Alternative matrices for cortisol measurement in fish.

Plasma cortisol is the most commonly used indicator of stress in fish but, as the blood sampling procedure itself can be a source of stress, it would be helpful to measure cortisol using less invasive matrices. It is also necessary to find alternative matrices as stress indicators in dead fish in which blood sampling is impossible. In the present study, we investigated transport stress in three aquaculture species, European sea bass (Dicentrarchus labrax L.), common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum), by cortisol determination (radioimmunoassay) in plasma and other matrices (skin mucus, gut content, lateral muscle and caudal fin). Cortisol significantly increased after transport in all species and matrices, except in the sea bass gut content, where it remained unchanged. The three species responded to transport stress by producing different cortisol levels. In conclusion, the significant correlation found between plasma cortisol and most of the other matrices opens up the possibility of using them to evaluate stress in fish: mucus sampling is a less invasive method than blood sampling, and in addition to muscle and fin sampling, it can be used in postmortem fish

BIOMONITORAGGIO DELLA LAGUNA DI VENEZIA: INDICATORI BIOLOGICI IN Zosterisessor ophiocephalus e Tapes philippinarum

Lo scopo del presente studio \ue8 stato quello di valutare l\u2019espressione di alcuni bioindicatori comunemente utilizzati in programmi di biomonitoraggio in pesci e molluschi prelevati in due periodi dell\u2019anno e in diversi siti della Laguna di Venezia che, per la loro collocazione e per le caratteristiche idrologiche, possono essere pi\uf9 o meno soggette a possibili fonti inquinanti. Le specie monitorate sono state il pesce bentonico Zosterisessor ophiocephalus (Teleostei: Gobiidae) e la vongola verace Tapes philippinarum, animali particolarmente adatti al biomonitoraggio in quanto stanziali, vivono a stretto contatto con il fondo e sono reperibili in Laguna di Venezia. Parallelamente alcuni individui di Z. ophiocephalus sono stati stabulati in vasche con acqua di mare per un periodo sufficiente detossificarsi, al fine di consentire una comparazione non solo tra i diversi siti di campionamento ma anche rispetto ad un controllo privo di contaminanti. I biomarcatori selezionati e analizzati sono stati: l\u2019espressione della vitellogenina (VTG), della citocromo P450 1A1 (CYP1A1) e dei principali marcatori dello stress ossidativo, quali 4-idrossi nonenale (HNE), nitrotirosina (NT), malondialdeide (MDA) e acroleina, considerati ottimi indicatori di inquinamento ambientale. Inoltre sono stati valutati: l\u2019espressione del messaggero delle Heat Shock Protein 70 (HSP70) e, in entrambe le specie monitorate, i livelli di steroidi sessuali (testosterone, estradiolo e progesterone) al fine di verificare eventuali variazioni imputabili alla presenza nell\u2019ambiente di distruttori endocrini cio\ue8 di sostanze inquinanti in grado di interferire con il sistema endocrino degli organismi. Mediante analisi di immunoistochimica, Western blot, saggio T-BARS e Real Time PCR abbiamo potuto confermare quanto gi\ue0 riportato in letteratura: il bacino di Lido \ue8 la zona maggiormente impattata della Laguna poich\ue9 influenzata dalla presenza di un\u2019estesa area industriale e della citt\ue0 di Venezia. Un minor impatto \ue8 stato invece riscontrato per le restanti aree monitorate. Tuttavia, al fine di di ottenere un quadro pi\uf9 completo dello stato dell\u2019intero ambiente lagunare, \ue8 necessario: identificare ulteriori ed efficaci bioindicatori, ampliare l\u2019area monitorata identificando altri siti di campionamento in tutti i bacini della Laguna di Venezia, aumentare il numero di esemplari da sottoporre a detossificazione utilizzando animali prelevati dai diversi bacini della Laguna di Venezia per allestire un esperimento di controllo completo

Biomonitoraggio della Laguna di Venezia: indicatori biologici in Zosterisessor ophiocephalus e Tapes philippinarum

