Mature Atlantic Salmon Research Articles

Temporal allele frequency changes in large-effect loci reveal potential fishing impacts on salmon life-history diversity.

Fishing has the potential to influence the life-history traits of exploited populations. However, our understanding of how fisheries can induce evolutionary genetic changes remains incomplete. The discovery of large-effect loci linked with ecologically important life-history traits, such as age at maturity in Atlantic salmon (Salmo salar), provides an opportunity to study the impacts of temporally varying fishing pressures on these traits. A 93-year archive of fish scales from wild Atlantic salmon catches from the northern Baltic Sea region allowed us to monitor variation in adaptive genetic diversity linked with age at maturity of wild Atlantic salmon populations. The dataset consisted of samples from both commercial and recreational fisheries that target salmon on their spawning migration. Using a genotyping-by-sequencing approach (GT-seq), we discovered strong within-season allele frequency changes at the vgll3 locus linked with Atlantic salmon age at maturity: fishing in the early season preferentially targeted the vgll3 variant linked with older maturation. We also found within-season temporal variation in catch proportions of different wild Atlantic salmon subpopulations. Therefore, selective pressures of harvesting may vary depending on the seasonal timing of fishing, which has the potential to cause evolutionary changes in key life-history traits and their diversity. This knowledge can be used to guide fisheries management to reduce the effects of fishing practices on salmon life-history diversity. Thus, this study provides a tangible example of using genomic approaches to infer, monitor and help mitigate human impacts on adaptively important genetic variation in nature.

Gene co-expression patterns in Atlantic salmon adipose tissue provide a molecular link among seasonal changes, energy balance and age at maturity.

Sexual maturation in many fishes requires a major physiological change that involves a rapid transition between energy storage and usage. In Atlantic salmon, this transition for the initiation of maturation is tightly controlled by seasonality and requires a high-energy status. Lipid metabolism is at the heart of this transition since lipids are the main energy storing molecules. The balance between lipogenesis (lipid accumulation) and lipolysis (lipid use) determines energy status transitions. A genomic region containing a transcription co-factor of the Hippo pathway, vgll3, is the main determinant of maturation timing in Atlantic salmon. Interestingly, vgll3 acts as an inhibitor of adipogenesis in mice and its genotypes are potentially associated with seasonal heterochrony in lipid storage and usage in juvenile Atlantic salmon. Here, we explored changes in expression of more than 300 genes directly involved in the processes of adipogenesis, lipogenesis and lipolysis, as well as the Hippo pathway in the adipose tissue of immature and mature Atlantic salmon with distinct vgll3 genotypes. We found molecular evidence consistent with a scenario in which immature males with different vgll3 genotypes exhibit contrasting seasonal dynamics in their lipid profiles. We also identified components of the Hippo signalling pathway as potential major drivers of vgll3 genotype-specific differences in adipose tissue gene expression. This study demonstrates the importance of adipose gene expression patterns for directly linking environmental changes with energy balance and age at maturity through genetic factors bridging lipid metabolism, seasonality and sexual maturation.

Loss of Fshr Prevents Testicular Maturation in Atlantic Salmon (Salmo salar L.).

Early puberty poses a significant challenge for male Atlantic salmon in aquaculture due to its negative impact on growth and welfare. The regulation of puberty in vertebrates involves 2 key reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their gonadal receptors. In male mice lacking FSH receptor, testes size is reduced, but fertility is maintained, while medaka and zebrafish with a disrupted fshr gene exhibit near normal testis size and fertility. In these fishes both Fsh and Lh are present during puberty and Lh may rescue fertility, while in salmonid fish only Fsh is present in the circulation during puberty. Using CRISPR-Cas9, we produced crispants with a high prevalence of fshr mutations at the target site, which remained fertile, although more than half showed a testis development deviating from wild-type (wt) males. Crossing out these F0 crispants to each other produced a viable F1 generation showing frameshift (fshr-/-) or in-frame mutations (fshrif/if). Nearly all wt males matured while all fshr-/- males remained immature with small testes containing A spermatogonia as the furthest developed germ cell type and prepubertal plasma androgen levels. Also, the pituitary transcript levels of gnrhr2bba and lhb, but not for fshb, were reduced in the fshr-/- males compared with maturing males. More than half of the fshrif/if mutant males showed no or a delayed maturation. In conclusion, Atlantic salmon show the unique characteristic that loss of Fshr function alone results in male infertility, offering new opportunities to control precocious puberty or fertility in salmon.

