Ecotoxicological evaluation of Harbour sediments using marine organisms from different trophic levels

Background, Aim and Scope  The toxicity of contaminated sediments should be evaluated considering the direct exposure of laboratory organisms to whole sediments and the indirect exposure to elutriates or extracts (Tay et al. 1992, Byrne and Halloran 1999, Nendza 2002). The alga Dunaliella tertiolecta is indicated for the use in toxicity bioassays because it is highly sensitive to several xenobiotics. Harpacticoid copepods have been already used for toxicity testing and Tigriopus fulvus is a promising Mediterranean target-species in ecotoxicology (Todaro et al. 2001, Faraponova et al. 2003, Pane et al. 2005a). In this study, the toxicity of sediments collected in harbour sites of the Northeastern Adriatic Sea was evaluated by growth inhibition test with free living and alginate-immobilized Dunaliella tertiolecta and acute toxicity test with nauplii and adult Tigriopus fulvus with the aim of pointing out the importance to utilize model organisms from different trophic levels in sediment ecotoxicology. Methodology  Elutriates and whole sediments were tested on free living and immobilized (Pane et al. 1998) algal cells, and on laboratory reared copepods. Free-living D. tertiolecta were exposed to diluted elutriates in a static, multi-well plate system. Naalginate immobilized D. tertiolecta were placed in polystyrene inserts fitted with polyester mesh bottoms and exposed to a thin layer (2 mm) of whole sediments in multi-well plates (EPS 1992, Pane and Bertino 1999). Toxicity tests with copepods were carried out on Tigriopus fulvus nauplii (elutriates) and adults (whole sediments and elutriates). Same-aged nauplii useful for toxicity tests were obtained by egg sac detaching and consequent hatching stimulation (Pane et al. 2006). Newborn nauplii (I–II stage) were exposed to elutriates in multi-well plates provided with polystyrene inserts. Adult T. fulvus maintained in polystyrene inserts fitted with polyester mesh bottoms were placed in contact with a thin layer (2 mm) of whole sediment placed on multi-well plate bottoms. All end-points were evaluated after 96 h. Results  In general, the effects increased with the increasing of elutriate concentration up to 50%; the stimulation or inhibition of algal growth was statistically significant in comparison to the control. The inhibiting elutriates induced EC₅₀ variations of algal growth ranging from 66.9% to 74.3%. The mortality of T. fulvus nauplii was always < 25% after treatment with 100% elutriates and < 10% after treatment with 50% dilution; no effect was shown up with 25% dilution; therefore LC₅₀ was not calculable. The effect of elutriates was negligible on adult copepods and LC₅₀ values were never calculable; percent mortality always resulted in < 10% after treatment with whole sediments. Discussion  Both experimental systems gave substantially similar results after exposition to whole sediments and elutriates. During the experiment with algal cells, the immobilization in Na-alginate and the employment of inserts which allowed the contact of organisms with sediments and their easy counting were particularly useful. Likewise, the employment of inserts of adequate mesh size in the tests with copepods allowed the contact of organisms with the sediment and made organism handling and counting easy, as well as the evaluation of mortality. The methodology here described and the utilization of the proposed test-species could have an importance also considering that the current trend in ecotoxicological research is towards finding the most appropriate organism for specific areas of concern by using indigenous species (Mariani et al. 2006) and towards the major significance of chronic and reproductive end-points. Conclusions  Based on the above results, it can be stated that the bioassay with Dunaliella tertiolecta could be a good estimation tool for the ecotoxicological assessment of marine sediments. The immobilization of algae in Na-alginate was seen to be useful to evaluate the toxicity of whole sediments; the employment of polystyrene inserts allowed an improvement of the procedures. T. fulvus nauplii and adults, as other harpacticoids such as Tigriopus japonicus (Yoon et al. 2006), satisfy the basic criteria for the employment of a standard species in marine bioassays. To date only pelagic Acartia tonsa are utilized in the standardized procedure to evaluate the risk assessment of chemicals or wastewaters (ISO 1999). As, on the contrary, the exposure of copepods to solid-phase contaminants it is not yet standardized, the employment of polystyrene inserts improved the procedures for T. fulvus too. So, the rapidity and the possibility to solve practical problems could be the main attractive features of this technique (Pane et al. 2005a) when applied to whole sediments. Recommendations and Perspectives  The methodology here developed being also applicable to long term and reproduction tests should be recommended because it provides relevant information in comparison with other frequently applied, standardized biotests with crustaceans (ISO 1999). The procedure has been shown to be easily applicable to selected marine organisms. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Determining the bioavailability of contaminants and assessing the quality of sediments

Background, Aims and Scope  

Our study determined if and when an avoidance or attraction behavioural response would be displayed by Ilyanassa obsoleta when presented with a choice between harbour and reference sediments. Our goal was to develop a non-lethal approach that could be used in future assessments of the environmental quality of field sediments.     

