Superoxidase dismutases (SODs) in the European eel: Gene characterization, expression response to temperature combined with hormonal maturation and possible migratory implications.

Superoxide dismutases (SODs) are antioxidant enzymes that protect cells from oxidation. Three SODs have been identified in mammals, but there is limited information in teleosts. This study investigates SODs in the European eel and their expression patterns during testis maturation. Phylogenetic and synteny analyses revealed SODs paralogs and their evolution in vertebrates. The eel possesses one SOD1 and two SOD2/3 (a and b), indicating SOD2 and SOD3 duplication in elopomorphs. SODs expression were then evaluated in various male and female tissues. SOD1 is more expressed in females, while SOD2a and SOD2b dominate brain-pituitary-gonad tissues in both sexes. SOD3a showed predominant expression in the ovary and the male livers, whereas SOD3b was found in the pituitary and brain of both sexes. The effects of different maturation protocols (standard hormonal treatment vs. same protocol preceded with cold seawater pre-treatment) on SODs expression during testis maturation were evaluated. Salinity increase at the onset of standard treatment at 20 °C, simulating early migration, upregulated SOD1, SOD2a, and SOD2b, coinciding with spermatogonia type A differentiated cells dominance. Thereafter, SOD2a and SOD3a decreased, while SOD2b increased during hormonal treatment-induced spermatogenesis. Pre-treatment with seawater at 10 °C, mimicking the conditions at the beginning of the seawater migration, downregulated SOD1 but increased SOD3a expression. Finally, the standard hormonal treatment, replicating spawning at higher temperatures, downregulated SOD1 in eels without any pre-treatment while SOD2a expression increased in pre-treated eels. This study revealed tissue-specific, sex-dependent, and maturation-related SOD expression patterns, predicting SODs dynamic expression profiles during their reproductive migration.

Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes.

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Ex vivo exposure to titanium dioxide and silver nanoparticles mildly affect sperm of gilthead seabream (Sparus aurata) - A multiparameter spermiotoxicity approach.

Nanoparticles (NP) are potentially reprotoxic, which may compromise the success of populations. However, the reprotoxicity of NP is still scarcely addressed in marine fish. Therefore, we evaluated the impacts of environmentally relevant and supra environmental concentrations of titanium dioxide (TiO2: 10 to 10,000 µg·L-1) and silver NP (Ag: 0.25 to 250 µg·L-1) on the sperm of gilthead seabream (Sparus aurata). We performed short-term direct exposures (ex vivo) and evaluated sperm motility, head morphometry, mitochondrial function, antioxidant responses and DNA integrity. No alteration in sperm motility (except for supra environmental Ag NP concentration), head morphometry, mitochondrial function, and DNA integrity occurred. However, depletion of all antioxidants occurred after exposure to TiO2 NP, whereas SOD decreased after exposure to Ag NP (lowest and intermediate concentration). Considering our results, the decrease in antioxidants did not indicate vulnerability towards oxidative stress. TiO2 NP and Ag NP induced low spermiotoxicity, without proven relevant ecological impacts.

Identification and stable expression of vitellogenin receptor through vitellogenesis in the European eel.

In teleosts, vitellogenin (Vtg) is a phospholipoglycoprotein synthesized by the liver, released into the blood circulation and incorporated into the oocytes via endocytosis mediated by the Vtg receptor (VTGR) to form the yolk granules. The VTGR is crucial for oocyte growth in egg-laying animals but is also present in non-oviparous vertebrates, such as human. The VTGR belongs to the low-density lipoprotein receptor superfamily (LDLR) and is also named very-low-density lipoprotein receptor (VLDLR). In this study, we identified and phylogenetically positioned the VTGR of a basal teleost, the European eel, Anguilla anguilla. We developed quantitative real-time PCR (qRT-PCR) and investigated the tissue distribution of vtgr transcripts. We compared by qRT-PCR the ovarian expression levels of vtgr in juvenile yellow eels and pre-pubertal silver eels. We also analyzed the regulation of ovarian vtgr expression throughout vitellogenesis in experimentally matured eels. The Vtg plasma level was measured by homologous ELISA experimental maturation. Our in silico search and phylogenetical analysis revealed a single vtgr in the European eel, orthologous to other vertebrate vtgr. The qRT-PCR studies revealed that vtgr is mainly expressed in the ovary and also detected in various other tissues such as brain, pituitary, gill, fat, heart, and testis, suggesting some extra-ovarian functions of VTGR. We showed that vtgr is expressed in ovaries of juvenile yellow eels with no higher expression in pre-pubertal silver eels nor in experimentally matured eels. This suggests that vtgr transcription already occurs during early pre-vitellogenesis of immature eels and is not further activated in vitellogenic oocytes. European eel Vtg plasma level increased throughout experimental maturation in agreement with previous studies. Taken together, these results suggest that vtgr transcript levels may not be a limiting step for the uptake of Vtg by the oocyte in the European eel.

