Sperm handling and management in the teleost model fish Japanese medaka (Oryzias latipes).

Japanese medaka (Oryzias latipes) has been used as a model organism in different research fields, including reproductive physiology. Sperm motility is the most important marker for male fertility in fish and, thus, reproduction success. However, because of small volume of ejaculate and short motility duration, it is still challenging to manage the sperm collection and analysis in small model fish. In the present study, we aimed to investigate sperm motility and to optimize sperm collection, short-term sperm storage, and cryopreservation in Japanese medaka (Oryzias latipes). Using two different approaches for sperm collection: testes dissection and abdominal massage, different housing conditions and activating the sperm with different activation solutions, we investigated immediate sperm motility. In the second part of this study, we used different osmolalities of immobilization solution, Hank's Balanced Salt Solution (HBSS) for sperm storage at 0, 2 and 3 h after sperm collection. Finally, the sperm were cryopreserved using methanol as cryoprotectant and HBSS as extender at two different osmolalities, and post-thaw sperm motility was investigated. The highest post-activating sperm motility was achieved in the groups activated by the extender at 300 mOsm/kg. The quality of sperm remained unaffected by co-housing with females or with males only. Furthermore, Hanks' Balanced Salt Solution (HBSS) with an osmolality of 600 mOsm/kg demonstrated its efficacy as a suitable extender for sperm storage, preserving motility and progressivity for 3 h. The highest post-thaw motility was around 35%. There were no significant differences between post-thaw motility in different groups. We also found that post-thaw incubation on ice can maintain the motility of the sperm for up to one hour after thawing.

Artificial light at night disrupts male dominance relationships and reproductive success in a model fish species.

Environmental light is perceived and anticipated by organisms to synchronize their biological cycles. Therefore, artificial light at night (ALAN) disrupts both diurnal and seasonal biological rhythms. Reproduction is a complex physiological process involving integration of environmental signals by the brain, and release of endocrine signals by the pituitary that regulate gametogenesis and spawning. In addition, males from many species form a dominance hierarchy that, through a combination of aggressive and protective behavior, influences their reproductive success. In this study, we investigated the effect of ALAN and continuous daylight on the behavior and fitness of male fish within a dominance hierarchy using a model fish, the Japanese medaka. In normal light/dark cycles, male medaka establish a hierarchy with the dominant males being more aggressive and remaining closer to the female thus limiting the access of subordinate males to females during spawning. However, determination of the paternity of the progeny revealed that even though subordinate males spend less time with the females, they are, in normal light conditions, equally successful at producing progeny due to an efficient sneaking behavior. Continuous daylight completely inhibited the establishment of male hierarchy, whereas ALAN did not affect it. Nonetheless, when exposed to ALAN, subordinate males fertilize far fewer eggs. Furthermore, we found that when exposed to ALAN, subordinate males produced lower quality sperm than dominant males. Surprisingly, we found no differences in circulating sex steroid levels, pituitary gonadotropin levels, or gonadosomatic index between dominant and subordinate males, neither in control nor ALAN condition. This study is the first to report an effect of ALAN on sperm quality leading to a modification of male fertilization success in any vertebrate. While this work was performed in a model fish species, our results suggest that in urban areas ALAN may impact the genetic diversity of species displaying dominance behavior.

Swimming exercise enhances brain plasticity in fish.

It is well-established that sustained exercise training can enhance brain plasticity and boost cognitive performance in mammals, but this phenomenon has not received much attention in fish. The aim of this study was to determine whether sustained swimming exercise can enhance brain plasticity in juvenile Atlantic salmon. Brain plasticity was assessed by both mapping the whole telencephalon transcriptome and conducting telencephalic region-specific microdissections on target genes. We found that 1772 transcripts were differentially expressed between the exercise and control groups. Gene ontology (GO) analysis identified 195 and 272 GO categories with a significant overrepresentation of up- or downregulated transcripts, respectively. A multitude of these GO categories was associated with neuronal excitability, neuronal signalling, cell proliferation and neurite outgrowth (i.e. cognition-related neuronal markers). Additionally, we found an increase in proliferating cell nuclear antigen (pcna) after both three and eight weeks of exercise in the equivalent to the hippocampus in fish. Furthermore, the expression of the neural plasticity markers synaptotagmin (syt) and brain-derived neurotrophic factor (bdnf) were also increased due to exercise in the equivalent to the lateral septum in fish. In conclusion, this is the first time that swimming exercise has been directly linked to increased telencephalic neurogenesis and neural plasticity in a teleost, and our results pave the way for future studies on exercise-induced neuroplasticity in fish.