Smoltification, seawater performance, and maturation in Atlantic salmon (Salmo salar) fed different fat levels

IntroductionThe use of recirculating aquaculture systems (RAS) to produce Atlantic salmon smolts has resulted in exceptionally high fish growth rates. However, there are potential negative trade-offs between fast growth and key physiological processes, such as inadequate smoltification and early sexual maturation, which can both be linked to body energy reserves.MethodsThis study determined the effect of i) dietary fat levels on Atlantic salmon whole-body fat and fatty acids composition, growth performance and smoltification and ii) a previous dietary regime on seawater growth performance and male early sexual maturation. In freshwater RAS, salmon parr (~19g) were fed 3 fat levels (20, 24, and 28%) over a 14-week period at 12°C. Subsequently, in seawater flow-through systems, smolt (~96g) were fed a control diet (26%) for 12 weeks at 2 temperatures (12 and 16°C).ResultsDietary fat levels resulted in differences in k-factor, fish whole-body fat and fatty acids composition; the low fat diet resulted in fish with a 23% lower whole-body fat when compared to the other two dietary groups at the end of the freshwater phase. These differences in whole-body fat faded at the end of the seawater phase. During the freshwater phase, all three feeds resulted in a comparable growth performance and smoltification indicator values, including k-factor, gill Na+, K+-ATPase, blood serum chloride and smolt index score. In contrast, water temperature resulted in fish body weight differences, where groups reared at 16°C were larger than those reared at 12°C. Nevertheless, both temperature regimes supported a similar thermal growth coefficient. Smolts grown at 16°C showed a higher level of the sex steroids androstenedione compared to those at 12°C. However, 11-ketotestosterone and testosterone levels did not differ despite a trend for higher levels at 16°C. Furthermore, testis histology at the final sampling indicated that some individuals showed initial signs of maturation (stage 3).DiscussionIn conclusion, varying dietary fat levels (20 - 28%) during the freshwater phase did not influence smoltification or male early sexual maturation during the subsequent grow-out phase. However, a temperature increase from 12 to 16°C resulted in larger fish and appeared to stimulate early male maturation in some fish individuals.

Probabilistic reaction norm reveals family-related variation in the association between size, condition, and sexual maturation onset in Atlantic salmon (Salmo salar).

This study investigated the relationship between the size, condition, year class, family, and sexual maturity of Atlantic salmon (Salmo salar) using data collected in an aquaculture selective breeding programme. Males that were sexually mature at 2 years of age (maiden spawn) have, on average, greater fork length and condition factor (K) at 1 year of age than their immature counterparts. For every 10-mm increase in fork length or 0.1 increase in K at 1 year of age, the odds of sexual maturity at 2 years of age increased by 1.48 or 1.22 times, respectively. Females that were sexually mature at 3 years of age (maiden spawn) have, on average, greater fork length and K at 2 years of age than their immature counterparts. For every 10-mm increase in fork length or 0.1 increase in K at 2 years of age, the odds of sexual maturity at 3 years of age increased by 1.06 or 1.44 times, respectively. The family explained 34.93% of the variation in sexual maturity among 2-year-old males that was not attributable to the average effects of fork length and K at 1 year of age and year class. The proportion of variation in sexual maturity among 3-year-old females explained by the family could not be investigated. These findings suggest that the onset of sexual maturation in Atlantic salmon is conditional on performance (with respect to energy availability) surpassing a threshold, the magnitude of which can vary between families and is determined by a genetic component. This could support the application of genetic selection to promote or inhibit the onset of sexual maturation in farmed stocks.

