In mammals, the STAT proteins (signal transducers and activators of transcription) are a family of cytoplasmic transcription factors mediating the actions of many peptide hormones and cytokines within target cells. In particular, STAT5A is a crucial mediator in the lactogenic hormone response being a candidate marker for milk traits in farm animals. In the present paper, the T→C nucleotide polymorphism at position 12743 in exon 16 of the bovine STAT5A gene was analyzed with PCR-RFLP in a sample of Jersey cows. The purposes of this investigation were to determine the frequencies of the variant alleles and the genotypes of this SNP in Jersey cows and to verify its association with some milk production traits. All the three possible genotypes were identified in the studied population. The observed frequencies of C and T alleles were 0.147 and 0.853 repectively. The TT genotype was the most frequent followed by TC and CC ones. No significant differences between the TT and TC genotypes were found considering MY, FY PC and PY. On the other side, the difference concerning the fat content of milk produced by cows belonging to TC and TT groups was found significant at the statistical analysis: in particular, milk from TT animals had a higher fat content in comparison with that of TC ones (4.55 vs. 4.14%, respectively;P<0.05). However it may be necessary to carry out further investigations about this SNP to better clarify its role on milk production traits in cattle.

Aflatoxins are one of the most widespread and worrisome sources of feed contamination worldwide, and have a considerable impact on fish farm production, leading to high mortality and a gradual decline in fish stock quality in aquaculture. In this study, we investigated, for the first time, the effects induced in vitro by aflatoxin B-1 (AFB(1)) on Sparus aurata hepatocyte culture and we compared our results with the Microtox (R) system using Vibrio fischeri. At AFB(1) doses ranging from 1 to 10 mu g.mL(-1), the results showed signs of primary necrotic cell death in hepatocytes and a very toxic evaluation with Microtox (R); between 0.005 and 1 mu g.mL(-1), the cytotoxic effects and apoptotic delayed death in eukaryotic cells corresponded with an evaluation of no toxicity or biostimulatory effect using V. fischeri. Overall, our results highlighted equivalent toxic responses and overlapped with values of EC50/IC50. Hence, these two in vitro systems could be considered as a useful starting point in the design of new batteries to evaluate the toxicity of potentially dangerous feed-borne substances.

Among all know mycotoxins, aflatoxin B1 is one of the most studied for its hepatotoxic, carcinogenic, mutagenic, teratogenic, and immunosuppressant effects. However, metabolic and toxicological studies on aflatoxins in farmed Sparus aurata are limited and restricted to in vivo trials. This work aimed to study the effects of AFB1 acute and chronic exposure on CYP1A and GST enzymes induced in vitro on S. aurata hepatocytes by immunoblot analysis, thus relating the cytotoxic effects leading to cell death by apoptotic studies. Immunofluorescence analysis revealed that cell damage was not recoverable but permanent, as the cellular repair systems were unable to recover the induced toxic insult. Our results showed detection of several CYP1A bands, enlightening an indirect correlation between induction of CYP1A with dose and time of exposure. The decreased expression of CYP1A over prolonged exposure times, along with high toxic concentration, could be related with lethal damage observed on hepatocytes by contrast phase and immunofluorescence analysis. A particular pattern of expression was found for GST isoforms upon AFB1 exposure, identifying each isoform profile two different kind of toxic insult. The 65 KDa and the 49 KDa bands being suggestive for markers of acute and chronic response respectively. Interestingly, apoptosis induction, considered an early lesion to DNA, was found associated with the chronic damage along with the low toxic concentration. The new cell model from S. aurata has been proven to be a useful and valid tool to further investigate the modulated response of liver phase I and II enzymes to AFB1.

