Management of broodstock and the control of spawning is an essential requirement for the intensive aquaculture of all finfish species.
A greater understanding of the environmental and neuroendocrine mechanisms underlying seasonal breeding in fish would allow the development of methods for the control of reproduction which could be used to improve the self-sufficiency of seed supplies and hence the profitability of farmed fish production in Europe. Hence the objectives of this proposal are as follows:
- To determine the stimulatory and inhibitory effects of photoperiod and temperature on the brain-pituitary-gonadal axis of the rainbow trout, Atlantic salmon, European sea bass and Atlantic cod.
- To investigate the neuroendocrinological mechanisms by which the daily and seasonal changes in daylength and temperature are perceived by fish and translated into hormonal or neural signals which act upon the brain-pituitary-gonadal axis, thereby controlling the onset and course of reproductive development.
- To establish management-tools for the environmental control of reproduction which could then be reliably used by commercial farms to improve the management of their broodstock, and to disseminate this information to the industry via technical advice sheets and workshops.
Environmental control of circulating melatonin levels
Melatonin analysis carried out on Atlantic salmon (in collaboration with partners 1 and 6) clearly showed that melatonin secretion was dependant on temperature. Further work carried out in collaboration with partner 6 at Matre Research Station on juvenile Atlantic salmon, which are to be maintained to maturity, confirmed that fish, maintained under constant light, display significantly reduced melatonin synthesis during the subjective dark phase compared to fish on a simulated natural photoperiod. Interestingly, the circulating plasma melatonin levels were higher during the subjective dark phase than the basal levels observed during the light phase suggesting an endogenous rhythm of melatonin production.
A similar experiment was carried out on a high grilsing stock on a commercial salmon farm in Scotland. This used night illumination on selected pens to mimic continuous daylight. Again the lights significantly reduced the night time levels of plasma melatonin when compared to fish under natural photoperiods, and resulted in a grilse rate of 6% compared to 61.5% in the fish under ambient daylength. Further trials are to take place over the 1997-98 season using a variety of lights and intensifies.
Melatonin rhythms in cod, sea bass and Atlantic salmon
A major aim of the project is to apply the technique of melatonin assay developed for use in trout and salmon to measure melatonin levels in cod and sea bass. Melatonin measurements provide a definitive assessment of how photic information is perceived by fish. Preliminary studies have shown a diel pattern in cod with higher levels at night than during the day. Blood samples for melatonin analysis have also been taken from cod maintained in sea pens under continuous illumination at Austevoll Research Station (partner 6). However these, together with cod samples from Bergen are still awaiting analysis by partner 1; these will be carried out when the assay for cod plasma has been fully validated.
Sea bass samples taken in collaboration with partners 1 and 4 during mid-light and mid-dark phase revealed a diel rhythm of melatonin secretion. This is exhibited by elevated levels during the dark phase when compared to light phase levels.
In vitro investigations by partner 1 into the presence of an endogenous rhythm of melatonin secretion in the Atlantic salmon suggest the presence of an endogenous circadian oscillator in the pineal gland. However, the maintenance of this oscillator after pineal removal may be dependant upon the preceding entraining photoperiod. Further studies will be carried out within the next 12 months.
Delayed puberty in rainbow trout
A commercially important finding has been the delay in puberty achieve under long days by trout at the farm of SME partner 2. This means that the egg supplies are being spread throughout the year. The larger eggs produced by first-spawning fish is also an important commercial advance.
Melatonin and estrogen receptor expression in rainbow trout
A major success in the first year of the project has been the development of probes for two of the melatonin receptors known to occur in fish. Investigations have been initiated with the aim of producing further information on the mechanism(s) by which photoperiodic change is transported to the reproductive axis. The development of probes for the melatonin receptors will significantly benefit this work together with ongoing studies to establish whether there are any effects of melatonin on ER expression.
Preliminary work carried out by partner 3 in conjunction with partner 1 has been looking at potential effects of melatonin on estrogen receptor (ER) expression in the liver has revealed no significant light/dark variation in ER expression in rainbow trout. However, an indication that melatonin may affect vitellogenin expression in the liver has prompted further investigations into the effects of pinealectomy and intra-muscular melatonin implantation on rainbow trout.
Partners 3 and 5 have also produced three separate sequences for the melatonin receptor, two of which appear to have a 90% homology with the Zebrafish ZEBIA2 receptor sequence (88.2%).The third sequence showed a 79.7% homology to the Zebrafish ZEB IB.
Studies into melatonin receptor expression have been undertaken by partners 3 and 5 using Northern blotting and in situ hybridisation. Northern blot analysis of brain, pituitary, liver and gonads detected a weak expression in the brain with probes corresponding to different melatonin receptor subtypes (two Mel1A and one Mel1B). In situ hybridisation using probes for the Mel 1 A subtype consistently detected MRNA in the pretectal and thalamic regions, the optic tectum, the cerebellum and the torus semi-circularis. Very low expression, if any, has been found to occur in the neuroendocrine regions controlling the pituitary gonadotrophic functions and in the pituitary itself. These results suggest that the primary targets of melatonin are components of the neurosensorial, in particular visual, systems confirming data obtained from iodinated melatonin binding studies.
Ontogenic variation in melatonin binding sites
A further important finding was the differential levels of melatonin binding found in immature and maturing fish. This may be related to the sites involved in the transduction of photoperiodic information to the reproductive axis.
This project involves the maintenance of stocks of trout, salmon, bass and cod under a range of different photoperiod and temperature regimes. From these experimental fish, samples will be taken for assessments of the changes in activity of the hypothalamic-pituitary-gonadal axis using specific assays for GnRH, GtH, vitellogenin and steroid hormones. Parallel experiments will be conducted on the farms of the SMEs involved in the project. In addition, studies will be made of the role of the pineal gland and melatonin in the transmission of the photoperiodic response to the reproductive axis. This will involve studies of the distribution of melatonin receptors in relation to that of GnRH and other hypothalamic neurones, which have been implicated in the control of reproductive function. Techniques to be utilised include autoradiography, immunohistochemistry and the development and use of a series of molecular biology probes.
Funding SchemeCSC - Cost-sharing contracts
Torre De La Sal (Castellón)
AB21 9SB Aberdeen
BT40 1JJ Larne