Relationships between environmental changes, maturity, growth rate and plasma insulin-like growth factor-I (IGF-I) in female rainbow trout.

Size reflecting growth rate, energy balance or nutritional status is regarded as an important determinant of the ability of trout to undergo puberty. The relationship of a change in photoperiod, either natural (SNP) or advancing (ADV), with growth, IGF-I and reproduction was investigated in virgin female rainbow trout. Under SNP 63% of the population attained maturity while only 29% spawned 6 months in advance in the ADV regime. Under SNP both size and growth rate in late spring-early summer appeared to determine whether an individual may initiate reproduction while condition factor appeared to be a better predictor in the ADV regime. A complete seasonal relationship between plasma IGF-I, daylength and temperature was demonstrated under natural conditions, and provides direct evidence for the relationship between reproduction and IGF-I. Conversely, trout maintained under ADV exhibited a significantly different plasma IGF-I profile relative to those under a natural photoperiod. Furthermore, IGF-I levels accurately reflected growth rate prior to elevations in sex steroids, suggesting that IGF-I may provide an endocrine signal between the somatotropic and reproductive axes that growth rate and/or size is sufficient to initiate gonad development. In addition, maturing individuals under SNP typically expressed higher circulating IGF-I levels than those that remained immature and may reflect a greater opportunity for IGF-I to act on the pituitary to stimulate gonadotropin production. We observed elevated levels in maturing fish for 3 months under SNP compared to only 1 month under ADV were observed. This may reflect a reduction in the window of opportunity to initiate reproduction under advancing photoperiods and hence explain the reduction in fish successfully recruited.

A comparative ex vivo and in vivo study of day and night perception in teleosts species using the melatonin rhythm.

The purpose of this study was to determine and compare the light sensitivity of two commercially important, phylogenetically different teleost species in terms of melatonin production. Three series of experiments were performed on both Atlantic salmon and European sea bass. First, a range of light intensities were tested ex vivo on pineal melatonin production in culture during the dark phase. Then, light transmission through the skull was investigated, and finally short-term in vivo light sensitivity trials were performed. Results showed that sea bass pineal gland ex vivo are at least 10 times more sensitive to light than that of the salmon. Light intensity threshold in sea bass appeared to be between 3.8 x 10(-5) and 3.8 x 10(-6) W/m2 in contrast to 3.8 x 10(-4) and 3.8 x 10(-5) W/m2 in salmon. These highlighted species-specific light sensitivities of pineal melatonin production that are likely to be the result of adaptation to particular photic niches. Light transmission results showed that a significantly higher percentage of light penetrates the sea bass pineal window relative to salmon, and confirmed that penetration is directly related to wavelength with higher penetration towards the red end of the visible spectrum. Although results obtained in vivo were comparable, large differences between ex vivo and in vivo were observed in both species. The pineal gland in isolation thus appeared to have different sensitivities as the whole animal, suggesting that retinal and/or deep brain photoreception may contribute, in vivo, to the control of melatonin production.

Photoperiod influences growth rate and plasma insulin-like growth factor-I levels in juvenile rainbow trout, Oncorhynchus mykiss.

The effect of different photoperiod regimes and the subsequent influence of melatonin on growth and insulin-like growth factor-I (IGF-I) were assessed in juvenile rainbow trout. In Experiment 1, triplicate groups of all female underyearling rainbow trout were exposed to one of three photoperiods; simulated natural photoperiod (SNP), constant short-days (LD 8:16), or constant long-days (LD 18:6) from June to December 2000 under ambient water temperatures. Fish exposed to LD 18:6 grew to a significantly heavier mean weight than the other treatments. Regression analysis showed a strong correlation between circulating plasma IGF-I, growth rate and temperature. Furthermore, it was apparent that fish exposed to LD 18:6 expressed significantly higher circulating levels of IGF-I. In a second experiment, duplicate groups of all female yearling trout were exposed to one of three photoperiods; SNP, LD 8:16, or constant light (LL), with sub groups receiving either a slow-release melatonin implant (18 mg), sham implant or left intact (control). LL increased growth rate in controls, reaching a significantly greater weight than SNP or LD 8:16 photoperiods but did not affect circulating IGF-I levels. Melatonin implants reduced growth rate in all photoperiod treatments below that of their respective controls but again did not affect circulating IGF-I levels. No differences in growth rate were found in implanted fish between photoperiods suggesting that a diel cycle of melatonin is necessary for the perception of daylength. These results would indicate that extended photoperiods (LD 18:6) may cause direct photostimulation of growth through up-regulation of IGF-I production. In contrast, in the absence of a changing diel melatonin signal, growth appeared to be maintained by a possible underlying endogenous rhythm, which was phase advanced under LL, as such plasma IGF-I levels simply reflected growth rate rather than photostimulation of the somatotropic axis. Overall, these findings indicate that measuring plasma IGF-I may be a useful tool for studying environmental influences on growth in rainbow trout.