In the present study, cytotoxic effects of the polycyclic aromatic hydrocarbons benzo[a] pyrene (B[a]P) were investigated in Sparus aurata hepatocytes primary cultures after acute and chronic exposure. Cells were treated with a wide range of B[a]P doses (1 pg/mL to 100 µg/mL) for 24, 48 and 72 h. B[a]P toxicity was quantified in sea bream hepatocytes by MTT assay and immunofluorescence analysis of apoptosis after the various exposure periods, in order to evaluate the hepatic damage and toxicity range. Results showed three cytotoxic responses: B[a]P cell death for primary necrosis after exposure to high concentrations for short times, apoptosis induction with the use of sublethal doses and cell proliferation allied with neoplastic foci formation after exposure to low concentrations for long times. This responses provided an interesting correlation between the damage caused on hepatocytes and the metabolism of this toxic compound, to date mainly studied in vivo. Additionally, the statistical analysis revealed that the effects of time and dose were significant for both parameters and especially the time was extremely significant (P<0.0001), in fact B[a]P induced damage that increased over time. Our findings demonstrated and confirmed that S. aurata is a very sensitive species to B[a]P exposure since adverse effects were found at all tested doses. Furthermore, the new in vitro animal model can be considered a useful tool for studying the cellular effects induced by any contaminant harmful for farmed fish.

The main goal of this work was to determine the effect of dietary live yeast Saccharomyces cerevisiae on the oxidative status of sea bass Dicentrarchus labrax juveniles. Fishes were fed on three diets: the GM group were fed a diet containing lyophilized yeast grown on grape must, the CS group were fed a diet containing lyophilized yeast grown on cornstarch, and the control group were fed a diet without yeast. The activity of the main antioxidative enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, glutathione S-transferase (GST), and glutathione (GSH) content, as well as lipid peroxidation, was measured in the liver of sea bass juveniles 90 days after hatching. Supplementation of the diet with S. cerevisiae significantly reduced the SOD and CAT activity, increased the GST activity, decreased the GSH content, and had no effect on lipid peroxidation. The results support the already reported radical-scavenging properties of yeast and usefulness of its employment as antiperoxidative agent in fish.

The effects of lycopene-enriched extenders on in vitro quality characteristics and lipid peroxidation of turkey semen after both chilled and frozen storage were evaluated. Five pools of semen diluted in extenders containing 0, 0.05 or 0.1 mg/mL lycopene were stored either at 5°C for 48 h or cryopreserved as pellets. Mobility, viability, osmotic resistance, DNA integrity as well as lipid peroxidation (malonaldehyde production) of spermatozoa were evaluated in fresh, chilled and frozen sperm. Semen quality was generally reduced after storage, especially post-freezing. However, in semen extended with the highest dose of lycopene, neither viability and osmotic-resistance of chilled sperm, nor DNA integrity of frozen sperm, differed significantly from those of fresh semen. Lipid peroxidation was higher in refrigerated than in fresh or cryopreserved spermatozoa. Anyway, spermatozoa chilled in lycopene-enriched extenders had significant lower malonaldehyde levels than those chilled without lycopene, whereas in frozen semen the addition of lycopene contributed to maintain the lipid peroxidation no different from that scored in fresh semen. In conclusion, lycopene improved the survival of turkey spermatozoa after liquid-storage and protected their DNA integrity against cryodamage. The positive effect of lycopene addition to extenders was likely due to its role in limiting the amount of sperm lipid peroxidation after both refrigeration and cryopreservation.

The development of primary cultures and cell lines from aquatic organisms is a valuable tool for a wide range of research activities applied to aquaculture. Despite several efforts, derivation and long-term culturing of primary hepatocytes from marine vertebrates are still rare and unsuccessful. This is the first report to fully characterize long-term cultures of primary hepatocytes from the European seabream, Sparus aurata L. (Osteichthyes, Sparidae) (SaHePs). In this new model, hepatocyte cells were long-term viable, active proliferating, and fully retained liver function up to 3 weeks. SaHePs expressed a differentiated phenotype, owing to the reacquisition of the peculiar cytoarchitecture with the complete assembly of cytoskeletal and junctional network, as shown by the production and immunolocalization of several polarity markers and cytoskeletal proteins (MDR1, ZO-2, C-CAM1, Vimentin, Cadherin, beta-Tubulin, beta-Catenin, beta-Actin). Cytostructural analysis to identify polarized expression and bile canaliculi formation was performed by immunofluorescence and contrast phase microscopy. Long cultured SaHePs also demonstrated evidence of Albumin, alpha 1-Antitrypsin (AAT) and alpha-Fetoprotein (AFP) synthesis, expression of the detoxifying metabolic enzyme cytochrome P-4501A (CYP 1A), and production of hepatocyte specific cytoskeleton proteins, such as Cytokeratin 8 (CK8) and Cytokeratin 18 (CK 18). The presence of specific markers for hepatic phenotype, detected by immunocytochemistry and Western blot analysis, is suggestive of the full maintenance of a highly differentiated phenotype and hepatic maturation. These data demonstrate that SaHePs can be long cultured without losing the hepatic functionality. This study provides a useful tool for innovative research applications in fish toxicological, pathological, and physiological studies, as one of the few hepatic, functionally active, in vitro model from marine fish.