Sperm motility in fish: technical applications and perspectives through CASA-Mot systems.

Although a relatively high number of sperm quality biomarkers have been reported over the years in several fish species, sperm motility is nowadays considered the best biomarker for fish spermatozoa. The first scientific reports focusing on fish sperm motility date from a century ago, but the objective assessment allowed by computer-aided sperm analysis (CASA-Mot) systems was not applied to fish species until the mid-1980s. Since then, a high number of sperm kinetic parameters from more than 170 fish species have been reported in more than 700 scientific articles, covering a wide range of topics, such as sperm physiology, sperm storage, broodstock management, the phenomenon of sperm competition, ecotoxicology and understanding the life cycle of the species. The sperm kinetic parameters provided by CASA-Mot systems can serve as powerful and useful tools for aquaculture and ecological purposes, and this review provides an overview of the major research areas in which fish sperm motility assessment by a CASA-Mot system has been used successfully.

Using specific recombinant gonadotropins to induce spermatogenesis and spermiation in the European eel (Anguilla anguilla).

New specific European eel (Anguilla anguilla) recombinant gonadotropins (aarGths) produced in the ovarian cells of Chinese hamsters (CHO) were used to induce maturation in captive male eels. In the first experiment, five different hormonal treatments were assayed: one group was given a constant dose of recombinant European eel follicle-stimulating hormone (aarFsh; 4 µg/fish) for 9 weeks, and the second group received a constant dose of recombinant European eel luteinizing hormone (aarLh; 2 µg/fish) also for 9 weeks. The other three groups were injected with different combinations of both aarGths (some doses constant, some variable). All five treatments stimulated androgen synthesis, but the increase was more pronounced in the fish treated with a combination of both aarGths. Unlike aarLh, aarFsh alone was able to induce spermiation, the best results were achieved in the fish that were treated with a constant dose of aarFSH and an increasing dose of aarLH, with spermiation being induced (20% motile cells) despite the fact that these fish were immature at the start of the experiment. In order to improve sperm quality, a second experiment was performed. Immature males received three constant doses of aarFsh (2.8, 1.4 or 0.7 µg/fish) and increasing doses of aarLh (every 3 weeks; 1, 2, 6 µg/fish). All the treatments induced spermiation, however the best sperm quality (with ≥50% motile cells) was observed in the males treated with the highest dose of aarFsh. In conclusion, these specific recombinant gonadotropins have demonstrated their capacity to induce spermatogenesis and spermiation in vivo in a teleost fish, the European eel.

Embryonic development of the grass pufferfish (Takifugu niphobles): From egg to larvae.

Tetraodontidae (pufferfish) family members carry the smallest genomes among vertebrates, and these pocket-sized genomes have directly contributed to our understanding of the structure and evolution of higher animals. The grass pufferfish (Takifugu niphobles) could be considered a potential new model organism for comparative genomics and development due to the potential access to embryos, and availability of sequence data for two similar genomes: that of spotted green pufferfish (Tetraodon nigroviridis) and Fugu (Takifugu rubripes). In this study, we provide the first description of the normal embryonic development of T. niphobles, by drawing comparisons with the closely related species cited above. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored at a constant temperature consistent with natural conditions. T. niphobles development was divided into seven periods of embryogenesis: the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods; and stages subdividing these periods are defined based on morphological characteristics. The developmental stage series described in this study aims to provide the utilization of T. niphobles as an experimental model organism for comparative developmental studies.

A comparison of techniques for studying oogenesis in the European eel Anguilla anguilla.

