A rapid, one-step agglutination assay has been developed, based on latex particles sensitized with antibodies against vitellogenin (Vtg), aimed at Atlantic bluefin tuna, Thunnus thynnus (Linnaeus) (ABFT), gender identification. The egg-yolk precursor protein Vtg was used as a gender marker for the assay as it is a female-specific protein synthesized during reproductive maturation. The presence of Vtg in the plasma was revealed in 60-120 s through an agglutination reaction by mixing small volumes of ABFT plasma and an anti-Vtg antibody-latex suspension on a microscope slide. The effectiveness of the present test was restricted to the months of May and June, concomitant with high circulating Vtg levels. Because of its rapidity and ease of performance in the field, the present gender identification assay could be useful for broodstock management in the aquaculture industry as well as in tagging studies on wild populations.

The knowledge of gametogenesis is of paramount importance to develop a reliable technology for Atlantic bluefin tuna (Thunnus thynnus L.) (ABFT) rearing in captivity. The aims of this study were: a) to evaluate the capacity of male ABFT, confined in captivity before puberty, to finalize spermatogenesis; b) to compare germ cell proliferation between wild and captive ABFT. Testis samples were taken from: a) 13 juvenile ABFT reared in the North Adriatic Sea (Croatia); b) 30 adult ABFT reared in the central and western Mediterranean (Spain, Malta and Italy); c) 20 adult wild ABFT captured by tuna traps in Italy and Morocco. Samples were fixed in 10% formalin, dehydrated in ethanol and embedded in paraffin. Proliferating germ cells were identified through the immunohistochemical detection of proliferating cell nuclear antigen (PCNA). The first spiniform ray of the first dorsal spine was taken from the juvenile fish in order to estimate the age through the count of annual discontinuities. Juvenile ABFT captured before puberty were able to finalize spermatogenesis starting from 3 years of age. Germ cell proliferation was delayed in captive-reared ABFT specimen compared to wild individuals. These results seem to indicate that testis maturation can be anticipated in ABFT caught before puberty, but spermatogenesis is somewhat damaged in adult fish reared in captivity compared to wild individuals.

The most commonly observed reproductive dysfunction in male fishes reared in captivity is reduction in sperm volume and quality. The Atlantic bluefin tuna Thunnus thynnus (Osteichthyes: Scombridae) is one of the few large pelagic and migratory marine fishes maintained in captivity with the purpose of establishing breeding populations to support an aquaculture industry. The objectives of the present study were to compare male germ cell proliferation and apoptosis between wild and captive individuals at two different phases of the spermatogenetic cycle, and to evaluate sperm motility characteristics of captive individuals. Histological observations were performed to analyze testicular activity, and germ cell proliferation and apoptosis were evaluated through the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferase mediated d’UTP nick end labeling (TUNEL) method,respectively. Computer-assisted sperm analysis (CASA) was used to evaluate sperm motility. Results showed that germ cell proliferation was delayed and germ cell apoptosis increased in captive animals relative to wild individuals. Sperm motility of samples obtained from captive individuals was anomalous, both in terms of motility duration and swimming efficiency. Thus it appears that rearing in captivity impairs male reproductive function through, at least, changes in germ cell roliferation and apoptosis.

The sequence of vitellogenin A (VgA) and vitellogenin B (VgB) cDNAs in Atlantic bluefin tuna (Thunnus thynnus L.) were determined, and vitellogenin expression levels in the liver and oocyte yolk accumulation were compared in wild and captive-reared individuals. Liver and ovary samples were taken from 31 individuals reared experimentally in three commercial Atlantic bluefin tuna fattening sites in the Mediterranean Sea and from 33 wild individuals caught by commercial traps during the fish’s migration towards their Mediterranean spawning grounds. The total length of VgA cDNA was 5585 nucleotides and that of VgB was 5267 nucleotides. The identity and similarity between deduced amino acid sequences of VgA and VgB were 60% and 78%, respectively. The Atlantic bluefin tuna VgA and VgB amino acid sequences have high similarities with those of other teleost fishes. Relative levels ofVgA and VgB mRNAs were low in April, increased significantly during the reproductive period in May and June, and declined in July. There was a trend towards higher relative levels of VgA and VgB mRNAs in captive fish compared to wild individuals during the reproductive period. The surface occupied by eosinophilic yolk granules in fully vitellogenic oocytes, as well as the frequency of oocytes in late vitellogenesis, was significantly higher in captive compared to wild individuals. The study suggests that the experimental conditions under which Atlantic bluefin tuna individuals were reared allowed the occurrence of normal vitellogenesis, based on gene expression of VgA and VgB in the liver and yolk accumulation in the oocytes. The higher yolk accumulation and frequency of vitellogenic oocytes observed in the ovaries of captive fish suggest that improvements in feeding practices may result in an improved vitellogenic process.