Hypothalamic expression analysis of m6A RNA methylation associated genes suggests a potential role of epitranscriptomics in sexual maturation of Atlantic salmon

Better understanding the molecular processes contributing to variation in maturation timing is important for Atlantic salmon (Salmo salar) aquaculture, as early maturation causes considerable financial losses. The m6A RNA methylation is a highly conserved and dynamically reversible mechanism controlling gene expression in a myriad of biological processes. The role of m6A methylation in sexual maturation, however, has remained largely unexplored and has never been studied in Atlantic salmon. While the maturation process is known to be affected by many genetic and environmental factors, the molecular mechanisms causing variation in the timing of maturation are still poorly understood. Hence, investigation of whether a widespread mechanism like m6A methylation could be involved in controlling of the maturation timing is warranted. In Atlantic salmon, two genes, also associated with age at maturity in humans, vgll3 and six6, have been shown to play an important role in maturation timing. In this study, we investigated the expression of 16 genes involved in the regulation of m6A RNA methylation in the hypothalamus of Atlantic salmon with different homozygous combinations of late (L) and early (E) alleles for vgll3 and six6 genes. We found differential expression of ythdf2.2, an m6A reader promoting mRNA degradation, with higher expression in six6*LL compared to other genotypes as well as in immature compared to mature males. In addition, we found that the expression levels of genes coding for an eraser, alkbh5, and for a reader, ythdf1, were higher in the hypothalamus of females than in males across all the different genotypes studied. However the total m6A levels between the whole hypothalamus of males and females were similar. Our results indicate a potential role of the m6A methylation process in sexual maturation of Atlantic salmon, and therefore, provide the first evidence for such regulatory mechanism in the hypothalamus of any vertebrate.

Sperm quality parameters, fertilizing potential, metabolites, and DNA methylation in cold-stored and cryopreserved milt from Atlantic salmon (Salmo salar L.).

Cold storage and freezing/thawing of milt may affect sperm functionality and the subsequent fertilization ability of milt. This study aimed to investigate sperm quality parameters and fertilization potential of Atlantic salmon milt, stored cold and subsequently cryopreserved, using different storage conditions. The objective was also to assess if analysis of milt metabolites and sperm DNA methylation signatures could be applicable to further elucidate sperm quality and fertilization following preservation. Milt samples were collected from eight mature Atlantic salmon males and stored for 4days at 2°C and 8°C. Samples were taken on day one of storage at 2°C and on day four of storage at 2°C and 8°C. Storage for 4days at 8°C is expected to be detrimental to sperm quality, and was included to create contrasts. Correspondingly, aliquots of cold-stored milt were prepared for cryopreservation, resulting in a total of six experimental conditions. Samples from all six experimental conditions were used in fertilization trials and analyzed for sperm viability, motility, ATP content, DNA fragmentation index, and High DNA stainability. In addition, milt samples from four of the males were analyzed for targeted metabolites and DNA methylation signatures by reduced representation bisulfite sequencing. The fertilization trials were performed using sperm:egg ratios of 75 × 103 and 500 × 103, respectively. Storage duration, temperature, and cryopreservation of cold-stored milt influenced several sperm quality parameters, metabolites, and DNA methylation signatures. The total motility, progressive motility, ATP, and velocity parameters were the sperm parameters with the strongest correlation to fertilization rates (p < 0.01). Several metabolites were correlated with fertility rates in both cold-stored and cryopreserved samples (p < 0.05). The fertilizing capacity of cold-stored milt was significantly reduced after 4days of storage at 8°C, while corresponding cryopreserved milt showed reduced fertilization at both storage temperatures (2°C and 8°C) (p < 0.05). The results indicate that cryopreservation of milt stored for 1day does not compromise either fertilization ability or DNA methylation signatures.

Interaction of temperature and photoperiod on male postsmolt maturation of Atlantic salmon (Salmo salar L.)