Neuroendocrine indicators of allostatic load reveal the impact of environmental acidification in fish.

When mobilized from surrounding soils and binding to gills at moderately low pH, aluminum (Al) cations can adversely affect fish populations. Furthermore, acidification may lead to allostatic overload, a situation in which the costs of coping with chronic stress affects long-term survival and reproductive output and, ultimately, ecosystem health. The brain's serotonergic system plays a key role in neuroendocrine stress responses and allostatic processes. Here, we explored whether sublethal effects of Al in acidified water affects serotonergic neurochemistry and stress coping ability in a unique land-locked salmon population from Lake Bygelandsfjorden, in southern Norway. Fish were exposed to untreated water with pH 6.5 and 74 µg Al l-1 or acidified (pH 5.5) water with different aluminum concentrations ([Al]; 74-148 µg l-1) for 5-6 days. Afterward, effects on stress coping ability were investigated by analyzing plasma cortisol levels and telencephalic serotonergic neurochemistry before and after a standardized acute stress test. Before the stress test, positive dose-response relationships existed between [Al], serotonergic turnover rate and plasma cortisol. However, in acutely stressed fish, exposure to the highest [Al] resulted in reduced cortisol values compared with those exposed to lower concentrations, while the positive dose-response relationship between Al concentrations and serotonergic turnover rate persisted in baseline conditions. This suggests that fish exposed to the highest Al concentration were unable to mount a proper cortisol response to further acute stress, demonstrating that neuroendocrine indicators of allostatic load can be used to reveal sublethal effects of water acidification-and potentially, the environmental impacts of other factors.

Effects of environmental enrichment on forebrain neural plasticity and survival success of stocked Atlantic salmon.

Fish reared for stocking programmes are severely stimulus deprived compared with their wild conspecifics raised under natural conditions. This leads to reduced behavioural plasticity and low post-release survival of stocked fish. Environmental enrichment can have positive effects on important life skills, such as predator avoidance and foraging behaviour, but the neural mechanisms underpinning these behavioural changes are still largely unknown. In this study, juvenile Atlantic salmon (Salmo salar) were reared in an enriched hatchery environment for 7 weeks, after which neurobiological characteristics and post-release survival were compared with those of fish reared under normal hatchery conditions. Using in situ hybridization and qPCR, we quantified the expression of brain-derived neurotrophic factor (bdnf) and the neural activity marker cfos in telencephalic subregions associated with relational memory, emotional learning and stress reactivity. Aside from lower expression of bdnf in the Dlv (a region associated with relational memory) of enriched salmon, we observed no other significant effects of enrichment in the studied regions. Exposure to an enriched environment increased post-release survival during a 5 month residence in a natural river by 51%. Thus, we demonstrate that environmental enrichment can improve stocking success of Atlantic salmon parr and that environmental enrichment is associated with changes in bdnf expression in the fish's hippocampus-equivalent structure.

The Role of Reproductive Sciences in the Preservation and Breeding of Commercial and Threatened Teleost Fishes.

The teleost fishes are the largest and most diverse vertebrate group, accounting for nearly half of all known vertebrate species. Teleost fish exhibit greater species diversity than any other group of vertebrates and this is reflected in the unique variety of different reproductive strategies displayed by fish. Fish have always been an important resource for humans worldwide, especially as food. While wild capture fisheries have historically been the main source of fish, the farming of fish (aquaculture) is increasingly becoming the more dominant source of food fish, and is predicted to account for 60% of total global fish production by 2030.Fishes are increasingly threatened by a wide range of anthropogenic impacts, including loss of habitat, pollution, invasive species and over-exploitation. In addition, climate change, especially the consequences of global warming, can impact fish at all levels of biological organization from the individual to the population level, influencing both physiological and ecological processes in a variety of direct and indirect ways. As such, there is an urgent need to protect and conserve the huge genetic diversity offered by this diverse vertebrate group, not just as a source of genes for contemporary breeding and for protection against the consequences of climate change and disease, but also as part of our national heritage. While the cryopreservation of reproductive cells is a means of achieving these objectives, currently only fish sperm can be successfully frozen. Due to their large size, large yolk compartment, low membrane permeability and high chilling sensitivity, successful and reproducible protocols for the cryopreservation of fish oocytes and embryos still remains elusive. However, significant advances have been made in the cryopreservation of primordial germ cells as an alternative means of conserving both paternal and maternal genomes. Although more research needs to be carried out on how these cells can be optimally applied to emerging reproductive technologies, including transplantation techniques and surrogate broodstock technologies, the successful cryopreservation of fish germ cells, and the establishment of genetic resource banks, offers the possibility of both conserving and restoring threatened species. Further, current and future conservation efforts need to consider the impact of climate change in both in situ conservation and reintroduction efforts.In conclusion, it is anticipated that the successful cryopreservation of fish germplasm will result in a range of economic, ecological and societal benefits. In partnership with emerging assisted reproductive technologies, the successful cryopreservation of fish germplasm will lead to more efficient reproduction in aquaculture, assist selective breeding programmes, and be of crucial importance to future species conservation actions.