Analysis of repetitive DNA sequences in the sex chromosomes of Oreochromis niloticus.

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.

Molecular-cytogenetic analysis reveals sequence differences between the sex chromosomes of Oreochromis niloticus: evidence for an early stage of sex-chromosome differentiation.

Sex determination in the Nile tilapia, Oreochromis niloticus, is primarily genetic, with XX females and XY males. A candidate sex-determining region in the terminal region of the largest chromosome pair has been identified by analysis of meiotic chromosomes. This region shows an inhibition of pairing and synapsis in the XY genotype, but not in XX or YY genotypes, suggesting that recombination is inhibited. Here we show that chromosome microdissection and subsequent amplification by degenerate oligonucleotide-primed PCR (DOP-PCR) can be used to produce in situ hybridization probes to this largest pair of O. niloticus chromosomes. Furthermore, analysis of the comparative hybridization of X and Y chromosome-derived probes to different genotypes provides the first demonstration that sequence differences exist between the sex chromosomes of O. niloticus. This provides further support for the theory that this chromosome pair is related to sex determination and further suggests that the sex chromosomes are at a very early stage of divergence.

Karyotype evolution in Tilapia: mitotic and meiotic chromosome analysis of Oreochromis karongae and O. niloticus x O. karongae hybrids.

The karyotype of Oreochromis species is considered to be highly conserved, with a diploid chromosome complement of 2n = 44. Here we show, by analysis of mitotic and meiotic chromosomes, that the karyotype of O. karongae, one of the Lake Malawi 'chambo' species, is 2n = 38. This difference in chromosome number does not prevent the production of inter-specific hybrids between O. niloticus (2n = 44) and O. karongae (2n = 38). Analysis of the meiotic chromosomes of the O. niloticus x O. karongae hybrids indicates that three separate chromosome fusion events have occurred in O. karongae. Comparison of the O. karongae and O. niloticus karyotypes suggests that these consist of one Robertsonian fusion and two fusions of a more complex nature.

Early origins of the X and Y chromosomes: lessons from tilapia.

Differentiated sex chromosome pairs in diverse species display certain common characteristics, normally comprising one largely heterochromatic genetically inactive chromosome and one euchromatic genetically active chromosome (e.g. the mammalian Y and X respectively). It is widely accepted that dimorphic sex chromosomes evolved from homologous pairs of autosomes. Although the exact mechanisms through which the pair diverged are not fully understood, an initial suppression of recombination in the sex-determining region is required by all of the major theories. Here we address the question of the mechanism by which this initial suppression of recombination occurs. Our model postulates that the stochastic, de novo accumulation of heterochromatin in the sex determining region can delay pairing of the sex chromosomes in meiosis, resulting in a decrease in recombination. Data to support this model is presented from the cichlid fish, Oreochromis niloticus. Although such a decrease would in most circumstances be evolutionarily disadvantageous, if the region concerned included the major sex determining gene and other gene(s) with sex-specific functions, then this would be selectively advantageous and could trigger the process(es) which, ultimately, lead to the differentiation of the sex chromosomes.

Identification of putative sex chromosomes in the blue tilapia, Oreochromis aureus, through synaptonemal complex and FISH analysis.

Sex determination in the blue tilapia, Oreochromis aureus, is primarily a ZW female-ZZ male system. Here, by analysis of the pachytene meiotic chromosomes of O. aureus, we demonstrate the presence of two distinct regions of restricted pairing present only in heterogametic fish. The first, a subterminal region of the largest bivalent is located near to the region of unpairing found in the closely related species O. niloticus, while the second is in a small bivalent, most of which was unpaired. These results suggest that O. aureus has two separate pairs of sex chromosomes.

Natural copepods are superior to enriched artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids.

