Since the early 2000s, unconventional natural gas development (UNGD) has rapidly grown throughout Pennsylvania. UNGD extracts natural gas using a relatively new method known as hydraulic fracturing (HF). Here we addressed the association of HF with asthma Hospitalization Admission Rates (HAR) using publicly available data. Using public county-level data from the Pennsylvania Department of Health (PA-DOH) and the Pennsylvania Department of Environmental Protection for the years 2001-2014, we constructed regression models to study the previously observed association between asthma exacerbation and HF. After considering multicollinearity, county-level demographics and area-level covariables were included to account for known asthma risk factors. We found a significant positive association between the asthma HAR and annual well density for all the counties in the state (3% increase in HAR attributable to HF, p<0.001). For a sensitivity analysis, we excluded urban counties (urban counties have higher asthma exacerbations) and focused on rural counties for the years 2005-2014 and found a significant association (3.31% increase in HAR attributable to HF in rural counties, p<0.001). An even stronger association was found between asthma hospitalization admission rates (HAR) and PM2.5 levels (7.52% increase in HAR attributable to PM2.5, p<0.001). As expected, asthma HAR was significantly higher in urban compared to rural counties and showed a significant racial disparity. We conclude that publicly available data at the county-level supports an association between an increase in asthma HAR and UNGD in rural counties in Pennsylvania.

Whole body cortisol levels were determined during early larval developmental stages of sea bass (Dicentrarchus labrax) subjected to a heat shock with the aim to investigate the correlation between the stress event and the activation of the hypothalamic-pituitary-interrenal axis. Moreover, the mRNA expression of inducible heat shock protein 70 (HSP70), insulin-like growth factor I (IGF-I) and myostatin (MSTN) was also detected. Whole body cortisol was determined by a radio-immunoassay (RIA) technique whereas the expression of HSP70, IGF-I and MSTN mRNAs was quantified by Real-Time PCR. Cortisol was detectable in all the larvae from hatching but its level increased significantly in larvae submitted to heat shock from 2-day post hatching onwards. An effect of the sole transfer on cortisol levels was detectable at day 10, indicating an increase of the hypothalamic-pituitary-interrenal axis sensitivity from this stage of sea bass development. In animals exposed to heat shock, the expression of inducible HSP70 resulted in a marked increase of mRNA levels already at hatching. This increase was significantly higher from 6 days onwards if compared to controls. Moreover, heat shock resulted in a decrease (although not significant) in IGF-I mRNA expression of stressed larvae if compared to controls. On the contrary, heat shock did not influence the expression of MSTN mRNA in all groups. The results indicate a very early activation of the hypothalamic-pituitary-interrenal axis and in general of the stress response during the development of European sea bass. Moreover, these results suggest the importance of cortisol and inducible HSP70 as bioindicators of stress in aquaculture and confirm the role of IGF-I and MSTN as regulatory factors during development and growth of 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.

The cellular localization of IGF-I, IGF-II and MSTN proteins was investigated during ontogenesis of triploid sea bass (Dicentrarchus labrax) by an immunohistochemical approach. The results were compared with those observed in diploids. IGF-I immunostaining was mainly observed in skin, skeletal muscle, intestine and gills of both diploids and triploids. From day 30 of larval life, IGF-I immunoreactivity observed in skeletal muscle, intestine, gills and kidney was stronger in triploids than in diploids. At day 30, triploids exhibited a standard length significantly higher than the one of diploids. Although IGF-II and MSTN immunoreactivity was detectable in different tissues and organs, no differences between diploids and triploids were observed. The spatial localization of IGF-I, IGF-II and MSTN proteins detected in this study is in agreement with previous findings on the distribution of these proteins in diploid larvae and fry. The highest IGF-I immunoreactivity observed in triploids suggests a possible involvement of ploidy in their growth performance.

Several studies carried out in the last years have demonstrated the presence of a wide range of contaminants in some areas of the Venice Lagoon. Many of these contaminants are able to drive free radical reactions, which lead to oxidative stress and can potentially affect fish health. In the present study, oxidative stress biomarkers were examined in three different sites (Porto Marghera, Val di Brenta and Caroman) of the Venice Lagoon and their levels monitored in Zosterisessor ophiocephalus, one of the most common fish species present in the lagoon. Schmorl's staining revealed the presence of melanomacrophage centres in spleen and head kidney, and the highest number of melanomacrophage centres was observed in the animals sampled at the Porto Marghera (Porto Marghera vs Val di brenta and Caroman: p<0.01). The cellular localization of HNE and NT, investigated through an immunohistochemical approach, showed that immunopositivity was mainly localized in melanomacrophage centres of spleen and kidney. It is relevant that the animals of the detoxified control group did not exhibit any immunoreactivity. By Western blot, the antibodies against HNE and NT recognized in the liver polypeptides damaged by oxidative stress with molecular weights under 66kDa. Comparing the relative densities, animals from the Val di Brenta site exhibited the lowest levels of HNE adducts (p<0.05), whereas animals from the Porto Marghera site exhibited the highest levels of NT adducts (p<0.05). MDA levels, measured spectrophotometrically by TBARS assay did not exhibit any statistical difference among sites.