Maturation of Atlantic salmon male postsmolts is a concern in aquaculture due to its increasing occurrence under intensive rearing conditions and its negative impact on growth, welfare and seawater readiness. The effect of temperature and photoperiod on maturation was assessed in male postsmolts kept in freshwater. We used a 2 × 2 factorial design with two temperatures (12.5 and 15 °C) and two photoperiods (a group in continuous light LD24:0 or LL, and another receiving a 5-week LD12:12 winter signal or WS). Salmon in the four resulting treatments (1000 parr, initial mean weight 52.1 ± 5.2 g) were reared in a flow-through system from 28 October 2019 to 30 May 2020. Morphology (body weight, condition factor), maturation indicators including gonadosomatic index (GSI), plasma 11-Ketotestosterone (11-KT), pituitary follicle-stimulating hormone β-subunit (fshb) and luteinizing hormone β-subunit (lhb) transcript levels, as well as smoltification markers (Na+, K+- ATPase activity) were assessed. Results revealed that rearing salmon at 15 °C was the most important factor promoting early maturation, leading to 100% of males maturing in late May in 15-WS, and 75% in 15-LL. However, the groups receiving a winter signal (WS) displayed a highly synchronized onset and progression of maturation specially at 15 °C, revealed by the low variability observed among individuals in GSI and fshb transcription after the WS. This evidences the role of the photoperiod switch from short to long day as zeitgeber for sexual maturation. On the contrary, under constant light (LL), entry into maturation was not synchronized among individuals, and onset of maturation occurred spontaneously in a proportion of males highly dependent upon temperature (75% in 15-LL, 25% in 12.5-LL). Signs of smoltification were poor at both temperatures, and the WS did not induce development of hypo-osmoregulatory abilities in any case. This suggests that a winter signal may not induce smoltification if introduced at high temperature or when fish have reached large size, and instead may increase the risk of a sexual maturation response. These findings are relevant for the aquaculture industry, since similar rearing conditions are currently used in the industry, including constant high water temperature and winter signal regimes. The use of such conditions can increase the risk of early maturation, as well as of poor hypo-osmoregulatory performance.

Dissecting the loci underlying maturation timing in Atlantic salmon using haplotype and multi-SNP based association methods

Characterizing the role of different mutational effect sizes in the evolution of fitness-related traits has been a major goal in evolutionary biology for a century. Such characterization in a diversity of systems, both model and non-model, will help to understand the genetic processes underlying fitness variation. However, well-characterized genetic architectures of such traits in wild populations remain uncommon. In this study, we used haplotype-based and multi-SNP Bayesian association methods with sequencing data for 313 individuals from wild populations to test the mutational composition of known candidate regions for sea age at maturation in Atlantic salmon (Salmo salar). We detected an association at five loci out of 116 candidates previously identified in an aquaculture strain with maturation timing in wild Atlantic salmon. We found that at four of these five loci, variation explained by the locus was predominantly driven by a single SNP suggesting the genetic architecture of this trait includes multiple loci with simple, non-clustered alleles and a locus with potentially more complex alleles. This highlights the diversity of genetic architectures that can exist for fitness-related traits. Furthermore, this study provides a useful multi-SNP framework for future work using sequencing data to characterize genetic variation underlying phenotypes in wild populations.

Malic enzyme, an “old-new player” in age at maturity in Atlantic salmon

Traditional genetic markers based on allozyme variation had indicated that in Atlantic salmon the genetic variation found for the locus coding for mMEP-2* could be adaptive, with the variation being related to river water temperature, growth, and maturation. More recently, SNP panels used to search for genes related to maturation in Atlantic salmon identified several genes, including the vgll3 gene related to early maturation. However, none of the analyses performed found an association between maturation and the already known locus coding for mMEP-2*. Recently, a method has been developed to characterize the different alleles of the locus coding for mMEP-2* using molecular techniques. In this work we use this method to study the salmon population of the Bidasoa River, located in the Iberian Peninsula, which is the limit of distribution of the species. We found a relationship between the presence of SNP G, equivalent to the 125* allele, and early maturation of Atlantic salmon in both males and females. It is possible that the relationship between the different alleles of this locus and maturation is not direct but is produced by differential growth during the juvenile and/or adult phase. It is also plausible that this association is exclusive to the salmon populations of the Iberian Peninsula.