Establishment of specific enzyme-linked immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins.

The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshß, mdLhß, mdFshßα, and mdLhßα by Pichia pastoris, generating specific antibodies against their respective ß subunits, and their use within the development of ELISAs. The advantages of this protocol include: •The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%).•The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity.•Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time.

Melatonin receptors in Atlantic salmon stimulate cAMP levels in heterologous cell lines and show season-dependent daily variations in pituitary expression levels.

The hormone melatonin connects environmental cues, such as photoperiod and temperature, with a number of physiological and behavioural processes, including seasonal reproduction, through binding to their cognate receptors. This study reports the structural, functional and physiological characterization of five high-affinity melatonin receptors (Mtnr1aaα, Mtnr1aaß, Mtnr1ab, Mtnr1al, Mtnr1b) in Atlantic salmon. Phylogenetic analysis clustered salmon melatonin receptors into three monophyletic groups, Mtnr1A, Mtnr1Al and Mtnr1B, but no functional representative of the Mtnr1C group. Contrary to previous studies in vertebrates, pharmacological characterization of four receptors in COS-7, CHO and SH-SY5Y cell lines (Mtnr1Aaα, Mtnr1Aaß, Mtnr1Ab, Mtnr1B) showed induction of intracellular cAMP levels following 2-iodomelatonin or melatonin exposure. No consistent response was measured after N-acetyl-serotonin or serotonin exposure. Melatonin receptor genes were expressed at all levels of the hypothalamo-pituitary-gonad axis, with three genes (mtnr1aaß, mtnr1ab and mtnr1b) detected in the pituitary. Pituitary receptors displayed daily fluctuations in mRNA levels during spring, prior to the onset of gonadal maturation, but not in autumn, strongly implying a direct involvement of melatonin in seasonal processes regulated by the pituitary. To the best of our knowledge, this is the first report of cAMP induction mediated via melatonin receptors in a teleost species.

Data on Western blot and ELISA analysis of medaka (Oryzias latipes) follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) using recombinant proteins expressed with Pichia pastoris.

The gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play essential roles in vertebrate reproduction. This article presents data on molecular weight validation of recombinant medaka (Oryzias latipes) (md) gonadotropins Fshß (mdFshß), Lhß (mdLhß), Fshßα (mdFshßα), and Lhßα (mdLhßα) generated by Pichia pastoris, as well as data on a validation of produced antibodies against Fshß and Lhß by Western blot analysis. Furthermore, the article includes data on Fsh and Lh protein levels in male medaka pituitaries using recombinant mdFshßα and mdLhßα within enzyme-linked immunosorbent assays (ELISAs), in which protein amounts were analyzed related to body weight and age of the fish. This dataset is associated with the research article entitled "Medaka Follicle-stimulating hormone (Fsh) and Luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression" (Burow et al., in press).

Medaka follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression levels.