Replicate groups of halibut larvae were fed to d 71 post-first feeding (PFF) either the marine copepod, Eurytemora velox, or Artemia nauplii doubly enriched with the marine chromist or golden algae, Schizochytrium sp., (Algamac 2000) and a commercial oil emulsion (SuperSelco). The fatty acid compositions of eyes, brains and livers from larvae fed the two diets were measured, and indices of growth, eye migration and skin pigmentation were recorded along with histological examinations of eye and liver. The docosahexaenoic acid [22:6(n-3); DHA]/eicosapentaenoic acid [20:5(n-3); EPA] ratios in Artemia nauplii enriched with the SuperSelco and Algamac 2000 were 0.4 and 1.0, respectively. The E. velox copepods were divided into two size ranges (125-250 and 250-400 microm) with the smaller size range containing the highest level of (n-3) highly unsaturated fatty acids (HUFA). The DHA/EPA ratios for the two size ranges of copepods were 2.0 and 0.9, respectively. The total lipids of eyes, brains and livers of larvae fed copepods had higher levels of DHA and lower levels of EPA than those of larvae fed enriched Artemia. The percentage of survival of the halibut larvae was significantly higher when copepods rather than enriched Artemia nauplii were fed, but larval specific growth rates did not differ. The indices of eye migration were high and not significantly different in larvae fed the two diets, but the percentage of larvae undergoing successful metamorphosis (complete eye migration and dorsal pigmentation) was higher in larvae fed copepods (40%) than in larvae fed enriched Artemia (4%). The rod/cone ratios in histological sections of the retina were 2.5 +/- 0.7 in larvae fed copepods and 1.3 +/- 0.6 in larvae fed enriched Artemia (P < 0.01). Histological examination of the livers and intestines of the larvae were consistent with better assimilation of lipid from copepods than lipid from Artemia nauplii up to 46 d post-first feeding. Thus, marine copepods are superior to enriched Artemia as food for halibut larvae in terms of survival, eye development and pigmentation, and this superiority can be related to the level of DHA in the feed.

Photoperiod-induced phase-shifts of the endogenous clock controlling reproduction in the rainbow trout: a circannual phase-response curve.

Different groups of winter-spawning female rainbow trout that had been maintained under seasonally changing daylength and temperature were exposed to 2 months of continuous light at different times of the year. The same photoperiod produced advances in the time of spawning of up to 232 days and delays of up to 80 days, depending upon the timing of exposure in relation to the phase of the reproductive cycle. The proportion of fish spawning in each group varied from 18% to 100%, again dependent on the timing of exposure to continuous light. The photoperiod-induced changes in spawning time can be interpreted as phase-dependent phase-shifts of an endogenous circannual clock controlling maturation. It is proposed that long days, occurring earlier or later than they would under a natural photoperiod, were perceived as indications that the clock was running slow or fast, thus initiating corrective forward adjustments (advance phase-shifts) or backward adjustments (delay phase-shifts), respectively. Collectively, these responses can be described in the form of a circannual phase-response curve.

Melatonin rhythms in Atlantic salmon (Salmo salar) maintained under natural and out-of-phase photoperiods.

Diel changes in circulating melatonin were measured in juvenile Atlantic salmon, Salmo salar, maintained under natural and out-of-phase seasonal photocycles. Under natural daylengths of autumn, winter, spring, and summer circulating melatonin levels were inversely related to light intensity, with levels low during the day and high at night. The duration of the nocturnal increase in circulating melatonin was related to the duration of darkness, i.e., longer in winter than in summer. Under simulated seasonal photocycles circulating melatonin concentrations measured in August, October, and December were also elevated for the duration of darkness, irrespective of whether the photoperiods were synchronized or 6 months out-of-phase with the natural light and temperature cycles. Circulating melatonin also provided an accurate representation of the prevailing photoperiod in fish initially maintained on simulated natural photocycles, either synchronized or 6 months out-of-phase with the natural light cycle, and then held for 3 months on daylengths approximating the summer and winter solstices. Well-defined melatonin rhythms were always present, irrespective of time of year, photoperiod, and temperature. The amplitude of the nocturnal increase in circulating melatonin was similar in groups of fish maintained under simulated seasonal photoperiods 6 months out-of-phase with each other, but otherwise identical conditions, indicating that daylength per se did not influence the amplitude of the melatonin rhythm. The amplitude of the melatonin rhythm was slightly higher during the summer months, suggesting that temperature may modify circulating melatonin levels. These results demonstrate that circulating melatonin profiles always reflect the prevailing daylength and hence have the potential to provide the Atlantic salmon with accurate information on daily and calendar time, which could be utilised to time daily and seasonal events.

Measurement and prediction of digestible energy values in feedstuffs for the herbivorous fish tilapia (Oreochromis niloticus Linn.).

Digestible energy (DE) values were measured in a selection of feedstuffs for the tilapia (Oreochromis niloticus Linn.) and used to develop equations for predicting DE values of a wider range of feedstuffs from chemical analyses. Preliminary work examined the influences of substitution level in a reference diet and adaptation over time on DE values for soya-bean meal. Length of adaptation period significantly affected DE values (P less than 0.01), but substitution level, over the range 200-600 g soya-bean meal/kg reference diet, did not. The DE values of sixteen feedstuffs, thirteen derived from plant sources and three animal by-products, were subsequently determined. DE values for plant-derived feedstuffs were found to be higher than those quoted in the literature for trout (Oncorhynchus mykiss) and catfish (Ictalurus punctatus), whereas DE values for animal-derived feedstuffs were lower than those for trout and pigs. It was concluded that energy values quoted in tables of feed composition for other species are inaccurate when used as proxy values for tilapia. Regression equations were therefore computed using data from the present study to provide a rapid means of predicting DE values of feedstuffs for tilapia. Equations using neutral-detergent fibre as an independent variable were found to predict DE values of plant-derived feedstuffs reliably. Where fibre values were not used as independent variables, available carbohydrate and crude protein (nitrogen x 6.25) were found to be useful predictors of DE values. These equations offer the possibility of reducing the need for time-consuming digestibility trials with tilapia when formulating least-cost production diets for this species.

