Anabolism,catabolism and proteomic analysis in the slow‐twitch muscle of pacu (Piaractus mesopotamicus) submitted to prolonged fasting followed by refeeding

Here, we aimed to study the slow muscle of the fish Piaractus mesopotamicus submitted to 30 days of fasting (D30) followed by 1 day (D31) or 30 days of refeeding (D60). Histological analysis of fibre diameter was performed in D30 and D60. The gene expression of parvalbumin (pvalb), atrogenes (murf1a, murf1b, mafbx) and anabolic genes (igf‐1, mtor) was analysed using RT‐qPCR in D30, D31 and D60. The proteome was obtained by shotgun proteomics at D30 and D60, and the set of differentially expressed proteins was analysed by bioinformatics. In all experiments, the control was regularly fed fish. The histological analysis showed no changes in muscle fibre diameter. The expression of catabolic and anabolic genes was not changed, except for the downregulation of igf‐1 in D30 and of mafbx in D31. The expression of pvalb was not changed in D30 and D60 but was decreased in D31. The proteomic analysis identified 169 proteins in D30 (24 upregulated and 18 downregulated) and 170 proteins in D60 (17 upregulated and 21 downregulated); many of them were related to energetic metabolism and intracellular Ca²⁺ homoeostasis. Overall, our results indicate that the slow‐twitch muscle presented few changes upon prolonged fasting and refeeding condition.     

Survey of polychlorinated dibenzo-p-dioxins (PCDDs), Polychlorinated Dibenzo-p-furans (PCDFs), polychlorinated biphenyls (PCBs), and mineral components in Italian citrus cold-pressed essential oils

Investigation of PCDDs, PCDFs, PCBs, Al, As, Pb, Ba, Co, Cu, Cr, Fe, Mn, Cd, Ag, Sn, Zn, and Hg contents in 60 samples of cold-pressed essential oils produced in Calabria and Sicily in 2003-2005 was carried out. PCDDs, PCDFs, and PCBs were analyzed by HRGC-HRMS techniques using U.S. EPA 1613/94 and U.S. EPA 1668/A (1999) analytical methods. Mineral components were determined through GFAAS techniques; Hg content was determined by FI-M/H-AAS. The results of this study showed that essential oil contamination was due to a widespread pollution, typical background of rural areas, with relatively higher concentrations of PCDDs compared to PCDFs and little presence of PeCDF. Congeners OCDD, HpCDF, and OCDF were found at high concentrations. Regarding mineral components, mean values of Cr, Fe, and Ni were in agreement with data reported in the literature. Concentrations of As and Pb were below the maximum limits accepted by the current legislation. Finally, none of the samples analyzed were contaminated with Hg.     

Changes in soil carbon and nitrogen stocks after conversion of subtropical natural forest to loblolly pine plantations

Pinus plantations have increased in Brazil, and native forest areas have been converted for timber production. The clearing and the long-term loblolly pine (Pinus taeda L.) land-use effects on soil carbon and nitrogen stocks were evaluated in a natural broadleaved forest and in loblolly pine sites cultivated for 29, 35, 38 and 49 years, as well the soil contribution as ecosystem carbon pool. According to the exponential-decay model fitted to changes in carbon stock, the initial soil carbon stock of 200 Mg ha^?1 to a depth of 100 cm in the natural forest decreased by 36% over 49 years of pine cultivation (around 72.4 Mg ha^?1 of C). Around two-thirds of this decrease occurred in the top 30 cm of the soil and intensively in the first 12 years of cultivation, but slowly faded as carbon stock tended to reach a new steady state after approximately 49 years of cultivation. The soil nitrogen stock in the natural forest was 14.2 Mg ha^?1 to a depth of 100 cm and decreased by 36% over the 49 years. This decrease was linear according to the fitted model, especially in the top 30 cm where nitrogen decline was 83% and was proportionally more intense than the carbon decline. Despite the soil carbon decrease, soil remained the largest carbon reservoir in the ecosystem for the growing rotation time of loblolly pine in this region.

     

Ultrastructural Morphology and Nuclear Maturation Rates of Immature Equine Oocytes Vitrified with Different Solutions and Exposure Times

Ultrastructural morphological injuries and maturation rates were investigated in equine oocytes exposed to vitrification solutions (VS) containing synthetic ice blockers (SIBs) during different exposure times. In experiment 1, compact cumulus-oocyte complexes (COCs; n = 30) were randomly allocated to treatments: (1) fresh fixed (control); (2) VS-1 (1.4 M dimethyl sulfoxide [DMSO] + 1.8 M ethylene glycol [EG] + 1% SIB) for 3 minutes of equilibrium time and VS-2 (2.8 M DMSO + 3.6 M EG + 0.6 M sucrose + 1% SIB) for 1 minute (Eq-long); and (3) VS-1 for 1.5 minutes and VS-2 for 30 seconds (Eq-short). In experiment 2, compact (n = 248) and expanded (n = 264) COCs were evenly distributed to the following treatments: (1) immediate maturation in vitro (control); (2) vitrification using the Eq-short protocol as in experiment 1; and (3) vitrification using a stock solution containing 2.8 M formamide, 2.8 M DMSO, 2.7 M EG, 7% polyvinylpyrrolidone, and 1% SIB (Eq-short-mod). More (P < .02) oocytes with normal ultrastructural morphology were seen in fresh control and Eq-short groups than in Eq-long group. Metaphase-II (MII) rates were higher (P < .05) for oocytes with expanded cumulus than compact cumulus in the control group, and higher (P < .05) for oocytes with expanded cumulus than compact cumulus in Eq-short and Eq-short-mod groups. No difference in MII rates was detected among groups within each type of COC. In conclusion, reduction of exposure time to VS better preserved oocyte ultrastructural features, and MII rates were higher for vitrified oocytes with expanded cumulus. This study advances our knowledge on potential alternatives for vitrification of immature equine oocytes.     