A multi-technique approach was used to study the changes occurring in European eel Anguilla anguilla ovaries during hormonally-induced vitellogenesis. Aside from classic techniques used to monitor the vitellogenic process, such as ovary histology, fat content analysis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and vitellogenin enzyme linked immunosorbent assay (ELISA), a new technique, Fourier-transform infrared (FT-IR) microspectroscopy, was used to analyse A. anguilla ovaries. The results from the different techniques provided different ways of approaching the same process. Although it is considered a time consuming approach, of all the employed techniques, histology provided the most direct evidences about vitellogenesis. SDS-PAGE and ELISA were also useful for studying vitellogenesis, whereas fat analysis cannot be used for this purpose. The FT-IR analysis provided a representative IR spectrum for each ovarian stage (previtellogenic stage, early vitellogenic stage, mid-vitellogenic stage and late vitellogenic stage), demonstrating that it is a valid method able to illustrate the distribution of the oocytes within the ovary slices. The chemical maps obtained confirmed changes in lipid concentrations and revealed their distribution within the oocytes at different maturational stages. When the results and the accuracy of the FT-IR analysis were compared with those of the traditional techniques commonly used to establish the vitellogenic stage, it became evident that FT-IR is a useful and reliable tool, with many advantages, including the fact that it requires little biological material, the costs involved are low, analysis times are short and last but not least, the fact that it offers the possibility of simultaneously analysing various biocomponents of the same oocyte.

First Production of Larvae Using Cryopreserved Sperm: Effects of Preservation Temperature and Cryopreservation on European Eel Sperm Fertilization Capacity.

Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20°C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20°C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae ('cryolarvae') were obtained 55 h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success.

Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen.

There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90s), the thermal regimen (i.e. 10°C (T10) or 15°C (T15) and up to 20°C, or constant at 20°C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30s is recommended as the standard time for motility acquisition. When eels were kept at 20°C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10, T15) delayed spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7-20), and the S3 yield was also higher overall (61.8±1.3%; mean ± s.e.m.) and more stable over time than the other hormonal treatments (averaging 53.0±1.4%). Using T20 and rhCG would be more economical and practical, allowing us to obtain a higher number of S3 spermatozoa over an extended time.

Relationship between sperm quality parameters and the fatty acid composition of the muscle, liver and testis of European eel.

This study looks at the correlations that fatty acids have with different tissues in the European eel (Anguilla anguilla L.) during hormonally-induced sexual maturation, with different sperm quality parameters. In order to evaluate the different dynamics of the use of fatty acids, a categorization of the results from each sperm quality parameter (volume, concentration, motility and velocity) was performed. Low and moderate correlations were observed between muscle tissue and some sperm quality parameters but no high correlations were found. Eicosapentaenoic acid (20:5n3, EPA) in the liver seems to have a role in determining the volume of sperm produced. This can be explained by the fact that EPA is a major requirement in the early phases of sperm production (probably as a component of the spermatozoal membrane). In addition, the levels of α-linolenic acid (18:3-n3, ALA) and linoleic acid (18:2-n6, LA) in the liver decreased when sperm motility increased. In all the tissues, a negative correlation was observed between arachidonic acid (20:4n-6, ARA) and the different sperm velocity parameters. The fact that an increase in the consumption of ARA coincides with an increase in the speed of spermatozoa, highlights the important role that this fatty acid plays not only in sperm production, but also in sperm velocity. All this information could prove useful in the development of suitable broodstock diets to improve sperm quality and subsequently, the larval development of this species.

Sperm motility parameters and spermatozoa morphometric characterization in marine species: a study of swimmer and sessile species.

The biodiversity of marine ecosystems is diverse and a high number of species coexist side by side. However, despite the fact that most of these species share a common fertilization strategy, a high variability in terms of the size, shape, and motion of spermatozoa can be found. In this study, we have analyzed both the sperm motion parameters and the spermatozoa morphometric features of two swimmer (pufferfish and European eel) and two sessile (sea urchin and ascidian) marine species. The most important differences in the sperm motion parameters were registered in the swimming period. Sessile species sperm displayed notably higher values than swimmer species sperm. In addition, the sperm motilities and velocities of the swimmer species decreased sharply once the sperm was activated, whereas the sessile species were able to maintain their initial values for a long time. These results are linked directly to the species-specific lifestyles. Although sessile organisms, which show limited or no movement, need sperm with a capacity to swim for long distances to find the oocytes, swimmer organisms can move toward the female and release gametes near it, and therefore the spermatozoa does not need to swim for such a long time. At the same time, sperm morphology is related to sperm motion parameters, and in this study an in-depth morphometric analysis of ascidian, sea urchin, and pufferfish spermatozoa, using computer-assisted sperm analysis software, has been carried out for the first time. A huge variability in shapes, sizes, and structures of the studied species was found using electron microscopy.