The neuroendocrine conditioning of reproduction in birds could perform a very important role in captive breeding, especially in endangered species. Whereas in domestic and wild mammals pharmacological reproductive conditioning is well developed, in birds an effective method is not available. The aim of this study was to test the influence of a new slow-release GnRH analogue (buserelin acetate) implant on the reproductive activity of the Budgerigar (Melopsittacus undulatus), used as model species for captive-bred endangered birds. The effects were assessed by looking at reproductive parameters (egg-laying rate, egg fertility rate) and measuring excreted sex steroid metabolite concentrations in male and female birds. Modification of reproductive parameters and steroid metabolites excretion patterns were observed among birds administered with a GnRH analogue implant and maintained under artificial photoperiod (group I; 16L:8D). Implanted birds showed higher rates of egg-laying, potentially a higher proportion of fertile eggs and higher excreted steroid metabolite concentrations than birds maintained under natural photoperiod (group II; 10L:14D) and birds maintained under artificial photoperiod (group III; 16L:8D). Thus, it is concluded that the new slow-release GnRH analogue implant may represent an innovative and practicable treatment to rapidly induce reproductive activity in the Budgerigar, and that excreted sex hormone metabolites detection permits to monitor male and female gonadal activity.

In general little is known about hormones and the ontogeny of the stress response in the early developmental stages of chondrostean fishes and in particular of white sturgeon (Acipenser transmontanus, Richardson 1836). In this study, we measured for the first time cortisol and sex steroids (testosterone and estradiol) in eggs, larvae, post-larvae, and fry of white sturgeon by radioimmunoassay (RIA), to elucidate some endocrine aspects of its development. The cortisol, testosterone, and 17β-estradiol of maternal origin found in unfertilized eggs of white sturgeon probably regulate both growth and development of the embryo. Cortisol decreased after fertilization, whereas testosterone and 17β-estradiol did not significantly change. During the late stages of embryo development and immediately after hatching, endogenous production of cortisol and sexual steroids, respectively, occurred. Sex steroids may be physiological inducers of gonad sex differentiation in sturgeon. All steroids showed an increase 10 days post-hatch (dph), near the transition from an endogenous to an exogenous energy source. Cortisol maintained the same basal levels even after metamorphosis, whereas testosterone and 17β-estradiol declined significantly in post-larvae at 35 and 45 days post-hatch. In addition, to evaluate the ontogeny of a functional hypothalamic-pituitary-interrenal (HPI) axis, larvae and fry were submitted to acute stress. The HPI axis did not seem to be functional on the first day post-hatch, but became so from the third day post-hatch onward.

Farmed fish are usually exposed to routine procedures which have strong effects on stress responses. Rodlet cells may represent an useful biomarker for studies on the presence of stressors in aquaculture. This work focused on the localization of rodlet cells by light and electron microscopy in gills of sea bass (Dicentrarchus labrax) subjected to different conditions of overcrowding. In general, a significant increase in number of rodlet cells has been observed in all animals subjected to overcrowding stress. In gills of control group rare rodlet cells were detected at the level of both primary and secondary lamellae, whereas in stressed group clusters of rodlet cells have been found in the epithelium of primary and secondary lamellae indicating that these cells are influenced by stocking density.

In the present work we investigated by immunohistochemistry the cellular localization of constitutive as well as inducible heat shock protein 70 in several tissues of common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) exposed to transport stress. In carp, the constitutive form (HSC70) was detected only in red skeletal muscle of both control and stressed animals. In the same species, the inducible form (HSP70) was evident in the epithelia of renal tubules, gills and skin of stressed animals, whereas in controls only red skeletal muscle exhibited an immunopositivity to HSP70 antibody. In trout, immunostaining to HSC70 antibody was found mainly in the epithelia of intestine, gills and skin of both control and stressed animals although the reactivity was generally higher in animals exposed to transport stress. In the same species immunostaining to HSP70 antibody was observed only in red skeletal muscle and epidermis of control animals.

Stress indicators (plasma cortisol, glucose and lactate) were evaluated in sea bass Dicentrarchus labrax submitted to different pre‐slaughter conditions, and cortisol levels in plasma were found to correlate with those detected in mucus, gut contents and muscle. The results demonstrate new possibilities for monitoring stress status in fishes in which blood sampling is difficult or impossible.

It is well known that a wide variety of endocrine-disrupting 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 beta-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 beta-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.

