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Analysis of fish behaviour is an effective way to indirectly identify the presence of environmental pollutants that negatively affect fish life, its production and quality. Monitoring individual and collective behaviours produces large amounts of non-linear data that require tailor-suited computational methods to interpret and manage the information. Fractal dimension (FD) and entropy are two groups of such non-linear analysing methods that serve as indicators of the complexity (FD) and predictability (entropy) of the behaviours. Since behavioural complexity and predictability may be modulated by contaminants, the changes in its FD and entropy values have a clear potential to be embedded in a biological early warning system (BEWS), which may be particularly useful in Precision Fish Farming settings and to monitor wild populations. This work presents a review of the effects of a wide range of environmental contaminants, including toxic compounds, cleaning and disinfecting agents, sti...

The present work explores the capacity of a machine vision system to detect deviations in the Shannon entropy (SE) of a European seabass (Dicentrarchus labrax) biological system in response to MeHg contaminated feed. Three groups of fish were exposed for 2 weeks to 0.5, 5 and 10 ppm of methylmercury (MeHg) in the feed and compared to a group whose feed was not artificially contaminated. No mortality was observed during the 2 weeks experimental period. The SE of the experimental cases was modified by the presence of MeHg, confirming our previous works. However, no clear dosedependent responses of the systems' SE to MeHg administration were detected during the 2 weeks period, although there was a different tendency in the evolution of SE during the experimental period between the non-exposed group and the exposed ones.

This review presents some applications of proteomics and selected spectroscopic methods to validate certain aspects of seafood traceability. After a general introduction to traceability and the initial applications of proteomics to authenticate traceability information, it addresses the application of proteomics to trace seafood exposure to some increasingly abundant emergent health hazards with the potential to indicate the geographic/environmental origin, such as microplastics, triclosan and human medicinal and recreational drugs. Thereafter, it shows the application of vibrational spectroscopy (Fourier-Transform Infrared Spectroscopy (FTIR) and Fourier-Transform Raman Spectroscopy (FT Raman)) and Low Field Nuclear Magnetic Resonance (LF-NMR) relaxometry to discriminate frozen fish from thawed fish and to estimate the time and temperature history of frozen fillets by monitoring protein modifications induced by processing and storage. The review concludes indicating near future tre...

Feeding 3.9 and 6.7 mg Hg/kg (Se/Hg molar ratios of 0.8 and 0.4, respectively) for 14 days negatively affected Dicentrarchus labrax growth and total DNTB- and thioredoxin-reductase (TrxR) activities and the transcription of four redox genes (txn1, gpx1, txnrd3, and txnrd2) in the liver, but a diet with 0.5 mg Hg/kg (Se/Hg molar ratio 6.6) slightly increased both reductase activities and the transcription of txn1, gpx1, and txnrd2. Feeding 6.7 mg Hg/kg for 53 days downregulated the genes of the thioredoxin system (txn1, txnrd3, and txnrd2) but upregulated gpx1, confirming the previously proposed complementarity among the antioxidant systems. Substitution of 20% of the feed by thawed white fish (hake) slightly counteracted the negative effects of Hg. The effects were not statistically significant and were dependent, in a non-linear manner, on the Se/Hg molar ratio of the feed but not on its Hg concentration. These results stress the need to consider the Se/Hg molar ratio of the feed/f...

Seafood is tasty, healthy, and a good source of fat, proteins and minerals. The welfare of the fish, which influences its quality, is of concern to consumers and farmers of seafoods, who are seeking to minimize the use of drugs and the infliction of stress. Processors of seafoods also need to develop new products, technologies, packaging materials and ways of thinking, to fulfill legislative and market demands for fresh, easy to prepare, safe food products, with consistent quality and price. Unfortunately, these new processes render the original species unidentifiable by eye in the final product, which opens the way for substitution of expensive species by cheaper ones of similar characteristics. New challenges to the consumer emerge from the use of molecular biology techniques for the production of novel species, and improvements in technologies for embryonic development. Molecular biology techniques, fortunately, will also help to identify the origin of these species. Our advice: ...

Tris and CHAPS–urea extracts from wild and farmed cod muscle and from rehydrated cod klipfish fillets were analyzed by one (1DE) and two-dimensional electrophoresis (2DE). 2DE maps of tris extracts from farmed cod differed from the wild in a series of spots of Mw 35 and 45kDa. The CHAPS–urea extracts from farmed cod had a several spots of Mw between

... [1998]; Bjerkeng et al. [1999] and Refsgaard et al. ... PC1 and PC2 explained 76% and 16% respectively of the total variability in the model. Data from Einen et al., [1998]; Bjerkeng et al., [1999] and Refsgaard et al. [1998] Page 29. SeafoodPlus. ...