Interaction of temperature and feed ration on male postsmolt maturation of Atlantic salmon (Salmo salar L.)

Early maturation of Atlantic salmon male postsmolts is undesirable in aquaculture due to its negative impact on growth, welfare and seawater adaptation, however it is an increasing problem under intensive rearing conditions. The effect of temperature and feeding ration on sexual maturation during the induction of smoltification was tested with a 3 × 2 factorial design including three temperatures (8, 12.5 and 18 °C) and two feeding rations (100% and 67%), from 28 October 2018 to 30 May 2019. Fish in the six resulting treatments (1800 parr, initial mean weight 23.1 ± 7.2 g) were reared in flow-through under continuous light (LD24:0) except for a 5-week winter signal (LD12:12) introduced on 4 February 2019 to induce smoltification. A major effect of temperature determining the life history pattern of each group was observed. At 18 °C, a majority of the males were maturing in May under both rations (100% males in 18–100% and 88.9% males in 18–67%). Fish at 18 °C experienced an early activation of the BPG (Brain-Pituitary-Gonad) axis and displayed very poor signs of smoltification. At 12.5 °C, early maturation was also present, although at lower proportion than at 18 °C and more dependent upon feed ration (40% of males maturing in 12.5–100%, 8.6% in 12.5–67%). Salmon at 12.5 °C displayed the best growth within each feed regime, some early signs of smolting but subsequent low Na+, K+- ATPase activity after the winter signal. At 8 °C, salmon showed best signs of smoltification and lowest growth, while no males matured. The feed restriction had a minor effect on maturation at 18 °C but a greater impact at 12.5 °C, evidenced by significant differences between 12.5–100% and 12.5–67% in gonadosomatic index, plasma 11-ketotestosterone, insulin-like growth factor-I, and pituitary fshb (follicle stimulating hormone β-subunit gene) transcription. Onset of maturation was associated with the photoperiod change from short to long daylength, which probably also induced a slight activation of the BPG axis in non-maturing groups. Our results suggest that postsmolt producers must be cautious when using sustained high water temperatures, or the risk of early maturation and poor hypo-osmoregulatory performance will increase significantly. In addition, reducing the feeding ration will not help decrease incidence of maturation without significantly affecting growth.

Mapping the Pattern of Essential Neuroendocrine Cells Related to Puberty and VA Opsin Expression Provides Further Insight in the Photoreceptive Regulation of the Brain-Pituitary-Gonadal Axis in Atlantic Salmon (Salmo salar)

In Atlantic salmon (Salmo salar), seasonal photoperiod is shown to regulate the onset of sexual maturation, yet which brain region(s) is involved, and how light information impacts the neuroendocrine system are still not fully understood in teleosts. Detailed knowledge about the photoperiodic regulation of maturation in fish is still missing. In birds, it is shown that gonadotropin-releasing hormone (Gnrh) is located in the same neurons as vertebrate ancient (VA) opsin, suggesting a direct photoreceptive regulation for the onset of sexual maturity. This study presents a comprehensive topographic mapping of gnrh2, gnrh3, kisspeptin 2 (kiss2), gonadotropin-inhibiting hormone (gnih), and VA opsin using in situ hybridization on mature Atlantic salmon brains. Neurons positive for gnrh3 are expressed in the olfactory bulb and ventral telencephalon, while gnrh2-positive neurons are located dorsally in the midbrain tegmentum. Gonadotropin-inhibiting hormone (Gnih)-expressing cell bodies are present in the ventral thalamus and extend caudally to the hypothalamus with kiss2-expressing cells appearing in a lateral position. VA opsin-positive cells are present in the telencephalon, the rostro-dorsal ring of the left habenula, the ventral thalamus, and the midbrain tegmentum. The results show no similar co-location as found in birds, hypothesizing that the photoreceptive modulation of Gnrh in salmon may interact through neuronal networks. The topography analyses of the essential neuroendocrine cells related to sexual maturation in the Atlantic salmon brain show that diencephalic (thalamus, hypothalamus) and midbrain (tegmentum) regions seem central for controlling sexual maturation.

