Deliverables Real time PCR methods for 14 key genes in salmon, sea bass and rainbow trout Real-time quantitative PCR assays have been developed, validated and applied for gonadotropin subunits (GTHa, FSHb and LHb) both in salmon and sea bass, for GnRH-R's in sea bass and trout (type I and type II), different ER's (ERa, ERb1 and ERb2) in trout, and FSHR, LHR and AR's (a and b) in salmon. The mRNA expression profiles of these genes have been put into the context of pubertal and/or seasonal development, as well as photoperiod treatments, in both male and female sea bass, rainbow trout and Atlantic salmon, by correlating the expression profiles with the stage of gonadal development (stage of spermatogenesis or oogenesis) and plasma sex steroid profiles. Established link between individual growth and puberty in sibling groups of salmon Monitoring growth performance and age at puberty in large groups of individually tagged salmon at different sites revealed that the bigger individuals with high condition factor around the time of winter solstice had a much higher likelihood of early maturation than their smaller and leaner siblings. This clearly suggests a link to body size, growth and adiposity for recruitment into puberty. However, restricted feeding prior to winter solstice has less effect than expected on age at puberty when studied on 3 sibling groups with individually tagged salmon in sea cages. This may imply that other factors than growth and adiposity modulates the outcome of a certain treatment. Individual sex hormone profiles in Atlantic salmon can potentially predict maturation later in season The hormonal events during onset and completion of puberty have been monitored b repeated blood sampling in 100 individually tagged salmon in sea cages and analysis of sex steroids. These results have been compared with gonad histology of sacrificed fish of same origin and indicate the temporal development of recruitment into puberty in one-sea-winter salmon. These results can be a basis for development for hormone masurement kits of on-farm assessment of maturity in salmon. Key components of brain-pituitary gonad-axis identified and characterized on molecular level in salmon, sea bass and trout A range of new hormone receptor variants were discovered and characterized, such as gonadotropin releasing hormone receptors (GnRH-R's) and estrogen receptors (ER's) in trout and sea bass, as well as follicle stimulating hormone receptor (FSHR), luteinising hormone receptor (LHR) and two androgen receptor forms (AR a and AR b) in Atlantic salmon. The localization of these receptors in tissues and cell types has been thoroughly investigated with real time quantitative PCR and in situ hybridization, along with pharmacological characterization to study how various ligands bind and activate these receptors. The mRNA expression profiles measure be-real time qPCR of these genes have been put into the context of pubertal and/or seasonal development, as well as photoperiod treatments, in both male and female sea bass, rainbow trout and Atlantic salmon, by correlating the expression profiles with the stage of gonadal development (stage of spermatogenesis or oogenesis) and plasma sex steroid profiles. Demonstrated interaction between growth, adiposity and response to continuous light treatments on age of puberty in salmon and rainbow trout Monitoring growth performance and age at puberty in large groups of individually tagged salmon at different sites revealed that the bigger individuals with high condition factor around the time of winter solstice had a much higher likelihood of early maturation than their smaller and leaner siblings. This clearly suggests a link to body size, growth and adiposity for recruitment into puberty. However, restricted feeding prior to winter solstice has less effect than expected on age at puberty when studied on 3 sibling groups with individually tagged salmon in sea cages. This may imply that other factors than growth and adiposity modulates the outcome of a certain treatment. In this particular experiment, most males and many females matured at the grilse stage (i.e. after one-sea winter), in spite of the sibling groups coming from a selected low grilsing strain (i.e. selected for high age at puberty). It may be that other environmental factors in the small sea cages (5x5x5m) applied in this experiment to some extent may have masked or overruled the effects of restricted feeding and stimulated most males to recruit into puberty. The plasma sex steroid profiles from January to August could imply that the low fed fish recruited into puberty (i.e. commenced rapid gonadal growth and full spermatogenesis or vitellogenesis) later in the spring than the normal fed fish. This implies some flexibility in the seasonal timing of recruitment to puberty, and that fish with a lower somatic status in terms of size and/or adiposity can catch up and commence maturation when offered surplus feed after January. New knowlegde for improved photoperiod protocols for 1) salmon, 2) sea bass and 3) rainbow trout A range of long-term experiments was conducted with different photoperiod treatments to affect puberty in Atlantic salmon, sea bass and rainbow trout. These experiments have been conducted in tanks for sea bass and trout, and in the case of salmon both in seawater tanks and sea cages of various sizes. In sea bass various photoperiod regimes were tested at different time-points in the lifecycle, in fish of differing size, and combined in one study with long-term sex steroid treatments. The sea bass trials show that continuous light treatment can be employed to delay or arrest early puberty in this species, in particular the precocious maturation in males at one-years of age. Long-term treatment with 11-ketotestosterone implants was able to cancel the inhibitory effect of LL on the precocious maturation in sea bass. An accelerating photoperiod regime was found to reduce the proportion of sexually mature rainbow trout, in line with previous studies in this species. The effects of photoperiod treatments have been closely examined as regards cell proliferation and apoptosis in testis in males and oogenesis and atresia in ovaries in females. Testis incubations in vitro has examined the