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Oppdrett av steril laks vil kunne redusere de negative interaksjonene mellom rømt oppdrettslaks og villaks, og dessuten forbedre fiskens velferd og kvalitet. Prosjektets hovedmål har vært å evaluere viktige produksjonsegenskaper hos steril laks etter å ha fjernet de embryonale kimcellene. Inaktivering av kimcellefaktoren Deadend ga livsvarig tap av kjønnsceller hos både hann- og hunnfisk. Steril laks viste normal osmoreguleringsevne, og sjøvannstoleransen var minst like god som hos fertil laks. Steril og fertil laks viste ingen konsistente forskjeller i stressrespons og immunstatus ved kortisolmåling og qPCR-analyse av stressrelaterte og pro-og anti-inflammatoriske faktorer. Steril laks viste robusthet også i sjø tross ekstreme værforhold og avlusing etter alvorlig luseangrep. Ytre sår, finneskader og rødme i buk ble registrert hos både steril og fertil laks med ubetydelige misdannelser. Steril og fertil laks viste samme vekstforløp i ferskvann, mens sammenlikning av vekst i sjø er usikker grunnet lite antall fertile fisk. Kjønnshormonene ble aktivert hos både sterile og fertile hannfisk i ferskvann, men kjønnssteroidnivået avtok hos de sterile hannene etter sjøutsetting. Prosjektet konkluderer at inaktivering av Deadend på embryostadiet gir en steril laks med samme fenotype som fertil laks, bortsett fra fravær av kjønnsceller, og anbefaler at nødvendig teknologi for storskala-produksjon av steril oppdrettslaks bør utvikles. Sterile farmed salmon would reduce the negative interactions between escapees and wild salmon and improve fish welfare and quality. The main goal of this project has been to evaluate important production traits in sterile salmon after ablation of the primordial germ cells. Transient inactivation of the embryonic germ cell factor Deadend caused lifelong loss of gametes in both males and females. The sterile salmon showed normal osmoregulatory ability and seawater tolerance. No consistent differences were shown in stress-response and immune status by cortisol measurement and qPCR quantification of multiple stress-related and pro-and anti-inflammatory factors. Sterile salmon showed robustness in seawater despite extreme weather conditions and serious sea lice attack. External wounds, fin damages and abdominal bleedings were registered in sterile and fertile salmon with only sporadic malformations. Sterile and fertile salmon showed similar growth performance in freshwater, but the comparison in sea water is uncertain due to few fertile fish left. Sex hormone synthesis was induced in both sterile and fertile males, but sex steroid levels decreased in sterile fish after seawater transfer. The project concludes that embryonic Deadend inactivation results in sterile salmon with the same phenotype as fertile fish, except for the missing gametes, and recommends that technology should be developed for large-scale production of sterile salmon in aquaculture.

Figure S1. Boxplot of lhb and fshb expression. Figure S2. Confocal images of Fshβ histochemical labeled of European eel male pituitaries. Figure S3. Heat map and 2D hierarchical clusters of all differentially expressed genes found between the testes samples. Figure S4. Heat map and 2D hierarchical clusters of all differentially expressed genes found between the pituitary samples. Figure S5. Heat map and 2D hierarchical clusters of all differentially expressed genes found between the brain samples. Figure S6. Group distribution of differentially expressed transcripts. Figure S7. Boxplot of testes expression of selected genes. Table S1. Biometric measurements. Table S2. Enriched GO terms from the differentially expressed genes found between T10 and Tvar, in the pituitary. Table S3. Enriched GO terms from the differentially expressed genes found between T10 and Control, in the brain. (DOCX 3120 kb)