Effects of laboratory salmon louse infection on mortality, growth, and sexual maturation in Atlantic salmon

Abstract Elevated salmon louse infection pressure generated by salmon farming represents a major threat for wild Atlantic salmon (Salmo salar). This study explored the effect of salmon lice on mortality, and body and gonad growth in F1 wild Atlantic salmon. Mature males (jacks) and immature fish were either infected with two different louse infection intensities (LIIs, 0.08 or 0.35 lice g−1) or kept as uninfected controls. Fish maturation was thereafter environmentally stimulated in seawater, followed by transfer to freshwater for 38 d to simulate river ascendance. No females matured, while 99% of the initially immature males started puberty. Jacks had high, and immature and maturing fish low, seawater mortality, independent of lice. The parasites had an LII-dependent negative effect on growth in length, weight, and condition factor in seawater. In freshwater, after the lice had detached, fish that were previously infected in seawater had reduced growth in length but not weight when compared to the uninfected control. The parasites did not affect relative gonad size in any fish phenotypes. The present results show that Atlantic salmon has a complex, and unexplored, regulation of growth when recovering from lice infection under laboratory settings. Further, the results suggest that possible negative effects of salmon louse on reproductive success in Atlantic salmon is most likely governed by the reduced body size and condition. However, further work on possible effects of salmon louse on semen quality is encouraged.

A long evolutionary reach for fishing nets.

Earlier maturation of Atlantic salmon is linked to indirect effects of fisheries on its prey.

Environment-driven reprogramming of gamete DNA methylation occurs during maturation and is transmitted intergenerationally in Atlantic Salmon.

An epigenetic basis for transgenerational plasticity in animals is widely theorized, but convincing empirical support is limited by taxa-specific differences in the presence and role of epigenetic mechanisms. In teleost fishes, DNA methylation generally does not undergo extensive reprogramming and has been linked with environmentally induced intergenerational effects, but solely in the context of early life environmental differences. Using whole-genome bisulfite sequencing, we demonstrate that differential methylation of sperm occurs in response to captivity during the maturation of Atlantic Salmon (Salmo salar), a species of major economic and conservation significance. We show that adult captive exposure further induces differential methylation in an F1 generation that is associated with fitness-related phenotypic differences. Some genes targeted with differential methylation were consistent with genes differential methylated in other salmonid fishes experiencing early-life hatchery rearing, as well as genes under selection in domesticated species. Our results support a mechanism of transgenerational plasticity mediated by intergenerational inheritance of DNA methylation acquired late in life for salmon. To our knowledge, this is the first-time environmental variation experienced later in life has been directly demonstrated to influence gamete DNA methylation in fish.

Large inter-stock differences in catch size-at-age of mature Atlantic salmon observed by using genetic individual origin assignment from catch data.

Genetic individual assignment of river stock of origin of mixed stock catch fish offers a tool to analyze size differences among river stocks. Data on the genetically identified river stock of origin of individual fish from commercial mixed stock catches were used to compare the catch size-at-age of mature Atlantic salmon catch fish (Salmo salar) from different rivers in the Baltic Sea. In this application of genetic mixed stock modeling, individual assignments of the river stock of origin were analyzed together with length- and weight-at-age data for individual catch fish. The use of four genetic stock identification based methods was compared for defining the length distributions of caught mature salmon in different river stocks. The catch data included information on maturing salmon in the northern Baltic Sea over the years 2000–2013. DNA microsatellite data on 17 loci and information on the smoltification age were used to assign spawners to their stock of origin. All of the compared methods for using probabilistic stock of origin data in our case yielded very similar estimates of the final mean length distributions of the stocks. The Bayesian mixture model yielded slightly more conservative estimates than the direct probability method, threshold method, or the modified probability method. The catch size between spawners of a same sex and age from river stocks differed significantly and the differences were large. The mean catch weight of 1-sea-winter old mature males in different rivers varied from 1.9 kg to 2.9 kg, from 5.1 kg to 7.5 kg for 2-sea-winter old males, from 5.0 kg to 7.2 kg for 2-sea-winter old females, and from 8.2 kg to 10.8 kg for 3-sea-winter-old females. The mean size of caught wild salmon spawners in each year-class was on average smaller than that of the hatchery-reared and sea ranched stocks.