The large majority of studies on the genotoxic hazard of PAHs polluted water widely applied the ENA assay as versatile tool in large number of wild and farmed aquatic species. Nuclear abnormalities are commonly considered to be a direct consequence of genotoxic lesions in DNA macromolecule, and such evaluation might be helpful in identifying the genotoxic damage induced by the most harmful PAHs such as B[a]P. Regarding at the fish species subjected to aquaculture, most of the toxicological data come from wild fish and mainly focus on freshwater fish, but very little is known for other marine major aquacultured species. The gilthead sea bream (Sparus aurata L.) is the most economically important sparid species cultured along the Mediterranean costs, and it has been proved a very sensitive species to acute B[a]P exposure. However, further investigation is needed on several other types of genotoxic assessments, especially for chronic effects. This work was totally based on an in vitro model for chronic toxicity, using long-term S. aurata hepatocytes in primary culture, continuously exposed to low levels of BaP, over a prolonged period of time, to provide evidences for latent toxicity response. We aimed to investigate the kind of nuclear damage in gilthead sea bream hepatocytes continuously exposed to B[a]P sublethal doses. Cells were exposed to several B[a]P concentrations (10 μg/mL, 1 μg/mL, 1 ng/mL, 1 pg/mL) for two exposure times (24 and 72 h), and then tested both for apoptosis induction and for nuclear abnormalities by immunofluorescence analysis. The presence of severe nuclear damage, revealed cells progressing towards abnormal genotypes, due to a series of aberrant mitosis followed by unequal distribution of chromosomal content. The nuclear atypia (NA) more frequently observed were: a) micronuclei (MN); b) nuclear buds or blebs (NBUDs); c) notched nuclei; d) lobed nuclei; e) nuclei with nucleoplasmic bridge (NPBs); f) nuclei squashed, with a residual nuclear membrane; g) open nuclei, with membrane tape unrolled; and h) apoptotic bodies. Our results showed at medium-low doses a sustained genotoxic response, whose potency increased with the exposure time, becoming apparent as apoptosis induction, both by cell surface and nuclear changes. At the lowest doses, the longer was B[a]P exposure, greater was the involvement on masses of replicating cells, establishing the connection between the escape from apoptosis and the selection of tumoral cell evolution. In view of these results, there is no evidence of a threshold dose below which B[a]P was found not to be genotoxic in sea bream cultured hepatocytes.

Benzo[a]pyrene (B[a]P) is the most studied dangerous polycyclic aromatic hydrocarbon for its hepatotoxic, carcinogenic, mutagenic, teratogenic, and immunosuppressant effects, which can affect both wild and farmed marine fish through the trophic chain. This study investigated, for the first time, the chronic effects induced in vitro by B[a]P prolonged exposure on gilthead sea bream (Sparus aurata L.) hepatocytes, evaluating the cellular and nuclear latent damage. The purpose was to characterize the kind of B[a]P cyto- and genotoxic damage by morphological and immunocytochemical parameters applied in combination with the use of multiple assay endpoints. In light of our results, the short-term effects at higher B[a]P doses were linked to higher cytotoxicities and necrotic lysis, whereas a sustained inflammatory response at medium-low doses was perceived as a mitochondria-mediated apoptosis, both by surface and nuclear morphological changes. The strong immunoreactivity for the cleaved caspase-3 showed that the labeled cells committed suicide by apoptosis. B[a]P involvement on carcinogenesis comes from prolonged exposure at lower doses, establishing the connection between the escape from apoptosis and the selection of a tumoral phenotype. Cells colabeled with proliferating cell nuclear antigen/caspase-3 within the proliferative foci, were proliferating transformed oval stem cells, which escaped the suicide by apoptosis allowing cancer development. Finally, it was established that sea bream cultured hepatocytes are highly sensitive to chronic B[a]P exposure, as serious genotoxic effects were found even at the lowest doses.