Venice Lagoon is a transitional environment on the Adriatic coast influenced by such human activities as agriculture, industry, and tourism. For this reason, in the last year, numerous national and international projects have been carried out in order to evaluate the quality status of the lagoon environment demonstrating the presence of a wide range of contaminants in the environment. The biomonitoring in the lagoon environment plays an important role in strategies and actions to identify, control and reduce the environmental problems. Biomonitoring programs usually involves the use of biomarkers, which represent biochemical, physiological or behavioural variation measured in tissues, biological fluids or the whole organism (Depledge and Fossi 1994). In the present study, the expression of vitellogenin, cytochrome P450 1A1, HSP70 and oxidative stress biomarkers and sex steroids concentrations (progesterone, 17β-estradiol and testosterone) have been examined in different sites (Val di Brenta, Porto Canale, Ca’ Roman and Porto Marghera) of the Venice Lagoon monitoring their levels in the grass goby Zosterisessor ophiocephalus e in the clam Tapes philippinarum. Sampling sites were selected on the basis of data reported in literature (Nesto et al. 2007; Losso and Volpi Ghirardini, 2010). Vitellogenin induction was detected in adult male of Z. ophiocephalus collected from the different sites evidencing high toxicological risk probably due to endocrine disrupting chemicals. Moreover, the highest levels of MDA and CYP1A1, measured spectrophotometrically by TBARS assay and by Real Time PCR respectively, were observed in animals sampled at the Porto Marghera site. The cellular localization of HNE and NT, investigated by an immunohistochemical approach, showed that immunopositivity was mainly localized in melanomacrophage-centres of spleen, kidney, liver and ovary. Both immunohistochemistry and Western blot analysis evidenced that HNE and NT were quantitatively higher in animals from Porto Marghera if compared with those sampled at the other sites. In conclusion, as reported in literature (Losso and Volpi Ghirardini, 2010), most indicators have shown that the Lido basin, influenced by the presence of the industrial area and the city of Venice, is the most highly impacted area suggesting that oxidative stress markers, vitellogenin and cytochrome P4501A1 in aquatic organisms can be useful in biomonitoring of environmental pollution. However, HSP70 expression and sex steroid concentrations are not fast and suitable tools to investigate environmental quality.Lo scopo del presente studio è stato quello di valutare l’espressione di alcuni bioindicatori comunemente utilizzati in programmi di biomonitoraggio in pesci e molluschi prelevati in due periodi dell’anno e in diversi siti della Laguna di Venezia che, per la loro collocazione e per le caratteristiche idrologiche, possono essere più o meno soggette a possibili fonti inquinanti. Le specie monitorate sono state il pesce bentonico Zosterisessor ophiocephalus (Teleostei: Gobiidae) e la vongola verace Tapes philippinarum, animali particolarmente adatti al biomonitoraggio in quanto stanziali, vivono a stretto contatto con il fondo e sono reperibili in Laguna di Venezia. Parallelamente alcuni individui di Z. ophiocephalus sono stati stabulati in vasche con acqua di mare per un periodo sufficiente a detossificarsi, al fine di consentire una comparazione non solo tra i diversi siti di campionamento ma anche rispetto ad un controllo privo di contaminanti. I biomarcatori selezionati e analizzati sono stati: l’espressione della vitellogenina (VTG), della citocromo P450 1A1 (CYP1A1) e dei principali marcatori dello stress ossidativo, quali 4-idrossi nonenale (HNE), nitrotirosina (NT), malondialdeide (MDA) e acroleina, considerati ottimi indicatori di inquinamento ambientale. Inoltre sono stati valutati: l’espressione del messaggero delle Heat Shock Protein 70 (HSP70) e, in entrambe le specie monitorate, i livelli di steroidi sessuali (testosterone, estradiolo e progesterone) al fine di verificare eventuali variazioni imputabili alla presenza nell’ambiente di distruttori endocrini cioè di sostanze inquinanti in grado di interferire con il sistema endocrino degli organismi. Mediante analisi di immunoistochimica, Western blot, saggio T-BARS e Real Time PCR abbiamo potuto confermare quanto già riportato in letteratura: il bacino di Lido è la zona maggiormente impattata della Laguna poiché influenzata dalla presenza di un’estesa area industriale e della città di Venezia. Un minor impatto è stato invece riscontrato per le restanti aree monitorate. Tuttavia, al fine di di ottenere un quadro più completo dello stato dell’intero ambiente lagunare, è necessario: identificare ulteriori ed efficaci bioindicatori, ampliare l’area monitorata identificando altri siti di campionamento in tutti i bacini della Laguna di Venezia, aumentare il numero di esemplari da sottoporre a detossificazione utilizzando animali prelevati dai diversi bacini della Laguna di Venezia per allestire un esperimento di controllo completo

