The Conservation of Atlantic Salmon in Scotland Through Gene Banking: Principles and Considerations

Supplementation/restoration using cryopreserved material

As with live gene banking, the aims of cryobanking are to conserve the maximum amount of existing genetic diversity within a population with the eventual goal of supplementation of and/or restoring wild populations. As such, there are numerous circumstances where cryobanking might be employed, and so it is vital that bespoke restocking plans are developed for each situation. These scenarios range from where a population has entirely disappeared from the wild and where restoration is required, to one in which the wild population is still viable, but cryobanking is being employed as a safety mechanism in the face of unknown future pressures. In each case, a detailed plan is required which, although bespoke, will have to include a number of analogous factors. These are described below, however this list may not be exhaustive as, while there is a growing body of literature on the use of gene banking to preserve material and the development of technical protocols to achieve this (see Martínez-Páramo et al. 2017, Diwan et al. 2020, Mooney et al. 2023), there is much less focusing on the reestablishment of the wild populations after stressors have been removed. An illustrative example of a restoration programme is outlined in more detail in Appendix 1.

When to restore?

An interplay of factors need to be taken into consideration when deciding when to restock using cryopreserved material. These will include the status of the wild population, the status of the external stressor/s, and the goals of the conservation programme. In the case of programmes where the aim is to act as a repository of material, stocking may not be required in the short or medium term, and this may also be the case if the wild population is still relatively healthy as all stocking, especially into a ‘healthy’ wild population brings risks (Gilbey et al. 2023). In the case where external stressors are strong and continuing, stocking may have to be delayed until they can be addressed. However, it may also be the case in such situations that stocking may still be performed in order to maintain some sort of live ‘wild’ population even in the face of pressures and/or high mortalities. Cryopreservation may also be used over a short timeframe with fertilisation and conservation stocking being performed even the same year of collection. This would be the case in programmes such as that seen with the Adriatic grayling (Thymallus thymallus) which is threatened by introgressive hybridization with non-native stocks, and where sperm is collected from potential broodstock, fin clips of the individuals genetically screened, and only sperm from native fish used for fertilisation and subsequent stocking (Horváth et al. 2012).

Number and ages of fish to replenish

Once it has been decided that stocking is to be carried out the question of how many and what ages will have to be addressed. The various factors to be taken into consideration in such a case are similar to those in any stocking programme using fish from regular hatcheries, or fish produced from cryopreserved sperm. Such factors will include the number of populations under consideration, the benchmark size of the wild population (i.e. how many would be expected in the absence of stressors), the remaining wild spawned individuals remaining, and the status of stressors acting on the population/s.

The crossing scheme

Perhaps the biggest difference between traditional hatchery maintenance of stocks and cryopreservation is the availability of a large repository of genetic material for a particular stock. If the correct collection SOP is being followed, a representative sample of the entire genetic diversity of the stock will have been collected, and this may also be enhanced by multiple collections spanning potentially many years or even decades. As such, the eventual use of this material for producing fish to be stocked out becomes a very complex process if the aim of the maintenance of the wild genetic diversity, and the avoidance of negative effects of inbreeding, are to be achieved. In order to achieve these aims, it is thus vital that a detailed bespoke crossing scheme is developed for each situation. Such a scheme will have to include a number of factors which are outlined below, and be shown to be statistically valid and capable of achieving the objectives.

Number of females

The number of females to include in any crossing scheme will depend on both the availability of females, the size of the stock in question, and the aims of the restocking programme (full replenishment of an extirpated population, supplementation of existing populations etc.). Further, the numbers should be chosen such that sufficient are utilised to avoid inbreeding depression. This would be achieved by genetically screening all fish and producing a crossing scheme taking into consideration relatedness and individual diversity (see below).

Female origins

Cryopreservation will provide sperm to a stocking programme and as such females will be required for egg production. It will obviously be favourable to have females native to the stock in question for this role. However, if the population is extirpated and/or the numbers are very low females from outwith the population will have to be used. Again a detailed multi-generational crossing scheme will be required to ensure the optimal removal of any non-native genetic material.

In the scenario where all native females are lost, ova will be sourced from females from the nearest available wild stocks that are most similar in habitat use and life-history composition. However, considering that there is likely to be spatial coherence in impacts on neighbouring populations, it may be difficult to remove adult females from similarly negatively impacted populations. In such a case, rather than removing adult female spawners to act as broodstock, juveniles and/or smolts could be captured and reared to maturity in a hatchery situation. All females (captured and/or reared) would then be retained in a hatchery until stripping, and as with males, genetically screened to identify farmed escapees, relatedness, and to determine their genomic utility in crossing schemes.

Number of male donors per female

In the same way as in the wild, and in a live hatchery situation, sperm from a number of males should be used to fertilise the eggs from a single female. The crossing scheme will define this

Number of females fertilised by each male

In the wild a male may be involved in more than one female fertilisation event and so again the crossing scheme should define how many batches of eggs from different females are use with each male.

Male genomic utility

Each sampled male will have a different genomic makeup. Genomic screening will allow this to be quantified across all samples held and for each male individually. Any crossing scheme should take the genomic characteristics of each male into account and be developed such that the maximum levels of diversity can be obtained using the optimal number of males. As each male will differ in their utility for such a crossing scheme, some having high utility and some low, differing fertilisation events will have to be incorporated into the crossing design with individual males identified and utilised in the optimal way.

Life history genotypes/phenotypes

Salmon have a number of life history phenotypes which need to be taken into consideration when stocking. Smolt age and growth, sea age, and male maturation have all, to a greater or lesser extent, an underlying genetic basis. As such it is vital that the diversity of these traits are maintained in any restocking programme. This will again require a bespoke crossing design which takes into consideration both phenotypic trait values (recorded when fish are sampled) and genetic trait characteristics defined by genetic screening at known genes associated with the various traits.

Relatedness

Of overriding importance to maintain the genetic diversity of a population is to ensure that closely related individuals are not crossed together. Such crossings will result in inbreeding depression and lower both overall genetic diversity in the stock and associated fitness of the population. It is thus paramount that the crossing design takes into consideration the relatedness of all fish used, both males from the cryobank, and the live females. This can only be done through genetic screening and the development of a crossing design utilising relatedness information to inform optimal crosses.

The above considerations are by no means an exhaustive list. It is of paramount importance that a crossing scheme is drawn up that maximises the genetic diversity within the reconstructed individuals/population, whilst at the same time addressing the differing life history genotypes/phenotypes within the stock. This is not a straightforward process, especially considering the various life history types within a salmon population, and so, whilst there are a considerable amount of online resources (e.g. Caballero et al. 2010, Judycka et al. 2019, Bøe et al. 2021, Boes et al. 2023), it is vital that a supplementation scheme is designed by fully qualified personnel with personal with expertise in quantitative genetics and crossing designs.

Time of operation

Apart from limited situations (for example where sperm is frozen while screening is performed to choose native fish and used the same year) it is likely that a cryobank will be required to operate for a number of generations (where each salmon generation is ~3 – 5 years). To replenish an extirpated population will mean many generations of inputs as non-native females will have to be used. Even with native females available, with stressors still active, many and/or continuous generations of fish will be required to be stocked. The advantage of a cryobank, however, is the storage of samples for an almost indefinite amount of time, and so there is no technical reasons why these requirements might not be met (although there is of course issues around available resources).