The cellular localization of IGF-II protein was investigated during larval and postlarval developmental stages of sea bass (Dicentrarchus labrax) by immunohistochemistry using antisera raised against Sparus aurata IGF-II. At hatching, IGF-II immunoreactivity was already present in the skin, developing intestine and skeletal muscle. During larval life IGF-II protein was also observed in heart musculature, in kidney and gill epithelia as well as in liver. In fry skeletal muscle a moderate IGF-II immunostaining was detected in red fibres, whereas white muscle fibres exhibited a faint immunoreactivity. In adults, a marked IGF-II immunostaining was observed in red muscle fibres. A moderate immunoreactivity was also present in white fibres as well as in heart striated myocardial fibres. These results are in agreement with previous findings on the spatial localization of IGF-II and IGF type 1 receptor in S. aurata and Umbrina cirrosa, confirming the role of IGF system during development and growth of fish.

This study was performed to evaluate the effects of high levels of inclusion of plant protein sources to replace fish meal in rainbow trout (O. mykiss) diets on growth performance and quality characteristics. Two isoproteic (44.8%) and isolipidic (19.6%) extruded diets were fed to eight groups of trout (IBW 106.6g) for 103 days. Diet FM, containing only fish meal as protein source, was used as control treatment and compared to diet PV80, where a plant protein mix (pea protein concentrate and wheat gluten) was included to replace 80% of fish meal protein. Growth performance (FBW: 318.5g; SGR: 1.06%) and feed to gain ratio (0.79) of the fish were not affected by dietary treatment (p>0.05). Dry matter and protein ADCs, measured in vivo after stripping, resulted higher in fish fed the high vegetable protein diet relative to the fish meal one (p<0.05). Trout fed diet PV80 were characterised by higher agility index (2.02 vs 1.72, p<0.05), carcass yield (91.95 vs 91.18%, p<0.05), lower hepatosomatic index (0.74 vs 1.07%, p<0.05) and viscerosomatic index (8.04 vs 8.82%, p<0.05) but higher mesenteric fat index (3.08 vs 2.88%, p<0.05) relative to FM fed trout. No significant effect was observed in terms of fillet yield (59.28 vs 6.41%, p<0.05). Feeding diet PV80 resulted in a significant reduction of fillet pH 48h post mortem (5.56 vs 6.44, p<0.05) while no reduction was observed in fillet of trout fed the FM diet (6.56, p>0.05). Higher dry matter (28.2 vs 26.8g/100g, p<0.01) and hydroxyproline (0.79 vs 1.31mg/g, p<0.01) content as a measure of total collagen, was observed in fillets of trout fed PV80 diet relative to the control ones. According to these data, also post mortem firmness measured by the Shear force of raw muscle resulted higher in fish fed the plant protein based diet (4.06 vs 3.30N, p<0.05). The firmness reduction observed 48h post mortem (4.24 vs 2.95N, p<0.05) resulted not affected by the dietary treatment.In summary, data show that even if the growth performance of rainbow trout fed a diet in which fish meal was largely replaced by high quality plant proteins resulted similar, some major quality traits such as dressing out, fillet composition and texture can be significantly affected by dietary treatment.

In aquaculture, fish are exposed to stressful conditions, which cause an increased synthesis of heat shock proteins (HSPs) at the cellular level. In this work we considered the expression of the constitutive and inducible forms of HSP70 as an indicator of stress caused by transport, during development of the sea bass (Dicentrarchus labrax), a teleost fish of high value for aquaculture. Qualitative RT-PCR analysis revealed expression of inducible HSP70 gene in larvae and fry (25, 40 and 80 days) as well as in adult tissues (liver, brain, muscle, gills, kidney, gonads, heart, spleen and skin) of both control and stressed animals. Expression of inducible HSP70 mRNA examined in different adult tissues by Real-Time PCR, was significantly higher in skin and skeletal muscle of stressed animals than in controls. Immunolocalization of inducible and constitutive forms of heat shock protein 70 (HSP70 and HSC70), reported here for the first time, demonstrated an ubiquitous distribution of HSC70 protein in several tissues of both stressed and control animals (at all stages), while inducible HSP70 protein was found only in skeletal muscle of stressed animals. In all stressed animals, regardless of their developmental stage, cortisol levels were higher than in control animals.

