triploidy in particular, resumed about 10 years ago with, in Scotland, the inception of the EU FP7 funded SALMOTRIP project from 2008 to 2011, which was subsequently followed by the Innovate UK funded Salmotrip+ project running from 2014 to 2018.
The requirement to disseminate the outcomes and findings of the project spawned the idea of a workshop as a useful way to achieve this and also include other technologies being developed to create sterile fish.
And so, on March 25 and 26, ARCH-UK, in collaboration with the University of Stirling, put together the International Finfish Sterility Workshop which attracted scientists and aquaculture practitioners from Norway, North America and the UK.
Following his welcome and introduction, Hervé Migaud of the Institue of Aquaculture went on to describe Salmotrip+, set up to establish ‘how to farm triploid salmon optimally’. It was hoped that a number of Key Performance Determinants (KPDs) would emerge which would form the basis of a ‘best practice’ guide for successfully culturing these fish; and this has indeed been achieved.
Perhaps the greatest differences that determine successful outcomes for triploid salmon compared to diploid ones come in the early part of the production cycle. In Tasmania, where triploid salmon are used instead of lights to combat the early maturation of diploid fish, egg incubation temperatures are held at 6°C to optimise outcomes.
Such outcomes can include the level of a variety of malformations of jaw, heart and vertebra, to which triploids appear to have a lower threshold of induction that diploids. But low incubation temperature successfully mitigates these without a concomitant reduction in performance; as Tom Fraser from IMR in Norway explained, triploids will actually out compete diploids at these temperatures.
In addition to incubation temperature, Mikey Clarkson, in pursuit of his PhD thesis at Stirling University, warned against performing triploidisation on eggs which are approaching over-ripeness. While never ideal, triploids will display a much greater variation in performance outcomes when compared with diploid eggs from a late stripping.
His findings were confirmed by Renee Contregas from Petuna Aquaculture in Tasmania, a production company that now only triploidise eggs from early hens (in their short stripping season) rather than the later ones.
Another KPD for triploids is appropriate nutrition. John Taylor’s work at the University of Stirling in cooperation with Salmotrip+ partner, BioMar, revealed that phosphorous must be included in the diet at higher levels for young triploid salmon compared with diploids- up from typically 13g/kg to over 19g/kg.
Higher inclusion in diets though is not the only way to achieve higher levels in fish. Probiotics could be deployed to improve both uptake and assimilation at existing dietary inclusion rates. Less than optimal levels in the early stages of triploids will pre-dispose them to an elevated risk of developing both jaw and trunk malformations following transfer to seawater.
The Salmotrip+ project invested a great deal of effort in determining whether routine onfarm health operations can be conducted on triploid stocks using accepted practices.
Vaccination is a key consideration here and much of Lynn Chalmers’ PhD focused on this aspect of husbandry as well as the use of therapeutants. In the case of the former, triploid stocks were not shown to be any more prone than diploids to developing side effects following intraperitoneal inoculation with a variety of oil adjuvanted vaccines provided by project partner Pharmaq. Efficacy, as measured both by survival as well antibody as response, was also found to be unaffected by ploidy.
Lynn went on to demonstrate that above 12°C triploid salmon can suffer higher mortalities when treated with H2O2 in seawater, confirming the accepted wisdom that these fish are less temperature tolerant than diploids. This is suggestive of a higher risk borne by these fish when subjected to routine bath treatments, particularly at higher temperatures.
A possible explanation for this temperature mediated sensitivity came from Tillman Benfey, of the University of New Brunswick, who demonstrated that triploids do not have the same aerobic scope as their diploid peers. This means that the difference between their
“Perhaps the greatest differences that determine successful outcomes come in the early part of the production cycle”
baseline and maximum level of metabolic rate is smaller than in diploids.
Thus if some of their aerobic capacity is required for, say, digestion, the balance which remains for coping with other stressors, applied concurrently, is less than for a diploid in the same circumstances. The greater the stressor the more evident and problematic this becomes.
Richard Hopewell of Dawnfresh, who spoke from the rare perspective of farming experience with both Atlantic salmon and rainbow trout, suggested that triploid trout are quite similar in their ‘farming temperament’ to diploid salmon, that is, less hardy than diploid trout but more so than triploid salmon.
Of course, triploidisation is not the only way to render fish sterile. A
number of new technologies are under investigation including gene silencing, a technique explained by Helge Tveiten of Nofima.
By silencing the so-called Vasa gene, which is crucial for germ cell development, offspring can be produced which are incapable of developing functional gonadal tissue. Research is underway to ‘vaccinate’ a female parent with a factor that will target her oocytes and cause them to develop into reproductively incompetent, but otherwise normal, offspring.
Another approach being investigated is to target the cells that are the precursors to gonadal tissue – the Primordial Germ Cells (PGCs). Yonathan Zohar from the University of Maryland and Anna Wargelius from the Institute of Marine Research in Norway suggested that these, too, could be knocked out, leading to sterility in their host.
The original drivers for the development of sterile farmed fish, including reproductive incompetence as a back-up to containment, and improved somatic performance, remain, but a new one has recently emerged.
Debbie Plouffe, of the Centre for Aquatic Technologies in Prince Edward Island, said that for AquaBounty’s ‘AquAdvantage’ salmon to be allowed to be reared in commercial conditions they must be sterile, according to the FDA which regulates these fish in a similar way to medicines!
Currently, triploidisation (in excess of 95 per cent) is deemed acceptable alongside total containment; however, PGC knock out is also being mooted as an alternative technology with sterility being linked to a pigmentation construct, such that sterile animals can be easily identified by eye.
So, the requirement for a simple and reliable method of producing sterile fish for aquaculture remains highly relevant across species, including tilapia, as explained by Yehwa Jin of the University of Stirling who is researching the use of CRISPR gene editing technology to induce sterility in Oreochromis niloticus.
And, as the conference demonstrated, the development of these techniques goes hand-in-hand with developing an understanding of how to optimally farm the fish which result. Chris Mitchell is sales manager of Pharmaq.
“Chalmers confirmed the accepted wisdom that these fish are less temperature tolerant diploids” than