responsiveness to gonadotropins at different stages of pubertal development in salmon, as well as following light treatments that either arrest or accelerate spermatogenesis. The efficiency of the light/photoperiod treatments has also been monitored by diel plasma melatonin profiles in salmon sampled in the cages, by assessment of sex steroid levels, gonad histology, growth performance and the final proportion of sexually maturing fish. In sea bass also LH levels in pituitary and plasma have been monitored following the photoperiod treatments along with expression profiles of pituitary mRNA levels of GTHa, FSHb and LHb. Continuous light treatment (LL) in sea cages from mid-winter onwards was found to reduce the proportion of sexually maturing salmon in line with previous experience. However, this reduction was less pronounced than expected, and varied between the experiments with different age groups, sibling groups and feeding history. Combination of restricted feeding during the autumn followed by LL treatment from January resulted in slightly fewer maturing females compared to LL treatment alone, but the restricted feeding had much less effect on age at puberty than expected in spite that the reduced feeding from September to January lead to a dramatic reduction of growth rate, condition factor and adiposity in fillet and viscera by January. Examination of 3 different sibling groups in this set-up revealed genetic differences in the response to both light treatment and reduced feeding, which to some extent may explain some of the variable results in the arresting effect of LL treatment observed in the different trials. New knowledge on testis development, and cell proliferation and apoptosis of germ cells and somatic cells in testis Germ and Sertoli cell proliferation and apoptosis in salmon testis under different experimental conditions. The incidence of apoptosis was high in animals that had been recruited into maturation but low in animals there were immature, irrespective of the light regimen the animals were exposed to. We conclude that changes in the incidence of apoptosis are secondary to changes in proliferation activity and do not reflect a direct effect of photoperiod manipulation. An antiserum against phosphorylated histone H3 (pH3; a protein present during the G2-phase and up until in the M-phase of the cell cycle) was found suitable as mitosis marker for salmon testis and was used on Atlantic salmon testis sections to study germ cell proliferation. We have shown that a very low incidence of proliferation was associated with LL-induced inhibition of testis growth. The limited numbers of cells that still were proliferating were mostly single spermatogonia and (rarely) Sertoli cells or extratubular somatic cells. In samples from LL-stimulated males collected in February and March, on the other hand, immunoreactive nuclei either belonged to single spermatogonia, or to small groups of spermatogonia. Moreover, Sertoli cell nuclei were labelled, indicating entry into the growth phase. Continued development resulted in the appearance of large groups of labelled cells, reflecting that the size of proliferating germ cell clones increased. This demonstrates the progress through the consecutive rounds of mitosis that is typical of the initial growth phase of the maturing testis. No differences were observed between maturing NL and LL males. However, a very interesting observation made in some samples of the NL group at the beginning of recruitment into maturation was that in February and March, a relatively high incidence of proliferation was found, predominantly single spermatogonia and Sertoli cells, while circulating androgen levels were still low. This indicates that the initial steps of proliferation are stimulated by another signal than androgens, possibly FSH. Subsequent to this initial step, however, elevated proliferation and 11KT plasma levels always were positively correlated in maturing animals in both NL- and LL-treated fish. We conclude that the initiation of spermatogenesis (ie. expansion of early spermatogonia) takes place while androgen levels are still low, possibly triggered by FSH, while the rapid spermatogonial proliferation leading to the generation of large cysts, is associated with high 11KT levels. This is supported by direct experimental evidence in other species, where treatment with 11KT in vivo (Cavaco et al., 2001) or in vitro (Miura et al., 1991) triggered spermatogonial proliferation. Altogether, the analysis of proliferation on the cellular level proved to be a very strong parameter in the evaluation of recruitment into maturation. Pineal sensitivity to light of different intensities and spectral quality in salmon and sea bass in vivo and in vitro Light perception and spectral sensitivity has been extensively studied in salmon and sea bass. This has been accomplished by using in vitro incubations of pineals exposed to different light regimes (photoperiods, intensities, and spectral compositions using narrow bandwidth filters) followed by assessment of melatonin production. In vivo studies have been conduced with different light intensities during the subjective day and night, as well as different spectral compositions of light using narrow bandwidth LED lights in small seawater tanks followed by assessment of the diel profiles of plasma melatonin. This has been extended with measurements of light transmission of different wavelengths in the skull (through the pineal window) in salmon and sea bass, and resulted in a model for light sensitivity in terms of melatonin production in these two species. This knowledge has been applied to develop and test narrow band-with lamps (green metal halogen and blue LED lamps) in full commercial scale salmon trials in sea cages. Method for in vitro assessment of pineal light sensitivity in relation to melatonin production in Atlantic salmon A method was developed to measure melatoning production in salmon pineal gland by in vitro incubations following exposure to different light regimes (photoperiods, intensities, and spectral compositions using narrow bandwidth filters). This method can be applied to investigate light sensitivity in fish species. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