The effects of different stressors on the atretic degeneration of ovarian vitellogenic follicles, as well as on the ovarian mass, were examined in female Atlantic bluefin tuna, Thunnus thynnus (L.), from the Mediterranean Sea. The stressors taken into consideration were short-term starvation (up to 14 days), long-term cage rearing (1 year) and crowding-induced severe panic frenzy. Wild-caught individuals were used as a control group. Fish subjected to either severe panic frenzy or starvation exhibited a decrease in gonad mass and had significantly higher intensity of a atresia in the vitellogenic follicles (means: 78% and 58%, respectively; range: 36–100%) than either wild or long-term caged individuals (means: 32% and 30%, respectively; range: 19–44%). The extensive atresia in fish stressed by severe panic frenzy was observed as early as 24 h after the stressing event. The present study represents the first evidence of the extreme susceptibility of Atlantic bluefin tuna to severe acute stress during vitellogenesis; it also shows that starvation is associated with progressive reabsorption of vitellogenic oocytes.

The cDNA sequences of vitellogenin receptor proteins (VgR+ and VgR−), containing or lacking the O-linked sugar domain, were determined in Atlantic bluefin tuna (Thunnus thynnus L.). VgR− gene expression in the ovary was compared in captive-reared and wild Atlantic bluefin tuna during the reproductive cycle. Gonad samples from adult fish were sampled from 2008 to 2010 from stocks reared in captivity at different commercial fattening operations in the Mediterranean Sea and from wild individuals caught either by traditional tuna traps during their migration towards the spawning grounds in the Mediterranean Sea or by the long-line artisanal fishery. In addition, juvenile male and female Atlantic bluefin tuna were sampled from a farming facility, to obtain baseline information and pre-adulthood amounts of VgR−. The total length of VgR+ cDNA was 4006 nucleotides (nt) and that of VgR− was 3946 nt. Relative amounts of VgR− were greater in juvenile females and in those adults having only previtellogenic oocytes (119 ± 55 and 146 ± 26 folds more than juvenile males, respectively). Amounts of VgR− were less in individuals with yolked oocytes (ripening stage, May–June) and increased after spawning in July (92 ± 20 and 113 ± 13 folds more than juvenile males in ripening and post-spawning fish, respectively). These data suggest that regulation of VgR− is not under oestrogen control. During the ripening period, greater VgR− gene expression was observed in wild fish than in fish reared in captivity, possibly because of (a) differences in water temperature exposure and/or energy storage, and/or (b) an inadequate diet in reared Atlantic bluefin tuna.

The European anchovy, Engraulis encrasicolus, is a multiple-spawning small pelagic fish with a comparatively long reproductive season. From April to October 2009, ovary samples were collected from individuals of the southwestern Adriatic Sea in order to examine ovarian histological changes and assess batch fecundity monthly variations throughout the whole reproductive season. To assess monthly variations of the relative batch fecundity, the correlation between batch fecundity (F) - i.e. the number of oocytes released at each spawning act - and ovary-free body mass (W*) was tested by four regression models; the power equation () was found to be the most suitable to describe correlations. The reproductive season of the anchovy of the central-southern Adriatic population lasts from May to September; in this period, all the oocyte development stages were observed, including hydrated oocytes and postovulatory follicles. In April, most fish had only unyolked oocytes; in October, an extensive atresia of yolked follicles was observed. The slope of all the on monthly regressions did not differ significantly from 1, which shows that relative batch fecundity is constant all over the anchovy size range, throughout the spawning season. In the central-southern Adriatic anchovy population, batch fecundity increased from May to July and then gradually decreased until September. Differences in batch fecundity of the anchovy from different areas of the eastern Atlantic and Mediterranean could possibly be due to both environmental parameters and genetic differences among the different populations.