Identification of fructooligosaccharides in different banana cultivars

Banana has been currently indicated as a good source of fructooligosaccharides (FOS), which are considered to be functional components of foods. However, significant differences in their amounts in bananas have been observed in the literature. This work aims to identify and quantify FOS during ripening in different banana cultivars belonging to the most common genomic groups cultivated in Brazil. Considering that these differences can be due to cultivar, stage of ripening, and the methodologies used for FOS analyses, sugar contents were analyzed by high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and gas chromatography-mass spectrometry (GC-MS). An initial screening of eight cultivars (Ouro, Nanic?o, Prata, Ma??, Mysore, Pacovan, Terra, and Figo) in a full-ripe stage showed that 1-kestose, the first member of the FOS series (amounts between 297 and 1600 microg/g of DM), was accumulated in all of them. Nystose, the second member, was detected only in Prata cultivar. Five of the cultivars were analyzed during ripening, and a strong correlation could be established with a specific sucrose level ( approximately 200 mg/g of DM), which seems to trigger the synthesis of 1-kestose (the low amounts of FOS, below the functional recommended dose, indicates that banana cannot be considered a good source of FOS).     

Classification of Peruvian highland maize races using plant traits

The maize of Latin America, with its enormous diversity, has played an important role in the development of modern maize cultivars of the American continent. Peruvian highland maize shows a high degree of variation stemming from its history of cultivation by Andean farmers. Multivariate statistical methods for classifying accessions have become powerful tools for classifying genetic resources conservation and the formation of core subsets. This study has two objectives: (1) to use a numerical classification strategy for classifying eight Peruvian highland races of maize based on six vegetative traits evaluated in two years and (2) to compare this classification with the existing racial classification. The numerical classification maintained the main structure of the eight races, but reclassified parts of the races into new groups (Gi). The new groups are more separated and well defined with a decreasing accession within group × environment interaction. Most of the accessions from G1 are from Cusco Gigante, all of the accessions from G3 (except one) are from Confite Morocho, and all of the accessions from G7 are from Chullpi. Group G2 has four accessions from Huayleño and four accessions from Paro, whereas G4 has four accessions from Huayleño and five accessions from San Geronimo. Group G5 has accessions from four races, and G6 and G8 formed small groups with two and one accession each, respectively. These groups can be used for forming core subsets for the purpose of germplasm enhancement and assembling gene pools for further breeding.     

Plasma lactate and purine derivatives accumulation after exercise of increasing intensity in standardbred horses

The aim of the experiment was to study the relationship between plasma lactate and allantoin accumulation in horses undergoing five exercises differing in intensity and length. Twenty-five adult trotter horses were used (18 males, two castrated, and five females), housed in three training centers. The horses were assigned to five groups: slow trot, over 2000 m (Group 1); slow trot over 1600 m (Group 2); fast trot over 1600 m (Group 3); fast trot over 2000 m (Group 4); fast trot over 2400 m (Group 5). Plasma was obtained from blood sampled at rest, at the end of the bout of exercise and after 15 and 45 minutes from the end of the bout of exercise and analyzed for glucose, lactate, uric acid, free fatty acids (FFA) and allantoin concentrations. Accumulations of plasma lactate and allantoin (mmol/sec) were calculated as difference between end of exercise and rest and between 45 minutes sample and rest, respectively.Ranking the intensity of exercise using the lactate concentrations at the end of exercise, the level of exertion was highest for Group 3 horses and lowest for Group 5 horses (20.9 and 2.8 mmol/l, respectively). At the end of exercise, glucose concentrations were much higher for horses undertaking the more intensive exercise (Groups 3 and 4 compared to Group 2). FFA concentrations were highest at the end of exercise for Groups 2 and 3 and after 15 minutes for Groups 4 and 5. Plasma uric acid and allantoin concentrations peaked 15 and 45 minutes from the end of exercise, respectively, independently of exercise intensity. The relationship between accumulation of plasma allantoin (y, dependent variables) and lactate (x, independent variable) was non-linear: y=0.15−2.61^*x+68.3^*x² (r²=0.900; se=0.19). This suggests that allantoin accumulation could be used together with plasma lactate to calibrate the workload to muscle conditions to prevent muscle injury.     

Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene

Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.     

Multiple feather follicle cysts in a Moroseta hen (Gallus gallus)

An 8-month-old white feathered, black skinned Moroseta hen was presented for examination because of numerous 2 mm- to 30 mm-diameter irregularly shaped, hard nodules in the skin of the head, wings, back, and abdomen. The nodules were confined to the skin and did not involve subcutaneous tissues. Nodules consisted of dilated feather follicles packed with a caseous tan-to-pale-yellow material admixed with feather remnants. Histologically, affected feather follicles were markedly dilated and filled with laminated keratin debris. Necrosis of the epidermis and perifollicular lymphocyte infiltration was also present. Bacteriologic investigation of internal organs was negative, while secondary bacteria, Proteus spp. and Bacillus spp., were isolated from skin nodules. A concomitant lice infestation of Menopon spp., as well as leg mange caused by Cnemidocoptes spp., were also present. These bacterial isolates and parasites were not related to the disease condition. The condition observed was differentiated from benign feather follicle tumors, and a diagnosis of multiple feather follicle cysts was made. In addition, a breed predisposition was hypothesized.     

