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 Atlantic bluefin tuna (Thunnus thynnus) is one of the tunas with the highest commercial value and it is supporting the capture-based tuna aquaculture industry in the Mediterranean Sea. This is a seasonal activity and it involves the capture of fish from the wild and their rearing in sea cages for periods ranging between 3 months to 2 years. Short-term rearing is done mainly to: (a) achieve a greater body fat percentage and (b) obtain a better price by not flooding the market in the brief fishing period. Due to the increasing fear of a collapse of the fishery, the International Commission for the Conservation of Atlantic Tunas currently reduced the total allowable catches for 2010 to 13,500 mtn from 32,000 mtn previously. Therefore, there is great interest in establishing a proper and sustainable tuna aquaculture industry. This necessitates the development of specific technologies for tuna aquaculture that will not rely on captured individuals from the wild, as it is practiced today. This article reviews the methods used for the farming and fattening

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.

Bone resorption in the first spine of the first dorsal fin of Atlantic bluefin tuna (ABFT) has long been considered for age estimation studies. In the present paper spine bone resorption was assessed in wild (aged 1 to 13 years) and captive-reared (aged 2 to 11 years) ABFT sampled from the Mediterranean Sea. Total surface (TS), solid surface (SS) and reabsorbed surface (RS) were measured in spine transverse sections in order to obtain proportions of SS and RS. The spine section surface was found to be isometrically correlated to the fish fork length by a power equation. The fraction of solid spine bone progressively decreased according to a logarithmic equation correlating SS/TS to both fish size and age. The values ranged from 57% in the smallest examined individuals to 37% in the largest specimens. This phenomenon was further enhanced in captive-reared ABFT where SS/TS was 22% in the largest measured specimen. The difference between the fraction of SS of wild and captive-reared ABFT was highly significant. In each year class from 1- to 7-year-old wild specimens, the fraction of spine reabsorbed surface was significantly higher in specimens collected from March to May than in those sampled during the rest of the year. In 4-year-old fish the normal SS increase during the summer did not occur, possibly coinciding with their first sexual maturity. According to the correlations between SS/TS and age, the rate of spine bone resorption was significantly higher, even almost double, in captive-reared specimens. This could be attributed to the wider context of systemic dysfunctions occurring in reared ABFT, and may be related to a number of factors, including nutritional deficiencies, alteration of endocrine profile, cortisol-induced stress, and loss of spine functions during locomotion in rearing conditions

The effect of a delivery system (implant) loaded with gonadotropin-releasing hormone agonist (GnRHa) on the spawning performance in captive-reared Atlantic bluefin tuna (Thunnus thynnus) was assessed by means of stereological quantification of ovarian post-ovulatory follicles (POF). Among untreated control fish no follicles were observed at the oocyte maturation (OM) stage; in contrast, 10 of the 16 GnRHa-treated fish showed follicles containing migratory-nucleus oocytes and/or hydrating oocytes. Post-ovulatory follicles were observed only in three of the 14 untreated individuals examined, while all but one GnRHa-induced fish showed POF in the ovary. Nevertheless, when POF were present in the ovary, the amount of ovulated oocytes was not found to be significantly different among GnRHa-treated and untreated control fish, and wild spawners. The relative batch fecundity (eggs g−1 of body mass) estimated from ovarian POF counts were 109.62±28.36 (control group), 68.92±13.05 (GnRHa-treated group) and 83.47±15.63 (wild group). It was concluded that treatment with GnRHa implant stimulated OM and spawning in captivity, thus enhancing the reproductive potential of the broodstocks; however, there appears to be a limitation in the number of eggs maturing in each batch, and the spawn fecundity was not increased by the hormonal treatment.

A controlled-release implant loaded with GnRH agonist (GnRHa) was used to induce spawning in Atlantic bluefin tuna (Thunnus thynnus) during two consecutive reproductive seasons. The fish were implanted underwater and sampled between days 2 and 8 after treatment. At the time of GnRHa treatment, females were in full vitellogenesis and males in spermiation. There was a rapid burst of pituitary luteinizing hormone (LH) release at day 2 after treatment in GnRHa-treated fish, and circulating LH remained elevated up to day 8 after treatment. In contrast, control fish had significantly lower levels in the plasma, but higher LH content in the pituitary, as observed in many other cultured fishes that fail to undergo oocyte maturation, ovulation and spawning unless induced by an exogenous GnRHa. Plasma testosterone (T) and 17b-estradiol (E2) were elevated in response to the GnRHa treatment in females, while 11-ketotestosterone (11-KT) but not T was elevated in males. Even though oocyte maturation and ovulation did occur in GnRHa-induced fish, no significant elevations in 17,20b-dihydroxy-4-pregnen-3-one (17,20b-P) or 17,20b,21-trihydroxy-4-pregnen-3-one (20b-S), in either the free, conjugated or 5b-reduced,3a-hydroxylated forms was observed in fish sampled within 6 days after treatment. Interestingly, a significant peak in plasma free 17,20b-P levels occurred in both males and females at day 8 after treatment. Histological sections of the ovaries in these females contained oocytes at the migrating germinal vesicle stage, suggesting the role of this hormone as a maturation-inducing steroid in Atlantic bluefin tuna. In conclusion, the GnRHa implants activated effectively the reproductive endocrine axis in captive Atlantic bluefin tuna broodstocks, through stimulation of sustained elevations in plasma LH, which in turn evoked the synthesis and secretion of the relevant sex steroids leading to gamete maturation and release.