The two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are of particular importance within the hypothalamic-pituitary-gonadal (HPG) axis of vertebrates. In the current study, we demonstrate the production and validation of Japanese medaka (Oryzias latipes) recombinant (md) gonadotropins Fshß (mdFshß), Lhß (mdLhß), Fshßα (mdFshßα), and Lhßα (mdLhßα) by Pichia pastoris, the generation of specific rabbit antibodies against their respective ß subunits, and their use within the development and validation of competitive enzyme-linked immunosorbent assays (ELISAs) for quantification of medaka Fsh and Lh. mdFsh and mdLh were produced as single-chain polypeptides by linking the α subunit with mdFshß or mdLhß mature protein coding sequences to produce a "tethered" polypeptide with the ß-chain at the N-terminal and the α-chain at the C-terminal. The specificity of the antibodies raised against mdFshß and mdLhß was determined by immunofluorescence (IF) for Fshß and Lhß on medaka pituitary tissue, while comparison with fluorescence in situ hybridization (FISH) for fshb and lhb mRNA was used for validation. Competitive ELISAs were developed using antibodies against mdFshß or mdLhß, and the tethered proteins mdFshßα or mdLhßα for standard curves. The standard curve for the Fsh ELISA ranged from 97.6 pg/ml to 50 ng/ml, and for the Lh ELISA from 12.21 pg/ml to 6.25 ng/ml. The sensitivity of the assays for Fsh and Lh was 44.7 and 70.8 pg/ml, respectively. A profile of pituitary protein levels of medaka Fsh and Lh comparing juveniles with adults showed significant increase of protein amount from juvenile group (body length from 12 mm to 16.5 mm) to adult group (body length from 21 mm to 26.5 mm) for both hormones in male medaka. Comparing these data to a developmental profile of pituitary mRNA expression of medaka fshb and lhb, the mRNA expression of lhb also increased during male maturation and a linear regression analysis revealed a significant increase of lhb expression with increased body length that proposes a linear model. However, fshb mRNA expression did not change significantly during male development and therefore was not correlated with body length. In summary, we have developed and validated homologous ELISA assays for medaka Fsh and Lh based on proteins produced in P. pastoris, assays that will be used to study the functions and regulations of Fsh and Lh in more detail.

Sub-lethal UV radiation during early life stages alters the behaviour, heart rate and oxidative stress parameters in zebrafish (Danio rerio).

Environmental UV radiation in sufficient doses, as a possible consequence of climate change, is potent enough to affect living organisms with different outcomes, depending on the exposure life stage. The aim of this project was to evaluate the potentially toxic effects of exposure to sub-lethal and environmentally relevant doses of UVA (9.4, 18. 7, 37.7 J/cm2) and UVB radiation (0.013, 0.025, 0.076 J/cm2) on the development and behaviour in early life stages (4.5-5.5 h post fertilization, hpf) of the zebrafish (Danio rerio). The used doses were all below the median lethal dose (LD50) and caused no significant difference in survival, deformities, or hatching between exposed and control groups. Compared to controls, there were transient UVA and UVB exposure effects on heart rate, with dose dependent reductions at 50 hpf, and at 60 hpf for UVA only. The UVB exposure caused an increasing trend in reactive oxygen species (ROS) formation at the two highest doses, even though only significant at 120 hpf for the second highest dose. Both UVA and UVB caused an increasing trend in lipid peroxidation (LPO) at the highest doses tested at 72 hpf. Furthermore, UVA exposure led to significant reductions in larval movement following exposure to the two highest doses of UVA, i.e., reduction in the time spent active and the total distance moved compared to control at 100 hpf, while no effect on the swimming speed was observed. The lowest dose of UVA had no effect on behaviour. In contrast, the highest dose of UVB led to a possible increase in the time spent active and a slower average swimming speed although these effects were not significant (p = 0.07). The obtained results show that UV doses below LD50 levels are able to cause changes in the behaviour and physiological parameters of zebrafish larvae, as well as oxidative stress in the form of ROS formation and LPO. Further testing is necessary to assess how this type of radiation and the effects observed could affect fish population dynamics.

Neurobiology of Wild and Hatchery-Reared Atlantic Salmon: How Nurture Drives Neuroplasticity.

Life experiences in the rearing environment shape the neural and behavioral plasticity of animals. In fish stocking practices, the hatchery environment is relatively stimulus-deprived and does not optimally prepare fish for release into the wild. While the behavioral differences between wild and hatchery-reared fish have been examined to some extent, few studies have compared neurobiological characteristics between wild and hatchery-reared individuals. Here, we compare the expression of immediate early gene cfos and neuroplasticity marker brain-derived neurotrophic factor (bdnf) in telencephalic subregions associated with processing of stimuli in wild and hatchery-reared Atlantic salmon at basal and 30 min post (acute) stress conditions. Using in situ hybridization, we found that the expression level of these markers is highly specific per neuronal region and affected by both the origin of the fish, and exposure to acute stress. Expression of cfos was increased by stress in all brain regions and cfos was more highly expressed in the Dlv (functional equivalent to the mammalian hippocampus) of hatchery-reared compared to wild fish. Expression of bdnf was higher overall in hatchery fish, while acute stress upregulated bdnf in the Dm (functional equivalent to the mammalian amygdala) of wild, but not hatchery individuals. Our findings demonstrate that the hatchery environment affects neuroplasticity and neural activation in brain regions that are important for learning processes and stress reactivity, providing a neuronal foundation for the behavioral differences observed between wild and hatchery-reared fish.