The regulation of aromatase and androgen receptor expression during gonad development in male and female European eel.

This research investigated the regulation of aromatase and androgen receptor gene expression in the brain-pituitary-gonad (BPG) axis of male and female European eels (Anguilla anguilla) during induced sexual maturation. Complete A. anguilla aromatase (aa-cyp19a1) and partial androgen receptor α and ß (aa-ara and aa-arb) sequences were isolated, and qPCR assays were validated and used for quantification of transcript levels for these three genes. Expression levels of the genes varied with sex, tissue and stage of maturation. aa-arb was expressed at higher levels than aa-ara in the pituitary and gonad in both sexes, suggesting aa-arb is the physiologically most important androgen receptor in these tissues. In the female brain, a decrease in aa-ara and an increase in aa-cyp19a1 were observed at the vitellogenic stage. In contrast, a progressive increase in all three genes was observed in the pituitary and ovaries throughout gonadal development, with aa-arb and aa-cyp19a1 reaching significantly higher levels at the vitellogenic stage. In the male pituitary, a decrease in aa-arb and an increase in aa-cyp19a1 were observed at the beginning of spermatogenesis, and thereafter remained low and high, respectively. In the testis, the transcript levels of androgen receptors and aa-cyp19a1 were higher during the early stages of spermatogenesis and decreased thereafter. These sex-dependent differences in the regulation of the expression of aa-ara, aa-arb and cyp19a1 are discussed in relation to the role of androgens and their potential aromatization in the European eel during gonadal maturation.

Evaluation of methods to determine sperm density for the European eel, Anguilla anguilla.

European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives were to evaluate methods to estimate European eel sperm density including spermatocrit, computer-assisted sperm analysis (CASA) and flow cytometry (FCM), using Neubauer Improved haemocytometer as benchmark. Initially, relationships between spermatocrit, haemocytometer counts and sperm motility were analysed, as well as the effect of sperm dilution on haemocytometer counts. Furthermore, accuracy and precision of spermatocrit, applying a range of G-forces, were tested and the best G-force used in method comparisons. We found no effect of dilution on haemocytometer sperm density estimates, whereas motility associated positively with haemocytometer counts, but not with spermatocrit. Results from all techniques, spermatocrit, CASA and FCM, showed significant positive correlations with haemocytometer counts. The best correlation between spermatocrit and haemocytometer counts was obtained at 6000 × g (r = 0.68). Of two CASA variants, one or three photographic fields (CASA-1 and CASA-2), CASA-2 showed a very high accuracy to haemocytometer counts (r = 0.93), but low precision (CV: CASA-2 = 28.4%). FCM was tested with and without microfluorospheres (FCM-1 and FCM-2), and relationships to haemocytometer counts were highly accurate (FCM-1: r = 0.94; FCM-2: r = 0.88) and precise (CV: FCM-1 = 2.5; FCM-2 = 2.7%). Overall, CASA-2 and FCM-1 feature reliable methods for quantification of European eel sperm, but FCM-1 has a clear advantage featuring highest precision and accuracy. Together, these results provide a useful basis for gamete management in fertilization protocols.

Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software.