ABSTRACT Myosin extracts of red and white muscles from mackerel (Scomber scombrus) were analyzed by native electrophoresis to investigate the existence of myosin isoforms in both kind of muscles, and by two-dimensional and SDS gel electrophoresis to study their subunit composition. Two isoforms were found in red muscle comprising one type of heavy chain and two light chains. Four isoforms were found in white muscle made up of one type of heavy chain and three types of light chains. The heavy chains from white muscle showed a higher electrophoretic mobility than that of red muscle in SDS-PAGE. Both heavy chains had an intermediate mobility between those of slow and fast myosins from rat diaphragm.

ABSTRACT This book provides readers with the recent advances and state-of-the-art in food proteomics, which constitutes one of the most relevant and rapidly developing areas in food science. The first part covers the principles of proteomics, including an in-depth discussion of the proteome, as well as the extraction and fractionation techniques for proteins and peptides, separation techniques like 2-D electrophoresis and chromatography, and mass spectrometry applications. The second part covers applications to foods, such as quality issues related with post-mortem processes in animal foods, and quality traits for a wide variety of foods like meat, fish, dairy, eggs, wine, beer, cereals, fruits and vegetables. Also discussed are the identification of bioactive peptides and proteins, crucial from a nutritional perspective, and safety issues like food authenticity, detection of animal species in the food, markers of pathogen microorganisms, and identification of prions.

This review presents the primary applications of various proteomic strategies to evaluate the impact of farming conditions on food quality and safety in aquaculture products. Aquaculture is a quickly growing sector that represents 47% of total fish production. Food quality, dietary management, fish welfare, the stress response, food safety, and antibiotic resistance, which are covered by this review, are among the primary topics in which proteomic techniques and strategies are being successfully applied. The review concludes by outlining future directions and potential perspectives.

This work investigates the suitability of (1)H NMR spectroscopy to identify the fate of some bioactive compounds in seafood submitted to several processing conditions and examines the possibility of using (1)H NMR spectroscopy profiling to classify such products. Perchloric acid extracts of cod white muscle from newly killed and (i) unprocessed, (ii) boiled, and (iii) fried fillets and from (iv) frozen fillets, (v) the frozen fillets after thawing, and (vi) their drip loss and from (vii) rehydrated cod klippfish (n = 5) were analyzed by 500 MHz (1)H NMR spectroscopy. It was possible to identify taurine, betaine, anserine, creatine, and trimethylamine oxide (TMAO) in all extracts examined, and frozen fish was recognizable by the presence of dimethylamine (DMA). None of the heating procedures seemed to induce the loss of bioactive compounds from the fillet, but freezing and thawing did: the compounds were lost in what is known as drip loss. About 80% of the samples were correctly classified using a probabilistic neural network procedure having as inputs the scores of the first 20 principal components of the principal component analysis of a selected region of the NMR spectra.

Burnt tuna (BT), or yake-niku, is a quality flaw of the muscle characterised by a pale colour and grainy and exudative texture. Cathepsin-L, water soluble and total protein components from normal and BT muscles, from three tropical tuna species - yellowfin (YFT, Thunnus albacares), bigeye (BET, Thunnus obesus) and skipjack (SKJ, Katsuwonus pelamis) - were compared by electrophoretic and western blot analyses to identify biomarkers for BT. As expected, SDS-PAGE patterns were species-specific but differences, due to BT, were observed only between some low ionic strength extracts of BET and YFT. Protein oxidation and cell proliferation analysed by immunoblotting did not show differences between BT and normal muscles. Gelatine zymography revealed different gelatinase activity patterns that, although not linked to BT, may affect the final texture of the muscle. A 43kDa band, identified as creatine kinase by proteomic analysis, showed the potential to be a good indicator for BT in BET and YFT.