The fate of Amazonian forest fragments: A 32-year investigation

We synthesize findings to date from the world’s largest and longest-running experimental study of habitat fragmentation, located in central Amazonia. Over the past 32 years, Amazonian forest fragments ranging from 1 to 100 ha have experienced a wide array of ecological changes. Edge effects have been a dominant driver of fragment dynamics, strongly affecting forest microclimate, tree mortality, carbon storage, fauna, and other aspects of fragment ecology. However, edge-effect intensity varies markedly in space and time, and is influenced by factors such as edge age, the number of nearby edges, and the adjoining matrix of modified vegetation surrounding fragments. In our study area, the matrix has changed markedly over the course of the study (evolving from large cattle pastures to mosaics of abandoned pasture and regrowth forest) and this in turn has strongly influenced fragment dynamics and faunal persistence. Rare weather events, especially windstorms and droughts, have further altered fragment ecology. In general, populations and communities of species in fragments are hyperdynamic relative to nearby intact forest. Some edge and fragment-isolation effects have declined with a partial recovery of secondary forests around fragments, but other changes, such as altered patterns of tree recruitment, are ongoing. Fragments are highly sensitive to external vicissitudes, and even small changes in local land-management practices may drive fragmented ecosystems in markedly different directions. The effects of fragmentation are likely to interact synergistically with other anthropogenic threats such as logging, hunting, and especially fire, creating an even greater peril for the Amazonian biota.     

Using BCG,MPT-51 and Ag85 as antigens in an indirect ELISA for the diagnosis of bovine tuberculosis

This study evaluated the Mycobacterium tuberculosis protein antigen MPT-51, the trimeric antigen 85 (Ag85) complex, and Bacillus Calmette-Guérin (BCG) in an indirect ELISA to diagnose bovine tuberculosis (TB) from serum samples. Serum was collected from 208 intra-dermal tuberculin test (ITT)-positive and 54 ITT-negative animals from a region where bovine TB is endemic. Using the Ag85 and BCG antigens, the indirect ELISA was able to discriminate ITT-positive from ITT-negative animals. This level of discrimination was not achieved when using the MPT-51 antigen. The highest sensitivity (Se) and specificity (Sp) of the test was found when BCG was used (Se, 82%; Sp, 91%). Further work in different epidemiological settings and with larger numbers of animals will be required to validate these findings.     

Leucocyte profile and growth rates as indicators of crowding stress in pejerrey fingerlings (Odontesthes bonariensis)

Crowding is one of the most common stressors found in intensive aquaculture, compromising growth rates and immune function. Plasmatic cortisol is a classic stress biomarker for fish, but its quantification is expensive and demands blood volumes that small individuals do not provide, constraining the usage of this technique to assess stress in fingerlings. The leucocyte profile is an alternative methodology to quantify stress with reduced costs and volumes of blood. Stress conditions promote neutrophilia and lymphocytopenia as response to elevated glucocorticoids levels. Considering the difficulties to assess stress imposed by intensive fish farming using measurement of glucocorticoid hormones, this study aimed to evaluate the stress‐induced changes in leucocyte profiles and growth rates imposed by crowding in fingerlings of Odontesthes bonariensis, a promising South‐American candidate for freshwater aquaculture. To meet these objectives, fingerlings (initial weight 0.05 ± 0.06 g and length 1.68 ± 0.13 cm) were reared for 45 days under three rearing densities (1, 5 and 10 fingerlings L^?1). At the end of this period, fish were anaesthetized and euthanized to obtain the leucocyte profile, neutrophil:lymphocyte ratio (N:L) and growth rate. Increasing density promoted: significant reduction in growth (final length, weight and specific growth rate); neutrophilia and lymphocytopenia; and increased N:L ratio. Concluding, the tested rearing densities imposed distinct levels of stress characterized by different N:L ratio, demonstrating that the leucocyte profile is a reliable alternative to measure stress levels in O. bonariensis fingerlings and probably in other fish species.     

Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum

Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities.     

Vibrio spp. Control at Brine Shrimp,Artemia, Hatching and Enrichment

Experiments were conducted to evaluate different prophylactic methods to control the bacterial load in brine shrimp, Artemia, hatching. The first experiment evaluated three treatments to control Vibrio spp. during the Artemia hatching: microalgae (Chaetoceros calcitrans), probiotic (Bacillus spp.), and antibiotic (Florfenicol). In the second experiment, Artemia metanauplius were enriched in distinct treatments with C. calcitrans, probiotic, and emulsion rich in docosahexaenoic and eicosapentaenoic fatty acids. Enriched Artemia metanauplius and nauplii (control) were offered to white shrimp, Litopenaeus vannamei, postlarvae (PL₇–PL₁₉). Presumptive Vibrio were quantified in Artemia, PL, and rearing water. Microalgae and probiotic were effective to control Vibrio spp. in Artemia nauplii. The enrichment process increased the Artemia bacterial load but did not affect Vibrio load in L. vannamei.