Owing to high cost, meat horse domestic production does not meet internal market demand, that is why such a livestock is mainly imported from Eastern Europe Countries. Fibre type composition deeply influences post-mortem changes in the conversion of muscle to meat, as well as the product quality. The aim of this study was to investigate histological characteristics of muscle fibres and adypocites related to two different horse genetic types (Abruzzese and Polish), sold in Italy. The two horse genetic types showed little variation in the fiber type of the meat, considering the least square means of all 3 fiber types (red, intermediate and white) found in the samples muscle. The alpha-white fibers were larger than beta-red and alpha-red (P<0.05). Significant differences in distribution were found for most fibre types within breeds. A higher proportion of slow fibers was found in the Longissimus muscle of the TPR x Abruzzese (P<0.05). The histological results showed that the two genetic types had small effect on the size of the LD fibers.

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.

As part of the endeavor aiming at the domestication of Atlantic bluefin tuna (BFT; Thunnus thynnus), first sexual maturity in captivity was studied by documenting its occurrence and by characterizing the key hormones of the reproductive axis: follicle stimulating hormone (FSH) and luteinizing hormone (LH). The full length sequence encoding for the related hormone β-subunits, bftFSHβ and bftLHβ, were determined, revealing two bftFSHβ mRNA variants, differing in their 5' untranslated region.A quantitative immuno-dot-blot assay to measure pituitary FSH content in BFT was developed and validated enabling, for the first time in this species, data sets for both LH and FSH to be compared. The expression and accumulation patterns of LH in the pituitary showed a steady increase of this hormone, concomitant with fish age, reaching higher levels in adult females compared to males of the same age class. Conversely, the pituitary FSH levels were elevated only in 2Y and adult fish. The pituitary FSH to LH ratio was consistently higher (>1) in immature than in maturing or pubertal fish, resembling the situation in mammals. Nevertheless, the results suggest that a rise in the LH storage level above a minimum threshold may be an indicator of the onset of puberty in BFT females. The higher pituitary LH levels in adult females over males may further support this notion.In contrast three year-old (3Y) males were pubertal while cognate females were still immature. However, it is not yet clear whether the advanced puberty in the 3Y males was a general feature typifying wild BFT populations or was induced by the culture conditions. Future studies testing the effects of captivity and hormonal treatments on precocious maturity may allow for improved handling of this species in a controlled environment which would lead to more cost-efficient farming.

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

Following the histological analysis of Arca noae samples from the south-western Adriatic Sea, five hermaphroditic specimens were found out of 168 sexed individuals (3.0%). The hermaphrodite gonads showed the co-occurrence of male and female germ cells within the same acini, i.e. both spermatozoa in the lumen and oogonia lining its wall. Gonia increased in size through winter, thus suggesting that the direction of sex change is from male to female. Both the biometrical analyses and theoretical considerations strongly suggest that A. noae is an obligate protandric species.

Understanding the reproductive biology of Atlantic bluefin tuna (ABFT) is important to both managing its fishery and developing hatchery technologies to close its life cycle in aquaculture. Globally, ABFT is comprised of two populations, the eastern and western stocks, with known breeding areas in the Mediterranean Sea and the Gulf of Mexico, respectively. Gametogenesis takes place during spring and early summer, and spawning usually occurs from May to July, coinciding with the rise of water temperature. Females display an asynchronous ovarian development, typical of a batch spawner. Comparing the endocrine-reproductive cycle in wild and captive ABFT led to the development of a hormone-based therapy to induce spawning in captive broodstock. While captivity affects gametogenesis in ABFT, at least some of the captive fish spawn spontaneously, which can be enhanced and prolonged using hormonal induction. Massive spawning of captive ABFT enabled the first aquaculture production of marketable fish, demonstrating the biological feasibility of this industry. Current research on hormonal regulation of its puberty may lead to the use of smaller ABFT broodstock, simplifying their husbandry and management. This, together with the establishment of land-based broodstock operations, will enable efficient and cost-effective on-demand and year-round production of ABFT seeds to drive the consistent farming of this fish