Gamma irradiation during gametogenesis in young adult zebrafish causes persistent genotoxicity and adverse reproductive effects.

The biological effects of gamma radiation may exert damage beyond that of the individual through its deleterious effects on reproductive function. Impaired reproductive performance can result in reduced population size over consecutive generations. In a continued effort to investigate reproductive and heritable effects of ionizing radiation, we recently demonstrated adverse effects and genomic instability in progeny of parents exposed to gamma radiation. In the present study, genotoxicity and effects on the reproduction following subchronic exposure during a gametogenesis cycle to 60Co gamma radiation (27 days, 8.7 and 53 mGy/h, total doses 5.2 and 31 Gy) were investigated in the adult wild-type zebrafish (Danio rerio). A significant reduction in embryo production was observed one month after exposure in the 53 mGy/h exposure group compared to control and 8.7 mGy/h. One year later, embryo production was significantly lower in the 53 mGy/h group compared only to control, with observed sterility, accompanied by a regression of reproductive organs in 100% of the fish 1.5 years after exposure. Histopathological examinations revealed no significant changes in the testis in the 8.7 mGy/h group, while in 62.5% of females exposed to this dose rate the oogenesis was found to be only at the early previtellogenic stage. The DNA damage determined in whole blood, 1.5 years after irradiation, using a high throughput Comet assay, was significantly higher in the exposed groups (1.2 and 3-fold increase in 8.7 and 53 mGy/h females respectively; 3-fold and 2-fold increase in 8.7 and 53 mGy/h males respectively) compared to controls. A significantly higher number of micronuclei (4-5%) was found in erythrocytes of both the 8.7 and 53 mGy/h fish compared to controls. This study shows that gamma radiation at a dose rate of ≥ 8.7 mGy/h during gametogenesis causes adverse reproductive effects and persistent genotoxicity (DNA damage and increased micronuclei) in adult zebrafish.

Hormonal changes over the spawning cycle in the female three-spined stickleback, Gasterosteus aculeatus.

Female three-spined sticklebacks are batch spawners laying eggs in a nest built by the male. We sampled female sticklebacks at different time points, when they were ready to spawn and 6, 24, 48 and 72h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72hps). At sampling, plasma, brain and pituitaries were collected, and the ovary and liver were weighed. Testosterone (T) and estradiol (E2) were measured by radioimmunoassay. Moreover, the mRNA levels of follicle-stimulating hormone (fsh-ß) and luteinizing hormone (lh-ß) in the pituitary, and of the gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain were measured by real-time qPCR. Ovarian weights peaked in "ready to spawn" females, dropped after spawning, before again progressively increasing from 6 to 72hps. Plasma T levels showed peaks at 24 and 48hps and decreased at 72hps, while E2 levels increased already at 6hps and remained at high levels up to 48hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-ß mRNA levels showed a peak at 48hps, while fsh-ß did not change. The neuropeptides and gpr54 did not show any changes. The changes in T and E2 over the stickleback spawning cycle were largely consistent with those found in other multiple-spawning fishes whereas the marked correlation between T and E2 does not support T having other major roles over the cycle than being a precursor for E2.

Dietary l-tryptophan leaves a lasting impression on the brain and the stress response.