Sex steroids in tapes philippinarum (Adams and Reeve 1850) during the gametogenic cycle: preliminary results.

It is well known that a wide variety of endocrinedisrupting chemicals (EDCs) (e.g. organotin compounds, heavy metals, herbicides, xenoestrogen compounds) can induce endocrine alterations in aquatic invertebrates, often causing reproductive disorders. Although these phenomena are of great concern, mostly in coastal and estuarine environments, few data are available about the mechanism of action of EDCs, mainly due to lack of knowledge about the invertebrate endocrine system. To understand the possible mechanism of action of EDCs better, the presence and patterns of variation of sexual steroids, progesterone, testosterone and 17\u3b2-estradiol were measured in the whole body of the clam Tapes philippinarum by specific and opportunely validated microtitre radioimmunoassays (RIAs). Female steroid concentrations showed similar trends, with higher values during spawning and lower ones in post-spawning and gametogenesis periods. Conversely, in males, sexual steroids varied over the year: progesterone remained unchanged, with values similar to those of females in gametogenesis, whereas testosterone and 17\u3b2-estradiol showed a significant decrease during gametogenesis only. Seasonal variations in clam steroid levels may reflect their role in modulating reproduction. All hormones were unexpectedly high in the resting/ early developing stage, during which gonadal tissue is scarce, indicating that digestive gland may have a biosynthetic steroid capacity or accumulation. Although these results are preliminary, evaluation of sexual steroids in T. philippinarum during the gametogenic cycle represent the first step for future research aimed at understanding the physiological role of these hormones and their potential interaction with EDCs

Behaviour and welfare of growing rabbits housed in cages and pens

The present study compared behaviour, fear and stress levels of 456 crossbred rabbits reared in bicellular wire-net cages (2 rabbits/cage) with top and wire floor or open-top collective pens (20 to 54 rabbits/pen) with wooden slatted floor. The effect of pen size (small vs. large) and stocking density (12 vs. 16 rabbits/m2) in collective pens was also evaluated. The rabbits kept in collective pens rested more than those in bicellular cages (82.1% vs. 77.6% of observed time; P<0.01), spent less time feeding (7.8% vs. 10.9%), allo-grooming (0.65% vs. 1.58%) and more time moving (0.81% vs. 0.35%) (P<0.01). The percentage of rabbits sensitive in the tonic immobility test was lower in rabbits kept collectively compared to those in bicellular cages (76.6% vs. 93.8%; P=0.03). In the open field test, the rabbits kept in collective pens moved less (40.4 s vs. 57.2 s; P<0.001) and explored the arena for a shorter time (345 s vs. 371 s; P<0.001) than rabbits housed in bicellular cages; instead, these rabbits stood still for a longer period of time (82.1 s vs. 38.7 s; P<0.001). The rabbits kept in collective pens displayed a higher hair corticosterone level (15.7 ng/g vs. 6.7 ng/g for rabbits from the cages; P<0.001). Within collective housing systems, stocking density had no effect whereas increasing pen size numerically reduced the time rabbits spent eating (8.30% vs. 6.55%), self-grooming (6.39% vs. 5.47%) and time spent resting (81.1% vs. 84.1%). In conclusion, rabbits housed in collective pens displayed a more complete behavioural pattern, despite resting more; they were more bold toward humans, but more fearful in a new environment than rabbits from cages. In the tested conditions, pen size and stocking density within collective systems exerted only a weak effect on behaviour, fear and stress levels of meat rabbits

Alternative matrices for cortisol measurement in fish.