The effects of lycopene-enriched extenders on motility, viability, osmotic resistance, DNA integrity and lipid peroxidation of rabbit sperm were examined after both chilled and frozen storage. Five pools of semen diluted in extenders containing 0, 0.05 or 0.1. mg/mL of lycopene were refrigerated at 5°C for 48. h or cryopreserved. Sperm quality was generally compromised after storage, especially post-freezing, however lycopene limited the amount of sperm lipid peroxidation after chilling and freezing. In chilled sperm, the highest dose of lycopene was provided to maintain the viability, acrosome and DNA intactness similar to that of fresh semen and contained the reduction of sperm motility, whereas in cryopreserved semen lycopene protected the DNA integrity of sperm, even if not in a dose-dependent way. So lycopene diminished sperm lipid peroxidation during refrigeration and cryopreservation, prolonging the survival of rabbit sperm after liquid storage, but it had a limited effect on sperm cryosurvival. © 2012 Elsevier B.V.

Neural Progenitor Cells (NPCs) have gathered more and more attention in the field of Neural Stem Cells (NSCs). However, the multilineage differentiating behavior of these cells and their contribution to tissue regeneration, almost in lower vertebrate taxa, remain unknown. Since the early 1970s, many comparative studies have been performed using immunocytochemical screening on the brains of several vertebrate taxa, including teleosts, in order to identify these cells, even if the data are sometimes contrasting. This study aims: (1) to investigate in vitro the potential proliferative role of NPCs and Radial Glia Progenitors (RGP) in seabream neurogenesis; (2) to reveal the strict ability of fish NSCs to undertake the multilineage development and differentiation in neurons, astrocytes and oligodendrocytes. By the use of double Immunofluorescence (IF) analysis and phase contrast microscopy, we identified the multilineage differentiation and the exact cell morphology. We demonstrated that NSC can self-renew and differentiate into different types of neurons or glial cells during extended culturing. Mature neurons expressed specific neuronal markers; they could differentiate during long term culturing, generating an extensive neurite growth. Glia was found highly mitotic and could developed mature astrocytes and oligodendrocytes. Glial cells were assessed by Glial Fibrillary Acidic Protein (GFAP) reactivity; neurons and myelinating oligodendrocytes were immunostained with cell-specific markers. This work provide that the multilineage differentiation potential of seabream neural cell progenitors might be a useful tool for neurodegenerative diseases, being a promising approach for repairing the CNS injuries, also in other animals, as a new coming strategy for function recovery of damaged nerves.

Neuroglia has been historically considered the “glue” of the nervous system, as the ancient Greek name suggests, being simply referred as non-neuronal cells, with supporting functions for neurons in the CNS of mammalian and lower vertebrates. All around the world, approximately 283 cell lines were obtained from fish, yet none of these was from the brain of Sparus aurata, neither in cell lines nor as primary culture. Here we describe a novel in vitro reproducible neuroglial marine model for establishing primary neuroglial cell cultures, by dissociating the whole brain of seabream juveniles. We showed that proliferating neural stem cells produced alongside three generating lineages, such as neuronal precursor cells, astroglial precursor cells and oligodendroglia precursor cells, which developed respectively neurons, astrocytes and oligodendrocytes. The radial glia, finely described by morphological studies and immunochemical antigen expression, showed a peculiar spatial distribution, giving rise simultaneously both to astrocytes and neuronal precursors within a highly proliferative assemblate. Radial glia cells were assessed by glial fibrillary acidic protein (GFAP) and vimentin reactivity, astrocytes by GFAP, neurons by the neuron-specific markers for ubiquitin carboxy-terminal hydrolase 1 (UCHL1) and intermediate filament associated protein (NF), whereas myelinating oligodendrocytes were immunostained with anti-myelin basic protein (MBP) and anti-O4. Our findings suggest that seabream neuroglial cells gain in 3-4 weeks of culturing proliferation, neuroglial differentiation, and oligodendrocyte maturation with myelination, thus disclosing on the possibility that mixed neuroglial cultures can accelerate the maturation of oligodendrocytes and the regeneration of CNS injury in fish.