Comparative models suggest that effects of dietary tryptophan (Trp) on brain serotonin (5-hydroxytryptamine; 5-HT) neurochemistry and stress responsiveness are present throughout the vertebrate lineage. Moreover, hypothalamic 5-HT seems to play a central role in control of the neuroendocrine stress axis in all vertebrates. Still, recent fish studies suggest long-term effects of dietary Trp on stress responsiveness, which are independent of hypothalamic 5-HT. Here, we investigated if dietary Trp treatment may result in long-lasting effects on stress responsiveness, including changes in plasma cortisol levels and 5-HT neurochemistry in the telencephalon and hypothalamus of Atlantic salmon. Fish were fed diets containing one, two or three times the Trp content in normal feed for 1 week. Subsequently, fish were reintroduced to control feed and were exposed to acute crowding stress for 1 h, 8 and 21 d post Trp treatment. Generally, acute crowding resulted in lower plasma cortisol levels in fish treated with 3×Trp compared with 1×Trp- and 2×Trp-treated fish. The same general pattern was reflected in telencephalic 5-HTergic turnover, for which 3×Trp-treated fish showed decreased values compared with 2×Trp-treated fish. These long-term effects on post-stress plasma cortisol levels and concomitant 5-HT turnover in the telencephalon lends further support to the fact that the extrahypothalamic control of the neuroendocrine stress response is conserved within the vertebrate lineage. Moreover, they indicate that trophic/structural effects in the brain underlie the effects of dietary Trp treatment on stress reactivity.

Effects of temperature on the final stages of sexual maturation in Atlantic salmon (Salmo salar L.).

Maturing male and female Atlantic salmon (Salmo salar L.) were held under three temperature regimes for 10 weeks between September and December: warm (constant 14-16 °C), ambient (decreasing from 11 to 5 °C), and cold (decreasing from 7 to 3 °C). Blood samples were analyzed for plasma steroid levels, and the fish were inspected for the presence of expressible milt (total volume and spermatocrit) and ovulation weekly. Samples of eggs were dry-fertilized with milt stripped from three males held at the same temperatures and incubated until the eyed stage. In females, levels of plasma testosterone (T) and 17ß-oestradiol (E2) dropped as ovulation approached, concurrent with a rapid increase in levels of plasma 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P). In males, levels of T and 11-ketotestosterone (11-KT) peaked 2-3 weeks after the first appearance of expressible milt, while levels of 17,20ß-P increased steadily and did not exhibit a definite peak. Exposure of females to cold water amplified and advanced the profiles of all three steroids compared with the ambient group, and increased the survival rates to the eyed egg stage. Cold water had no immediate effect on the male steroid profiles, but later, higher levels of 17,20ß-P were evident compared with both the ambient controls and the warm water group, while the effects on 11-KT and T were more variable. Exposure to warm water completely inhibited both milt production and ovulation. Moreover, warm water modulated the steroid profiles of the males with lower 11-KT levels compared with ambient controls and lower 17,20ß-P level compared with cold-water-treated males. In females, warm water resulted in total inhibition of the peri-ovulatory peak in 17,20ß-P and prevented the normal decline of T and E2 levels associated with ovulation. The findings of the present study are highly relevant for broodstock management in aquaculture, as well in understanding the impact of climate change/temperature variability on wild salmon spawning.

Increased reactivity and monoamine dysregulation following stress in triploid Atlantic salmon (Salmo salar).

Artificial triploid salmonids are sterile and therefore commercially bred to prevent genetic interactions between wild and domestic fish strains. The full biological effects of having an extra chromosome set are largely unknown, but triploids are considered to be more sensitive to sub-optimal environmental conditions and to be stressed by the presence of diploid conspecifics. Brain serotonergic and dopaminergic activity are known to regulate the stress response in vertebrates, but monoamine systems in diploid and triploid fish have yet to be compared. Here we study monoamine neurochemistry in the telencephalon and brain stem of juvenile diploid and triploid Atlantic salmon (Salmo salar) in response to stress (unstressed vs stressed individuals) and holding (separate- vs mixed-ploidy) conditions. Both diploids and triploids showed an increase in serotonergic activity following stress, but the increase was significantly greater in the telencephalon of triploids compared to diploids. Furthermore, while telencephalic dopaminergic activity was significantly increased in diploids following stress, there was no response in triploids. Holding conditions had a significant effect on dopaminergic activity in the brain stem of diploids only, with lower values in mixed- compared to separate-ploidy conditions. These results suggest artificially produced triploids experience increased reactivity and monoaminergic dysregulation following stress that may impede their welfare and performance.

In vivo endocrine effects of naphthenic acids in fish.