Characterization of miRNAs in Cultured Atlantic Salmon Head Kidney Monocyte-Like and Macrophage-Like Cells.

Macrophages are among the first cells to respond to infection and disease. While microRNAs (miRNAs) are involved in the process of monocyte-to-macrophage differentiation in mammals, less is known in teleost fish. Here, Atlantic salmon head kidney leukocytes (HKLs) were used to study the expression of miRNAs in response to in vitro culture. The morphological analysis of cultures showed predominantly monocyte-like cells on Day 1 and macrophage-like cells on Day 5, suggesting that the HKLs had differentiated from monocytes to macrophages. Day 5 HKLs also contained a higher percentage of phagocytic cells. Small RNA sequencing and qPCR analysis were applied to examine the miRNA diversity and expression. There were 370 known mature Atlantic salmon miRNAs in HKLs. Twenty-two miRNAs (15 families) were downregulated while 44 miRNAs (25 families) were upregulated on Day 5 vs. Day 1. Mammalian orthologs of many of the differentially expressed (DE) miRNAs are known to regulate macrophage activation and differentiation, while the teleost-specific miR-2188, miR-462 and miR-731 were also DE and are associated with immune responses in fish. In silico predictions identified several putative target genes of qPCR-validated miRNAs associated with vertebrate macrophage differentiation. This study identified Atlantic salmon miRNAs likely to influence macrophage differentiation, providing important knowledge for future functional studies.

Phospholipid and LC-PUFA metabolism in Atlantic salmon (Salmo salar) testes during sexual maturation.

The importance of dietary lipids in male reproduction are not as well understood as in females, in which dietary lipids, such as phospholipids (PL) and associated fatty acids (FA), are important structural components of the eggs and provide energy for their offspring. In mammals, lipids are suggested to be important for spermatogenesis and to structural components of the spermatozoa that could improve fertilization rates. New knowledge of how lipids affect sexual maturation in male Atlantic salmon (Salmo salar), an important global aquaculture species, could provide tools to delay maturation and/or improve reproductive success. Therefore, changes in testicular composition of lipids and gene transcripts associated with spermatogenesis and lipid metabolism were studied in sexually maturing male salmon compared to immature males and females. An increase in total testis content of FA and PL, and a shift to higher PL composition was observed in maturing males, concomitant with increases in mRNA levels for genes involved in spermatogenesis, FA uptake and synthesis, and production of long chain-polyunsaturated fatty acids (LC-PUFA) and PL. A particularly interesting finding was elevated testis expression of acyl-CoA synthetase 4 (acsl4), and acyl-CoA thioesterase 2 (acot2), critical enzymes that regulate intra-mitochondrial levels of 20:4n-6 FA (arachidonic acid), which have been associated with improved cholesterol transport during steroidogenesis. This suggested that FA may have direct effects on sex steroid production in salmon. Furthermore, we observed increased testis expression of genes for endogenous synthesis of 16:0 and elongation/desaturation to 22:6n-3 (docosahexaenoic acid) in sexually maturing males relative to immature fish. Both of these FA are important structural components of the PL, phosphatidylcholine (PC), and were elevated concomitant with increases in the content of phosphatidic acid, an important precursor for PC, in maturing males compared to immature fish. Overall, this study suggests that, similar to mammals, lipids are important to spermatogenesis and serve as structural components during testicular growth and maturation in Atlantic salmon.