ABSTRACT Introduction Mercury is a persistent contaminant, widely distributed in the environment, originating from both natural and anthropogenic sources that accumulates and bioamplifies in the trophic chain. Methylmercury (MeHg) is one of the most readily absorbed and bioavailable forms in nature and comprises about 96% of the Hg in fish muscle. The harmful effects of Hg on animal health are well documented and have been attributed to the inactivation of selenoenzymes. Indeed, selenoenzymes have been proposed as the molecular targets of methylmercury toxicity and it has been postulated that it is the ratio Se:Hg what has to be taken into account in the evaluation of the potential toxicity of foodstuffs and that the amount of Hg alone is not an indicator of their potential toxicity (Branco et al., 2012, Ralston et al., 2008; Yamashita et al., 2010). Simultaneous exposition to sodium selenite (NaSe) and MeHg has shown to decrease or eliminate the negative effect of MeHg on the activity of some enzymes (Branco et al., 2012). However we have not been able to find works examining whether pre-exposure to NaSe might mitigate the harmful effect of MeHg. This was worth considering in our opinion since some feeds might contain NaSe (and indeed some people take mineral supplements with NaSe) which may help to build up protective effect against MeHg toxicity. The aim of this work was to identify the effect that prior exposure of Dicentrarchus labrax to 10μg of sodium selenite per litre seawater may exert when the fish is later exposed to seawater contaminated with 4μg of MeHg per litre. Materials and Methods 150 Dicentrarchus labrax of about 5g kindly provided by Tinamenor (Spain) were treated according to Branco et al (2012) with the following modifications: 75 fish were placed in each of two 800 L tanks. The fish in tank 1 were marked with an elastomer and fish in tank 2 were punctured in the same method but without administering any solution. Fish in tank 1 were kept in 10μg of sodium selenite per litre of seawater for a week. After this period, 10 fish from each tank were sacrificed; the administration of NaSe was discontinued, 30 fish from tank 1 were then placed in tank 2, and 30 fish from tank 2 were placed in tank 1. Then 4μg of MeHg /litre were added to tank 1. After 1 and 2 additional weeks, 10 fish from each group were sacrificed. The administration of MeHg was then be discontinued and 10 fish of each group were sacrificed after one extra week (total=5 weeks from the beginning of the experiment). Skeletal muscle, heart, liver and brain were sampled and prepared for histology and biochemical analyses. Fulton’s Condition Index (K = 100(W/L^3)) was calculated for the fish sacrificed at each period. This presentation reports preliminary result on the effect of this treatment on white muscle proteins: 50mg of white muscle were mixed with 200μl of Laemmli buffer (Laemmli, 1970): 65mm Tris-HCl, pH 6.8, 5% SDS, 100mM DTT, finely cut with scissors, incubated at 95oC, cut again and centrifuged at 14,000 rpm for 10min. The supernatants were collected and the proteins were determined by measuring the optical density at 280nm. They were separated by 5-15% gradient SDSPAGE (Laemmli, 1970). The gels were transferred to nitrocellulose membranes and probed with anti-myosin heavy chain and anti-actin antibodies. Results Given the small number of sampled fish (n=10) and the very large standard deviation in the weight of all experimental groups at all sampling periods, the results of the effect of the treatments on the fish K-factor have to be interpreted with care. NaSe (shown as ‘Se’ in the following Figures) did not seem to affect the K-facto during the first week, while treatment with MeHg induced a sharp decrease in the K-index of all fish regardless of previous exposure to NaSe, but immediately after the removal of the MeHg the K-value increased in all treated groups although those that had previously exposed to NaSe only recovered partially. These preliminary results do not show clear differences between the treatments, contrast with the differences observed in the fishes behavior immediately after the MeHg treatment. We are currently performing further analysis (ubiquitination and degree of protein oxidation) that will be reported at the conference. References Branco, V, Canário, J, Lu, J, Holmgren, A, Carvalho, C (2012) Mercury and selenium interaction in vivo: Effects on thioredoxin reductase and glutathione peroxidase. Free Radical Biology & Medicine 52, 781–793. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Ralston, NVC, Ralston CR, Blackwell L III, Raymond, LJ. (2008) Dietary and tissue selenium in relation to methylmercury toxicity. NeuroToxicology, 29: 802–811. Wilber, CG (1980) Toxicology of selenium: A review. Clin Toxicol, 17: 171-230. Yamashita, Y, Yabu, T and Yamashita, M (2010) Discovery of the strong antioxidant…

... Myosin heavy chain (MHC) from salmon was selected because we wished to detect its fragments after enzyme hydrolysis using a rabbit polyclonal anti-MHC preparation raised against a salmonid, Arctic charr (Salvelinus alpinus), MHC (Martinez & Pettersen, 1992). ...

... farmed (AquaGen strain) and wild parr was achieved by examining the body form, shape of the ... In contrast, N2O and CH4 exhibit wide isotopic variation, and they reflect both significant isotopic ... a distinct isotopic fingerprint giving information on the frequency of each isotope in a ...