Oil pollution from various sources, including exploration, production and transportation, is a growing global concern. The highest toxicity of hydrocarbon pollutants is associated with the water-soluble phase compounds, including naphthenic acids, a known component found in all hydrocarbon deposits. Recently, naphthenic acids (NAs) have shown estrogenic and anti-androgenic effects in vitro. For this reason we investigated the potential effects of two commercial mixtures of naphthenic acids on fish in vivo, using the three-spined stickleback (Gasterosteus aculeatus) as a model species. Anti-androgenic and estrogenic properties of tested compounds were evaluated using the androgenized female stickleback screen (AFSS) and a variant of the 21-d fish screen (TG230) respectively. One-dimensional gas chromatography-mass spectrometry (GC-MS) showed that the complex commercial NAs mixtures were dominated by acyclic carboxylic acids. In one experiment (freshwater) we found a clear effect of NA exposure on spiggin levels; this was contrary to our hypothesis since NAs enhanced the androgenic potency of DHT (when co-administered) without inducing spiggin when tested in the absence of DHT. Exposure to NAs did not have a statistically significant effect on vitellogenin (Vtg) production in male stickleback, although the Vtg responses were increasing with increasing exposure concentrations. This study shows that in contrast to previous in vitro data, NAs did not exhibit either estrogenic or anti-androgenic properties in vivo, at the concentrations tested. On the contrary, at least in freshwater, NAs appear to have an overall androgenic effect that is not mediated via the androgen receptor involved in spiggin synthesis. Possible reasons for this discrepancy between in vitro and in vivo results as well as between our studies are discussed.

Effects of polar oil related hydrocarbons on steroidogenesis in vitro in H295R cells.

Oil pollution from various sources, including exploration, production and transportation, is a growing global concern. Of particular concern is the environmental impact of produced water (PW), the main waste discharge from oil and gas platforms. In this study, we have investigated the potential of polar hydrocarbon pollutants to disrupt or modulate steroidogenesis in vitro, using a human adrenocortical carcinoma cell line, the H295R assay. Effects of two of the major groups of compounds found in the polar fraction of crude oil and PW; alkylphenols (C(2)- and C(3)-AP) and naphthenic acids (NAs), as well as the polar fraction of PW as a whole has been assessed. Endpoints include hormone (cortisol, estradiol, progesterone, testosterone) production at the functional level and key genes for steroidogenesis (17ß-HSD1, 17ß-HSD4, 3ß-HSD2, ACTHR, CYP11A1, CYP11B1, CYP11B2, CYP17, CYP19, CYP21, DAX1, EPHX, HMGR, SF1, STAR) and metabolism (CYP1A) at the molecular level. All compounds induced the production of both estradiol and progesterone in exposed H295R cells, while the C(3)-AP and NAs decreased the production of testosterone. Exposure to C(2)-AP caused an up-regulation of DAX1 and EPHX, while exposure to NAs caused an up-regulation of ACTHR. All compounds caused an up-regulation of CYP1A1. The results indicated that these hydrocarbon pollutants, including PW, have the potential to disrupt the vitally important process of steroidogenesis.

Changes in regional brain monoaminergic activity and temporary down-regulation in stress response from dietary supplementation with l-tryptophan in Atlantic cod (Gadus morhua).

The brain monoamines serotonin (5-hydroxytryptamine; 5-HT) and dopamine (DA) both play an integrative role in behavioural and neuroendocrine responses to challenges, and comparative models suggest common mechanisms for dietary modulation of transmission by these signal substances in vertebrates. Previous studies in teleosts demonstrate that 7 d of dietary administration with L-tryptophan (Trp), the direct precursor of 5-HT, suppresses the endocrine stress response. The present study investigated how long the suppressive effects of a Trp-enriched feed regimen, at doses corresponding to two, three or four times the Trp levels in commercial feed, last in juvenile Atlantic cod (Gadus morhua) when the fish are reintroduced to a diet with standard amino acid composition. We also wanted to determine whether Trp supplementation induced changes in brain monoaminergic neurochemistry in those forebrain structures innervated by DA and 5-HTergic neurons, by measuring regional activity of DA and 5-HT in the lateral pallial regions (Dl) of the telencephalon and nucleus lateralis tuberis (NLT) of the hypothalamus. Dietary Trp resulted in a dose-dependent suppression in plasma cortisol among fish exposed to confinement stress on the first day following experimental diet; however, such an effect was not observed at 2 or 6 d after Trp treatment. Feeding the fish with moderate Trp doses also evoked a general increase in DA and 5-HT-ergic activity, suggesting that these neural circuits within the NLT and Dl may be indirectly involved in regulating the acute stress response.