Seasonal patterns in serum levels of thyroid hormones and sex steroids in relation to photoperiod-induced changes in spawning time in rainbow trout, Salmo gairdneri.

Serum levels of thyroid hormones (T4 = L-thyroxine and T3 = 3,5,3'-triiodo-L-thyronine) were measured and correlated with previously published levels of 17 beta-estradiol, testosterone (T), and Ca2+ (index of vitellogenin) in four groups of female trout held for 1-3 years at 8.5-9.0 degrees on a ration of 0.5% of body weight day-1 under different constant photoperiod regimes. In group 1, trout under a regimen of 18L:6D became sexually mature (SM) in April/May and then SM again in September/October; in group 2, trout under a regimen of 6L:18D became SM in January/February; in group 3, trout under a regimen of 6L:18D became SM in March/April; in group 4 trout under a regimen of 18L:6D followed by 10L:14D became SM in September/October. In all groups, regardless of the photoperiod-induced changes in spawning time, serum T3 exceeded T4 and both serum T3 and T4 patterns showed a consistent relationship with the sequence of steroid hormone changes and spawning time. Thyroid hormone levels were high during previtellogenesis but fell as sex steroids and serum Ca2+ increased. T3 and T4 were lowest before spawning when sex steroids were at their peak and then increased sharply following spawning as sex steroid levels declined. Peak serum T coincided with ovulation and usually preceded the postreproductive surge in serum T3 and T4. The hypothesis is discussed that T3 enhances early ovarian development, but as energy-demanding vitellogenesis proceeds T3 formation is suppressed, thereby curtailing growth and favoring energy partition to the ova.

Thyroxine binding globulin (TBG) and thyroxine binding prealbumin (TBPA) measurement, compared with the conventional T3 uptake in the diagnosis of thyroid disease.

An electroimmunoassay for the determination of thyroxine binding prealbumin is described. The diagnostic efficiency of the assay when used in conjunction with the serum thyroxine as a thyroxine: thyroxine binding prealbumin ratio, is compared with the conventional free thyroxine index and the more recently developed thyroxine: thyroxine binding globulin ratio. The population studied included euthyroid, hypothyroid and hyperthyroid patients and also those who were either pregnant or receiving oral contraceptive therapy. Despite recent evidence establishing the theoretical/practical advantages of using a direct measurement for thyroid binding proteins rather than an indirect method (tri-iodothyronine uptake), results obtained from this study suggest that, for the majority of patients requiring biochemical assessment, the free thyroxine index is still the superior discriminator of thyroid abnormality.

Inhibition of thyroidal iodine release by oestrogens in euthyroid subjects.

The effects of oestrogens on the release of thyroxine iodide (T4 I) and non-T4 I by the thyroid gland of 6 normal subjects and 3 thyrotrophin (TSH)-treated patients with hypopituitarism were assessed before, during and after treatment with 25 mg of Dienoestrol (Glaxo Lab.), orally, daily, during 8 days. Each subject received an iv injection of 150 microCi of 125I followed 6 days later by an iv injection of 75 microCi of [131I]T4. Blood samples and collections of urine were obtained every 12 h. From plasma and urinary (U) radioactivity, the ratio U[125I]/U[131I], U[125I]/PB[125I] and U[131I]/PB[131I] were obtained and served to calculate thyroidal iodine release and non-T4 I secretion by the thyroid gland. T4 I secretion was equivalent to T4 I disposal rate obtained by the semilogarithmic regression line of PB[131I]. In normal subjects, oestrogens decreased T4 I secretion from 491 +/- 57 (SD) nmol/day to 451 +/- 60 nmol (P less than 0.025), while in the same subjects serum TSH increased with oestrogens from 3.3 +/- 1.1 to 6.6 +/- 2.1 mU/l (P less than 0.01). The non-T4 I secretion had a mean of 585 +/- 307 nmol/day during control, 324 +/- 199 nmol during oestrogens (P less than 0.01) and 480 +/- 286 nmol oestrogens (P less than 0.05 vs oestrogens). In the TSH-treated patients oestrogens induced non-significant reductions in T4 I and non-T4 I secretion, although total iodine secretion decreased significantly (P less than 0.05). The data is consonant with a partial inhibition by oestrogens of thyroid gland release.