Marinelli, L., Gabai, G., Simontacchi, C. and Bono, G., 2007. Effect of aging and reproductive condition on dehydroepiandrosterone plasma levels in the bitch. Veterinary Research Communications, 31(Suppl. 1), 169–172

The fish stunning/slaughtering procedure has an important role both for the ethical aspect related to animal welfare and for the potential quality of the final products. Stressful harvest procedure and killing methods can negatively influence the post mortem biochemical processes with a consequent faster fish freshness loss. In particular, killing procedures causing a long agony are not humane and can shorten fish shelf life; others, more humane, can have risks for the consumers health (anaesthetics or chemical substances) or are not feasible in small/medium size fish industry (spiking and knocking). The aim of this study was to compare the traditional killing method (asphyxia - A) used for rainbow trout (Onchorynchus mykiss) with an innovative stunning/slaughtering method (two-stage electric stun: 2s at a 500Hz electric field of 2.5V/cm and then 58s at a 50Hz electric field of 0.75V/cm - E), through the study of their effects on stress and quality indicators in fish fed with traditional fish meal (FM) or innovative 80% plant protein diet (PV80). Fish (n=30) of each group (FMA, FME, PV80A, PV80E) were stored at 1°C with ice covering and analysed from 0h to 192h from death. The following analyses were done: blood parameters (only at death); rigor index (RI%) and sensorial evaluation with EU Scheme Rule 2406/96 EEC (performed by 5 judge); eye liquor and muscular pH (pHe, pHm); ATP and its degradation products in muscle; lactic acid in muscle and fillet length (FLC) and height contraction % (FHC) (at 0, 3, 6, 24h). Fillet quality appearance was evaluated by absence/presence and size of bloodstains along the midline on the left fillet. Data were analyzed by ANOVA (killing method, diet and their interaction). Asphyxiated fish (A) died within 15-30’ after prolonged struggling and agony, while innovative method stunned/killed fish (E) were immediately stunned and died in 1 minute. Asphyxiated group showed: higher values of blood glucose and muscle lactic acid; lower values of pHe and pHm at 3, 6 and 24h (P<0.01); earlier full rigor (RI 100% at 1h). RI, significantly different (P<0.001) between A and E fish until 6h after death, in E group reached 100% only at 8h. In agreement with this result, FLC of E fish showed lesser contraction both at 3 h (E 6.9 vs A 18.4%, P<0.001) and at 6h (E 13.9 vs A 18.4%, P<0.05). The same emerged for FHC at 3 h (E 0.9 vs 5.3%, P<0.001). The lower values of ATP/IMP ratio at 0, 3 (P<0.001) and 6h (P<0.01) in A fish than in E fish confirmed the stress condition of the first ones. Differences due to the diet were scarce: PV80 fish had higher pHe, pHm and inosine values at 72h (P<0.001). Fish sensorial evaluation indicated A class until 144h in all groups and B class at 192h after death, except in PV80A fish (75% in B class at 144h). As waited, some bloodstains were found in FME (26.7%) and PV80E (53.3%) fillets while A fish had negligible damages (1.7%). The bloodstains should downgraded the fillet acceptance. However, because the portion size trout are traded wholes/gutted by Italian fish industry, the problem seems less serious. The innovative killing procedure, really a fast and humane procedure, need to be improved for fillet appearance.

Previous studies reported that diets can increase tolerance to various stressful conditions in different fish species. Consequently, the present work evaluated the effects of replacing fish meal (FM group) with an 80% of plant protein source (PV 80 group) in the diet of farmed rainbow trout (Oncorhynchus mykiss) on stress response caused by two different slaughter methods (electrical stunning and asphyxia). Plasma cortisol levels are widely employed as quantitative measure of stress (Wendelaar Bonga, 1997). However, to avoid the blood sample, it would be useful to test less invasive alternative biological matrixes such as skin mucus, faeces and caudal fin to use them especially in live fish and also invasive matrixes as muscle for post-slaughter analysis. From each experimental group twelve fish were netted, six were slaughtered by electrical stunning and six by asphyxia. From each fish, plasma, skin mucus, intestinal content, caudal fin tip and muscle were rapidly collected and frozen for cortisol evaluation by RIA (radioimmunoassay). Data on plasma cortisol were submitted to analysis of variance using the GLM procedure (STATISTICA, 2006) according to a bi-factorial arrangement (2x2) with slaughter methods and diets as main variability factors. Pearson’s linear regression was used to correlate cortisol values in different matrixes. Statistical significance was taken as P<0.05. Results showed that plasma cortisol was significantly higher in groups slaughtered by asphyxia (P=0.002) and no differences were found in plasma cortisol between the different diets (P=0.34). Plasma cortisol was compared with the level in the other matrixes showing a positive correlation with skin mucus and intestinal content (r=0.57 and r=0.58, respectively). The low correlation of plasma cortisol with muscle and fin (r=0.2 and r=0.1, respectively) is in contrast with previous study on trout transport stress (unpublished data). This is probably due to the reduced vitality of fish during asphyxia or to insufficient time (20’) for the steroid to reach the highest levels that easily diffuse into muscle and fin. In summary, the electrical stunning is preferable as regard fish welfare and a diet in which fish meal is replaced by plant proteins could not influence the stress response to slaughter. Moreover less invasive matrixes such as mucus and faeces could be good indicators of the stress level in fish.