[EN] BackgroundThe impossibility of closing the life cycle of the European eel (Anguilla anguilla) in captivity troubles the future of this critically endangered species. In addition, the European eel is a highly valued and demanded resource, thus the successful closing of its life cycle would have a substantial economic and ecological impact. With the aim of obtaining the highest gamete quality, the study of the effects of environmental factors, such as temperature, on reproductive performance may prove valuable. This is especially true for the exposure to cold water, which has been reported to improve sexual development in multiple other Actinopterygii species.ResultsEuropean eel males treated with cold seawater (10 degrees C, T10) for 2weeks showed an increase in the proliferation and differentiation of spermatogonial cells until the differentiated spermatogonial type A cell stage, and elevated testosterone and 11-ketotestosterone plasma levels. Transcriptomes from the tissues of the brain-pituitary-gonad (BPG) axis of T10 samples revealed a differential gene expression profile compared to the other experimental groups, with clustering in a principal component analysis and in heat maps of all differentially expressed genes. Furthermore, a functional analysis of differentially expressed genes revealed enriched gene ontology terms involved in the regulation of circadian rhythm, histone modification, meiotic nuclear division, and others.ConclusionsCold seawater treatment had a clear effect on the activity of the BPG-axis of European eel males. In particular, our cold seawater treatment induces the synchronization and increased proliferation and differentiation of specific spermatogonial cells. In the transcriptomic results, genes related to thermoception were observed. This thermoception may have caused the observed effects through epigenetic mechanisms, since all analysed tissues further revealed differentially expressed genes involved in histone modification. The presented results support our hypothesis that a low temperature seawater treatment induces an early sexual developmental stage in European eels. This hypothesis is logical given that the average temperature experienced by eels in the early stages of their oceanic reproductive migration is highly similar to that of this cold seawater treatment. Further studies are needed to test whether a cold seawater treatment can improve the response of European eels to artificial hormonal treatment, as the results suggest., This work was subsidized by the Spanish Ministry of Science and Innovation (REPRO-TEMP project, AGL2013-41646-R), the Spanish Ministry of Innovation, Science and Universities (EELGONIA project, RTI2018-096413-B-I00), and by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), including the CR and JGHJ predoctoral contracts. JGHJ was granted a Short Term Scientific Mission by the COST Office (COST Action AQUAGAMETE: Assessing and improving the quality of aquatic animal gametes to enhance aquatic resources. The need to harmonize and standardize evolving methodologies, and improve transfer from academia to industry). The funding bodies had no role in the study, analysis, interpretation of data, or in writing the manuscript.

Accession numbers of vertebrate Buc and BucL proteins. (DOCX 17Â kb)

Primer list for real-time qPCR (F-forward, R-reverse). (DOCX 16Â kb)

Partial Mexican axolotl Buc predicted from two contigs identified in the genome by Blasting against Xenopus Velo1. Sequence alignment revealed a single base deletion introducing a premature translation stop. The partial axolotl Buc shares 52 % identity with Xenopus Velo1. (DOCX 15 kb)

Conservation of the N-terminal end in A. Buc of various vertebrates, and B. BucL (−like) of zebrafish, salmon and spotted gar. Well conserved positions are shown in bold. (DOCX 15 kb)

Chromosome localization (A) and sequence alignment (B) of buc and buc-like (bucL) genes in cow and dog. Putative pseudogenes (Ψ) contain premature stop codons (*), which are ignored in the protein prediction. (DOCX 70 kb)

Partial buc genes from eight primates including with predicted human N-terminus (above) compared with ferret buc and predicted protein (below). The inserted A in primates results in a premature stop, while the deletion of the inserted base would introduce another stop as shown in italics on top. Translation start codon is mutated in gorilla. Chromosomal positions of the primate buc genes: Human; Chr5:129170048, Chimpaneze; Chr5:129813080, Gorilla; Chr5:112518743, Orangutan; Chr5:130553556, Baboon; Chr6:122901841, Gibbon; Chr1:28463206, Macaque; Chr6:125569040, Vervet; Chr23:32116145. (DOCX 14Â kb)

A. Negative control (secondary antibody alone) of immunostained salmon embryo at 4-cell stage shown in Fig. 4. B. Negative control (secondary antibody alone) and C. Positive control (collagen staining) of immunohistochemical staining of ovarian sections from maturing 2-year old salmon shown in Fig. 6. (DOCX 1930 kb)