Viability, motility, ATP content and fertilizing potential of sperm from Atlantic salmon (Salmo salar L.) in milt stored before cryopreservation

Artificial fertilization is increasingly used in aquaculture, mostly applying short-term cold stored milt. Large scale cryopreservation of milt could be valuable for increased flexibility and acceleration of breeding progress. The aim of this study was to assess viability, motility and ATP content of sperm from Atlantic salmon as a function of storage time, before and after cryopreservation. The objective was also to investigate whether in vitro parameters were associated with sperm fertilizing ability after cryopreservation. Milt from six mature Atlantic salmon males were collected twice, one week apart. The milt was stored undiluted at 5 °C in cell culture flasks for six days. Samples were taken on days 1, 3 and 6 of storage for cryopreservation. In total, 36 batches were diluted to a standardized sperm concentration of 2 × 109 spermatozoa/mL, filled into 0.5 mL French medium straws and cryopreserved. In vitro analyses were assessed on the same sample for the 72 combinations of male, collection week, days of storage and cold stored or frozen-thawed. Fertilization trials with cryopreserved milt were carried out for all 36 batches in triplicate for each combination of male, collection week, storage time and sperm:egg ratios of either 2 or 4 × 106 sperm per egg, respectively, totally 218 experimental units, including two egg controls. There was a significant influence of storage and collection week on sperm quality parameters, both cold stored and cryopreserved, and cryopreservation had a significant effect on all tested sperm quality parameters. High correlations for cold stored vs cryopreserved samples was demonstrated for ATP content (p < 0.00001), motility and velocity parameters (p < 0.001), but not for viability, straightness and linearity. The overall percentage of fertilization achieved was 73.9 ± 1.7%. Sperm collected in week 2 showed significantly lower fertility when cryopreserved after six days of storage than after 1 or 3 days for sperm to egg ratios of 2 × 106 (p < 0.005), while there was no such effect for milt collected in week 1. Several post-thaw sperm parameters were correlated to fertilization rates, while curvilinear velocity best explained variations in fertilization by modelling. Our results suggest that cryopreservation of Atlantic salmon milt should be performed soon after milt collection to maximize the cryopreserved sperm quality. Fertilization results seems not to be compromised by storage for three days before cryopreservation.

Beyond large-effect loci: large-scale GWAS reveals a mixed large-effect and polygenic architecture for age at maturity of Atlantic salmon

BackgroundUnderstanding genetic architecture is essential for determining how traits will change in response to evolutionary processes such as selection, genetic drift and/or gene flow. In Atlantic salmon, age at maturity is an important life history trait that affects factors such as survival, reproductive success, and growth. Furthermore, age at maturity can seriously impact aquaculture production. Therefore, characterizing the genetic architecture that underlies variation in age at maturity is of key interest.ResultsHere, we refine our understanding of the genetic architecture for age at maturity of male Atlantic salmon using a genome-wide association study of 11,166 males from a single aquaculture strain, using imputed genotypes at 512,397 single nucleotide polymorphisms (SNPs). All individuals were genotyped with a 50K SNP array and imputed to higher density using parents genotyped with a 930K SNP array and pedigree information. We found significant association signals on 28 of 29 chromosomes (P-values: 8.7 × 10−133–9.8 × 10−8), including two very strong signals spanning the six6 and vgll3 gene regions on chromosomes 9 and 25, respectively. Furthermore, we identified 116 independent signals that tagged 120 candidate genes with varying effect sizes. Five of the candidate genes found here were previously associated with age at maturity in other vertebrates, including humans.DiscussionThese results reveal a mixed architecture of large-effect loci and a polygenic component that consists of multiple smaller-effect loci, suggesting a more complex genetic architecture of Atlantic salmon age at maturity than previously thought. This more complex architecture will have implications for selection on this key trait in aquaculture and for management of wild salmon populations.