Contractions of ovarian tunica albuginea, the teleostean cystovary wall layer containing smooth muscle fibres, facilitate oocytes and fluids movements within the ovary, oocytes ovulation and spawning. Fish isotocin, the homologue hormone of mammalian oxytocin, plays a significant role in ovulation, oviduct contraction and spawning. In the present study, ovarian wall spontaneous contraction, as well as isotocin in vitro effect on tunica albuginea contractility, was analysed in female seabream in different reproductive conditions: vitellogenesis, regressing (post-spawning) and extensive atresia. Tunica albuginea spontaneous contractility was recorded using ovary wall strips mounted in an organ bath containing modified Ringer's solution. The strips were then exposed to cumulative doses of isotocin (6, 30, 60 μg/ml). Female seabream in regressing condition exhibited the highest level of tunica albuginea spontaneous contraction amplitude compared with the other two groups. Only fish in vitellogenesis state showed a significant increase in contraction amplitude after isotocin administration at the dose of 30 μg/ml. The same group exhibited also a significant isotocin dose-dependent decrease in the contractile frequency. These results confirm the involvement of isotocin in stimulating tunica albuginea contractile activity during the oestrogen-regulated phase of vitellogenesis, whereas the absence of significant effects of isotocin on ovarian contractility in fish at the regressing state might be ascribed to the occurrence of a contractile activity autonomously regulated by the internal pacemaker system. The absence of exposed isotocin receptors could explain the lack of effects of the isotocin administration in seabream showed extensive atresia of the follicular cells.

The Atlantic bluefin tuna Thunnus thynnus (ABFT) is intensely fished in the Mediterranean Sea to supply a prosperous capture-based mariculture industry. Liver apoptotic structures and tumor necrosis factor (TNF) gene expression were determined in: wild ABFT caught in the eastern Atlantic; juvenile ABFT reared in the central Adriatic Sea; juvenile ABFT reared in the northern Adriatic Sea; adult ABFT reared in the western Mediterranean. The highest density of liver apoptotic structures was found in the juveniles from the northern Adriatic. Two partial TNF cDNAs (TNF1 and TNF2) were cloned and sequenced. TNF1 gene expression was higher in juveniles than in adults. The highest expression of TNF2 was found in the juveniles from the northern Adriatic. These findings might be related to the juvenile exposure to environmental pollutants.

Melanomacrophage centres (MMCs), located in different organs of non-mammalian vertebrates, play a role in the destruction, detoxification or recycling of endogenous and exogenous materials. Cytochrome P450 monoxygenase 1A (CYP1A) is involved in xenobiotics biotransformation, and its liver expression is considered as a biomarker for detecting exposure to environmental pollutants. Atlantic bluefin tuna (ABFT), Thunnus thynnus L., liver samples were collected from: wild animals caught in the eastern Atlantic; juveniles reared in the central Adriatic; juveniles reared in the northern Adriatic; adults reared in the western Mediterranean. The samples were processed for basic histology, histochemistry and for CYP1A immunodetection. An unexpected high density of MMCs, containing ferric iron and lipofuscin-ceroids, was detected in the juveniles sampled in the northern Adriatic Sea. These individuals showed also a strong anti-CYP1A immunopositivity in hepatocytes and in the epithelium of bile ducts. This study supports the utility of MMCs as biomarkers of fish 'health status' and gives concern for a potential contaminant accumulation in ABFT.

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Fish liver is constituted of hepatocyte cords pervaded by a network of sinusoids. In fish liver, macrophages tend to give rise to melano-macrophage centres (MMCs). The aim of this study was to: a) characterize histochemically Atlantic bluefin tuna (Thynnus thynnus L.) (ABFT) MMCs; b) evaluate the use of MMCs as indicator of health status. Liver samples were taken from: a) wild adult males captured by traditional traps in Sardinia and Morocco; b) captive adult males experimentally reared in sea cages in Spain; c) captive juvenile males commercially reared in Croatia. The samples were fixed in 10% formalin, dehydrated in ethanol and embedded in paraffin wax. Sections were stained with: haematoxylin-eosin, α-naphtyl acetate esterase (ANAE) for macrophages; Mallory's method for lipofuscin/ ceroid, Perl’s stain for hemosiderin; antibodies against vitellogenin (VTG) and cytocrome 450P1A (CYP1A) mono-ossigenase. MMCs showed lysosomial activity and contained lipofuscins/ ceroids and hemosiderin. MMCs density was higher in the Croatian group in comparison to the other two fish groups. Moreover, individuals with hepatocytes immunopositive to VTG and CYP1A were found only in the Croatian group, thus indicating the exposure of these fish to environmental pollutants. This study indicates a role of MMCs as metabolic dumps and confirmed their utility as biomarker of fish health state