Page 1. Part I Omega-3 Fatty Acid Content of Intact Muscle of Farmed Atlantic Salmon (Salmosalar) Examined by 1 H MAS NMR Spectroscopy ... About 20% of the muscle lipids of farmed Atlantic salmon are n-3 FAs, with some variation due to the FA composition of the fish feed. ...

Fish welfare refers to its ability to enjoy freedom from hunger; discomfort; pain, injury and disease; fear and distress and to express normal behavior. Fish welfare keeps a positive relationship with its health, growth and the quality and safety of the products. Fish behavior is known to be altered by some contaminants. Thus the aim of this was to develop a non-invasive methodology for image acquisition, processing and nonlinear trajectory analysis of the collective fish response to a stochastic event. The method should have the potential to be applicable to monitor stressors, ie., contaminants in fish farming. The experimental was approved by The Ethical Committee for Animal Welfare No. CEBA/285/2013MG. European sea bass (Dicentrarchus labrax, 4±2 g, 8±1 cm) were provided by Grupo Tinamenor (Cantabria, Spain). Three experimental cases were used to test the methodology: C1 and C2, consisting of 81 fish each and differing only in that C2 fish were tagged with Visual Implant Elastome...

1. White skeletal muscle myosin of four marine teleost fish species (cod, blue whiting, Norway haddock, and spotted wolf-fish) was analyzed by native, SDS-PAGE, and 2-dimensional electrophoresis. 2. Four types of native myosin were present in cod, blue whiting and Norway haddock. The second fastest migrating form was predominant. 3. Myosin from spotted wolf-fish also resolved into four forms. The fastest migrating form was hardly noticeable. The other three were present in apparently similar amounts. 4. In the myosin from each species there were three types of light chains. The pattern of light chains was species specific. 5. Apparently, there was only one type of heavy chain in myosin from cod, Norway haddock and spotted wolf-fish. One preparation of cod showed an extra band of higher electrophoretic mobility than the main band. In blue whiting we found two bands present in approximately equal amounts.

The expression of myosin isoforms and their subunit composition in the white skeletal body musculature of Arctic charr (Salvelinus alpinus) of different ages (from 77-day embryos until about 5 years old) was studied at the protein level by means of electrophoretic techniques. Myosin from the white muscle displayed three types of light chain during all the developmental stages examined: two myosin light chains type 1 (LC1F) differing in both apparent molecular mass and pI, one myosin light chain type 2 (LC2F) and one myosin light chain type 3 (LC3F). The fastest-migrating form of LC1F seemed to be predominant during the embryonic and eleutheroembryonic periods. The slowest-migrating form of LC1F was predominant in the 5-year-old fish. Between 1 year and 4 years, both types of LC1F were present in similar amounts. Cardiac as well as red muscle myosin from 3-year-old fish had two types of light chain. The myosin light chains from atria and ventriculi were indistinguishable by two-dimen...

Samples from pre- and post-rigor cod mince, surimi (a concentrate of fish myofibrillar proteins obtained after washing and dewatering the fish mince) and water from the first wash in the surimi manufacture, processed with and without the addition of 7.5 mM CaCl2 and 15 mM MgCl2, were analyzed by two-dimensional electrophoresis. The results showed that the main myofibrillar proteins, including myosin, actin and tropomyosin, remained in the surimi. Several other proteins were selectively removed during the washing procedure. Some additional major spots were detected in the two-dimensional gels containing samples of the wash water and surimi processed with the addition of Ca2+ and Mg2+ salts. These spots were either absent or present in minor amounts in the samples of post-rigor cod mince, wash water and surimi processed without Ca2+ and Mg2+ salts and in all the pre-rigor samples. This induced us to suggest that the new additional spots may constitute fragments of proteins originated ...

ABSTRACT The annual global increase in the production, particularly from aquaculture, and consumption of seafood is expected to continue in the future. One of the main worldwide concerns from the point of view of seafood safety is the increasing number of novel and unexpected chemical substances that contaminate the aquatic environment. Currently, there is a lack of cost effective, user-friendly methods to detect many of these contaminants and there is no method to detect unknown contaminants. A paradigm shift is necessary in the seafood production industry from contaminant detection only, to monitoring the effects of the contaminants as well. This can be achieved by introducing a systems approach and using a biological warning system (BWS) specifically adapted to fish farming. In this manner, it would be possible to develop affordable, on-line identification of production units displaying atypical responses or behaviour – and therefore potentially contaminated – regardless of whether the contaminant is an identified or an unknown substance. Once developed, the BWS should be implemented within the HACCP plans and the results accompany the traceability documentation of the products.