Plasma cortisol is the most commonly used indicator of stress in fish but, as the blood sampling procedure itself can be a source of stress, it would be helpful to measure cortisol using less invasive matrices. It is also necessary to find alternative matrices as stress indicators in dead fish inwhich blood sampling is impossible. In the present study, we investigated transport stress in three aquaculture species, European sea bass (Dicentrarchus labrax L.), common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum), by cortisol determination (radioimmunoassay) in plasma and other matrices (skin mucus, gut content, lateral muscle and caudal fin). Cortisol signi\ua2cantly increased after transport in all species and matrices, except in the sea bass gut content, where it remained unchanged. The three species responded to transport stress by producing di\ua1erent cortisol levels. In conclusion, the significant correlation found between plasma cortisol and most of the other matrices opens up the possibility of using them to evaluate stress in fish: mucus sampling is a less invasive method than blood sampling, and in addition to muscle and fin sampling, it can be used in postmortem fish

SEX STEROIDS IN TAPES PHILIPPINARUM (ADAMS AND REEVE, 1850) DURING THE GAMETOGENIC CYCLE : PRELIMINARY RESULTS.

It is well known that a wide variety of endocrine disruptor chemicals (EDCs) (e.g., organotin compounds, heavy metals, herbicides, and xenoestrogenic compounds) can induce endocrine abnormalities in marine invertebrates often causing reproductive disorders. Despite these phenomena are of great concern mostly in coastal and estuarine environments, only few data are available about the mechanism of action of EDCs, mainly due to a lack of knowledge on the invertebrate endocrine system. In particular, in the clam Tapes philippinarum estrogenic effects of nonylphenol (NP) were recently demonstrated, they resulting in vitellogenin-like protein induction, mostly in male clams (Matozzo and Marin, 2005), as NP probably mimics endogenous estrogens. To better understand the possible mechanism of action of NP, as well as of other EDCs, the presence and patterns of variation of sexual steroids were evaluated during the various phases of the gametogenic cycle of T. philippinarum. Adult clams were collected from two licensed areas located in the Lagoon of Venice (Italy) from July 2005 to May 2006, corresponding to following stages: spawning (July), post-spawning (October), spent (January) and gametogenesis (May). The clams were sexed by microscopic observation (400 7) of a smear of gonadal tissue with the exception of the spent stage in which no gametes were detectable. Progesterone, testosterone and estradiol-17b were measured in the whole-body clam homogenate using specific and opportunely validated microtitre RIAs. Differences in hormone concentrations during annual cycle were assessed by analysis of variance (ANOVA) followed by a Tukey\u2019s Post hoc test (P<0.05). In females, all the hormones showed the same profile, with higher concentrations during the spawning period and lower during post-spawning and gametogenesis. In males, progesterone and estradiol-17b never differed among stages and the values were very similar to those of females in non spawning periods. Conversely, testosterone statistically decreased during gametogenesis. All the hormones were unexpectedly high in the spent stage in which no gonadal tissue was observable and females and males were thus jointly processed. Progesterone was the most representative sexual steroid showing levels (about 1 ng/g) six to seven times higher than those of estradiol-17b and testosterone. Although the results obtained are preliminary and need further validation, the protocols set up to evaluate sexual steroids in T. philippinarum represent the first step for future research aimed at understanding the physiological role of these hormones and their potential interaction with EDCs. Matozzo V., Marin M.G., 2005. Can 4-nonylphenol induce vitellogenin-like proteins in the clam Tapes philippinarum? Environ. Res., 91: 179-185