Effects of duration of zilpaterol hydrochloride feeding and days on the finishing diet on feedlot cattle performance and carcass traits

British and British x Continental steers (n = 560; initial BW = 339.4 +/- 1.76 kg) were used in a serial slaughter study with a completely random design to evaluate effects of zilpaterol hydrochloride (ZH; 8.33 mg/kg of dietary DM basis) on performance and carcass characteristics. Treatments were arranged in a 4 x 4 factorial (112 pens; 7 pens/treatment; 5 steers/pen) and included duration of ZH feeding (0, 20, 30, or 40 d before slaughter plus a 3-d ZH withdrawal period) and days on feed (DOF) before slaughter (136, 157, 177, and 198 d). No duration of ZH feeding x slaughter group interactions were detected for the performance measurements (P > 0.10). Final BW did not differ (P = 0.15) between the 0-d group and the average of the 3 ZH groups, but ADG was greater for the average of the 3 ZH groups during the period in which ZH diets were fed (P < 0.01) and for the overall feeding period (P = 0.05). As duration of ZH feeding increased, DMI decreased (P = 0.01) and G:F increased linearly (P < 0.01). With the exception of KPH (P = 0.022), no duration of ZH feeding x slaughter group interactions (P > 0.10) were detected for carcass characteristics. Regardless of the duration of ZH feeding, cattle fed ZH had greater HCW (P < 0.01), greater dressing percent (P < 0.01), less 12th-rib fat (P < 0.01), larger LM area (P < 0.01), less KPH (P = 0.03), and lower yield grade (P < 0.01) than the 0-d cattle. The 0-d group had greater marbling scores (P < 0.01) than cattle fed ZH diets, with a tendency for a linear decrease in marbling score (P = 0.10) as duration of ZH feeding was extended. A greater percentage of carcasses in the 0-d group graded USDA Choice or greater (P < 0.01) than in the 3 ZH groups, whereas the percentage of Select carcasses was greater (P = 0.01) for the 3 ZH groups. From d 0 to end (P = 0.04) and during the last 43 d on feed (P < 0.01), ADG responded quadratically to DOF before slaughter. No differences were detected among slaughter groups for DMI for the entire trial period; however, a quadratic response (P = 0.02) was observed for the final 43 d before slaughter. A quadratic response was also detected for the final 43 d before slaughter (P < 0.01) and from d 0 to end (P = 0.02) for G:F. Final BW, HCW, dressing percent, and 12th-rib fat increased linearly (P < 0.01) as DOF before slaughter increased. Our results indicate that no substantial effects on performance and carcass measurements were observed when ZH was fed for 30 or 40 d as opposed to 20 d, and that effects of ZH generally did not interact with DOF before slaughter.     

ASAS Centennial Paper: contributions in the Journal of Animal Science to understanding cattle metabolic and digestive disorders

Acute and subacute ruminal acidosis, bloat, liver abscesses, and polioencephalomalacia (PEM) were reviewed with respect to contributions published in the Journal of Animal Science (JAS) regarding these metabolic and digestive disorders in beef cattle. Increased grain feeding and expansion of the feedlot industry in the 1960s led to considerable research on acidosis, and early publications defined ruminal changes with acute acidosis. The concept of subacute acidosis was developed in the 1970s. Significant research was published during the 1980s and 1990s on adaptation to high-grain diets, effects of ionophores, and the development of model systems to study ruminal and metabolic changes in acidosis. Since 2000, JAS publications on acidosis have largely focused on individual animal variability in response to acid loads and the role of management strategies in controlling acidosis. Increased grain feeding also was associated with an increase in the incidence of liver abscesses, which were quickly linked to insults to the ruminal epithelium associated with acidosis. The role of antibiotics, particularly tylosin, in decreasing the incidence and severity of liver abscesses was a significant contribution of JAS publications during the 1970s and 1980s. Papers on bloat were among the earliest published in JAS related to metabolic and digestive disorders in cattle. Noteworthy accomplishments in bloat research chronicled in JAS include the nature of ruminal contents in legume and feedlot bloat, the role of plant fractions and microbial populations in the development of bloat, and the efficacy of poloxalene, ionophores, and, more recently, condensed tannins in decreasing the incidence and severity of bloat. Although less research has been published on PEM in JAS, early publications highlighting the association between PEM and ruminal acidity and the role of thiaminase in certain forms of the disorder, as well as more recent publications related to the role of sulfur in the development of PEM, are noteworthy contributions. Since the 1940s, outstanding and often-cited review articles have made JAS a highly visible source of information on these disorders. Thus, JAS has played a significant role as a repository for information pertaining to metabolic and digestive disorders in cattle and other ruminants, and it will no doubt continue to be a premier resource for information on these conditions during the second century of the American Society of Animal Science.     

Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8 kDa and from 57.8 to 73.3 kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.