The development of powerful computer-assisted sperm analysis software has made kinetic studies of spermatozoa possible. This system has been used and validated for several species, but some technical questions have emerged regarding fish sample evaluations (i.e., frame rate, sperm dilution, chamber model, time of analysis, magnification lens, etc.). In the present study, we have evaluated the effects of different procedural and biological settings with the aim to correctly measure sperm quality parameters of the European eel. The use of different chambers did not affect the sperm motility parameters. However, regarding lens magnification, 10× was the most accurate lens, showing the least variation in the acquired data. Similarly, the frame rate setting resulted in a dramatic effect in some sperm kinetic parameters, primarily in terms of curvilinear velocity; we therefore recommend using the camera's highest available frame rate setting. Finally, the reduction in sperm motility over postactivation times suggests that sperm analysis should be performed within the first 60 seconds after activation of the European eel sperm. In conclusion, some protocol variables of sperm analysis by computer-assisted sperm analysis software can affect the measurement of eel sperm quality parameters, and should be considered before directly comparing results obtained by different laboratories. Moreover, because marine fish species show relatively similar features of sperm kinetic parameters, these results could be considered in the evaluation of the motility of sperm from other fish species.

Variations in the gene expression of zona pellucida proteins, zpb and zpc, in female European eel (Anguilla anguilla) during induced sexual maturation.

Vertebrate eggs are surrounded by an extracellular glycoprotein coat termed zona pellucida (ZP). Integrity of ZP is critical for a correct embryo development. Two zona pellucida protein genes (zpb and zpc) from European eel were characterized, specific qPCR assays developed and their expression in immature males and females carried out. An experimental group of silver-stage eel females was maintained at 18 °C and hormonally induced to sexual maturation by weekly injections of carp pituitary extract during 12 weeks. Changes in zpb and zpc expression during sexual maturation were studied in liver and ovary by qPCR. In liver, no changes were recorded during hormonal treatment, while in ovary expression of both genes decreased during sexual development. These results are a first step in the characterization of ZP in European eel and in the understanding of the mechanism underlying egg envelope formation.

Comparison of two techniques for the morphometry study on gilthead seabream (Sparus aurata) spermatozoa and evaluation of changes induced by cryopreservation.

The development of powerful software has made possible spermatozoa morphology studies. However, some problems have emerged in relation to protocol standardization to compare results from different laboratories. This study was carried out to compare two techniques commonly used (staining vs phase contrast technique) for the morphometry study of gilthead sea bream spermatozoa using an integrated sperm analysis system (ISAS). Spermatozoa morphometry values were significantly affected by the technique used, and phase contrast technique was found to be the more accurate method, showing lower coefficients of variation on spermatozoa morphometry parameters measurements. Moreover, it has been shown that cryopreservation process produces damage in gilthead sea bream spermatozoa, causing negative effects in sperm parameters as spermatozoa morphometry (a decrease in cell volume), motility (from 95 to 68% motile cells) and viability (from 95 to 87% of live cells), being the addition of freezing medium containing cryoprotectant (DMSO) an important factor that caused the morphometry changes.

Heterozygosity-fitness correlations in the gilthead sea bream Sparus aurata using microsatellite loci from unknown and gene-rich genomic locations.

Heterozygosity-fitness correlations (HFC) were assessed for a sample of a gilthead sea bream Sparus aurata population. Two hundred and seventy-one fish were genotyped at 22 known and novel microsatellite loci, from which correlations between the multilocus heterozygosity index (I(MLH) ) and various fitness traits (fork length, mass and specific growth rates) were calculated. Significant global HFCs were found in this sample (0·02 ≤r(2) ≤ 0·08). In addition, all the significant correlations found in this work were negative, indicating that heterozygotes had lower fitness than their homozygote counterparts. Marker location could not explain the observed HFCs. Evidence of inbreeding, outbreeding or population and family structuring was not found in this work. The presence of undetected general effects that may lead to the appearance of HFCs, however, cannot be ruled out. These results seem to be best explained by the occurrence of local effects (due to linkage) or even by possible direct locus advantages.

Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females.

We examined the effect of temperature in European silver eels during their maturation induced by injections of carp pituitary extract on endocrine parameters: pituitary fshß and lhß expression, plasma 17ß-estradiol (E2) and vitellogenin, estrogen receptor 1 (esr1), and vitellogenin 2 (vtg2) expression in liver. A variable thermal regime (T10) that increased from 10° to 14° and 17°C was compared with a constant 20°C regime (T20) during 12 weeks. T10 caused a faster development until week 8, higher fshß, lhß, esr1 expression, and higher E2 levels. The results strongly suggest that T10 is inducing a higher endogenous FSH level which increases the E2 circulating level during vitellogenesis. A variable thermal regime induced an fshß expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.