Influencia de factores endógenos (edad y sexo) en los niveles de biomarcadores de estrés oxidativo en alcatraz (Morus bassanus) / Influence of endogenous factors (age and sex) on the levels of oxidative stress biomarkers in Northern gannet (Morus bassanus)

Las aves marinas pueden ser utilizadas como bioindicadoras de las alteraciones del ambiente en que residen, identificándose en ellas distintos biomarcadores, que no informen sobre los niveles cuantitativos de contaminantes sino sobre los efectos adversos subclínicos que dichos agentes pueden causar. En el presente trabajo se han analizado los niveles de Malondialdehído (MDA) y de actividad Glutatión-S-Transferasa (GST) en muestras de hígado y riñón de 30 alcatraces (Morus bassanus) procedentes de las costas de Galicia, como posibles indicadores de estrés oxidativo en las aves. Además, se ha determinado la influencia de dos factores endógenos (sexo y edad) sobre los niveles de estos biomarcadores. En hígado los valores medios de MDA obtenidos fueron de 0,508±0,502 nmol/mg proteína, mientras que en riñón fueron de 15,67±12,18 nmol/mg proteína. La actividad GST media en hígado fue de 10,93±7,067 nmol/min/mg proteína, mucho menor que la media de los valores en riñón, que fue 62,30±26,97 nmol/min/mg proteína. En cuanto a las variaciones respecto a la edad, solo se encontraron diferencias estadísticamente significativas en riñón en la actividad GST, siendo mayor la actividad de esta enzima en alcatraces adultos que en inmaduros. Por su parte, el factor sexo no influyó en los niveles de ninguno de los biomarcadores considerados. Los resultados obtenidos apuntan a que ambos biomarcadores podrían ser de utilidad en programas de biomonitorización de contaminación ambiental centrados en el alcatraz, aunque serían necesarios estudios que consideren un mayor número de animales, incorporando un abanico más amplio de biomarcadores. (AU)

Seabirds can be used as bioindicators of changes in the environment in which they live, identifying different biomarkers in them, which do not provide information on the quantitative levels of contaminants, but on the subclinical adverse effects that these agents can cause. At the present study, the levels of Malondialdehyde (MDA) and glutathione-S-transferase (GST) activity in liver and kidney samples of 30 gannets (Morus bassanus) from the coast of Galicia have been determined, as suitable biomarkers of oxidative stress on birds. In addition, the influence of two endogenous factors (sex and age) on the levels of these biomarkers has been determined. In the liver, the mean values of MDA were 0.508 ± 0.502 nmol/mg protein, while in kidney they were 15.67 ± 12.18 nmol/mg protein. On the other hand, mean GST activity in the liver was 10.93 ± 7.067 nmol/min/mg protein, much lower than the mean activity values found in kidney, which were 62.30 ± 26.97 nmol/min/mg protein. Regarding the variations with respect to age, statistically significant differences were only found in the kidney in GST activity, the activity of this enzyme being higher in adult gannets than in immature ones. Secondly, the gender factor did not influence the levels of any of the biomarkers considered. The obtained results indicate that both biomarkers could be useful in biomonitoring programs of environmental contamination focused on the gannet, although studies that consider a larger number of animals, incorporating a broader range of biomarkers, would be necessary. (AU)

Concentrations of chlorinated pollutants in adipose tissue of yellow-legged gulls (Larus michahellis) from Spain: Role of gender and age.

Concentrations of 7 different polychlorinated biphenyl (PCB) congeners, and eleven organochlorine pesticides (OCPs) and metabolites, including DDTs (dichlorodiphenyltrichloroethane), HCHs (hexachlorocyclohexane isomers), Endosulfan, Endosulfan sulfate, Endrin, Dieldrin and HCB (hexachlorobenzene), were determined in adipose tissue of 57 yellow-legged gulls collected from NW and N Spain. Furthermore, the possible differences due to two endogenous factors, age and gender, were determined. All the analyzed PCBs were detected in over 66% of the samples, with levels of 291.9 (PCB 180), 34.5 (PCB 118), 0.7 (PCB 28), 432.6 (PCB 153), 225.5 (PCB 138), 1.3 (PCB 101) and 0.4 (PCB 52) µg/kg of adipose tissue. With respect to the OCPs and metabolites, only 4,4'-DDE and HCB were detected in more than 50% of the samples, with means of 360.6 and 2.5 µg/kg of adipose tissue, respectively. From all the considered contaminants, only 4,4'-DDE levels presented significant differences depending on the gender, with females showing higher values than males (p < 0.01). Significant differences (p < 0.001) were also found related to age for the levels of PCBs 180, 138, 101, 28 and 153, as well as 4,4'-DDE, with adult levels being higher than those in young birds. The results of the present study constitute a baseline to better assess the environmental impacts of PCB and OCP contamination at other coastal sites for future biomonitoring studies, with particular emphasis on gender- and age-related differences.

The American mink (Neovison vison) is a competent host for native European parasites.

The American mink (Neovison vison) is a mustelid native to North America that was introduced in Europe and the former USSR for fur farming. Throughout the last century, accidental or deliberate escapes of mink from farms caused the establishment of stable feral populations. In fact, the American mink is considered an invasive alien species in 28 European countries. The present study evaluates the gastrointestinal and cardiopulmonary helminth fauna of the American mink in Galicia (NW Spain) to understand its role as a potential reservoir for parasites affecting other autochthonous mustelids. In the period 2008-2014, fifty American mink (35 males and 15 females) of different ages (22 immature and 28 adults) from the provinces of Lugo, Ourense and Pontevedra were captured and sacrificed. Eight parasite species were found (6 nematodes and 2 trematodes) with the following prevalences: Molineus patens (68%), Aonchotheca putorii (54%), Crenosoma melesi (10%), Aonchotheca annulosa (8%), Angiostrongylus daskalovi (6%), Aelurostrongylus spp. (2%), Troglotrema acutum (2%) and an unidentified trematode (2%). Eighty-two per cent of the mink harboured helminths, including 15 animals (30%) infected by only one parasite species, 19 (38%) by two species, 5 (10%) by three species and 2 mink (4%) by four species. All helminth species identified are native to European mustelids. Statistical models were used to evaluate if animal characteristics (age, sex and weight), date and capture area influenced the prevalence, intensity or parasite richness. Statistical differences were detected only in models for intensity of M. patens, A. putorii and C. melesi. This is the first report of Angiostrongylus daskalovi, a cardiopulmonary nematode, and A. annulosa, a gastrointestinal nematode specific of rodents, in American mink. Moreover, although the fluke T. acutum has already been cited in American mink, to our knowledge, the present study represents the first report of this trematode in the lung.

Does gender influence the levels of heavy metals in liver of wild boar?

The aim of this study was to determine heavy metal reference levels for risk assessment studies. For this purpose, the levels of lead, cadmium, copper and zinc were determined in liver tissues of wild boars sampled in NW Spain. The mean values were 0.383, 0.326, 23.50 and 56.86mg/kg dried weight, respectively. In general, the levels detected were similar to or lower than the levels reported in literature. This study not only provides a useful baseline for biomonitoring the levels of the analyzed contaminants in wildlife in NW Spain, it also helps to understand the effects of gender on the levels of these elements. Similar to studies performed in other geographical regions, no significant gender-related differences could be detected. Although differences were not significant, the levels of zinc, cadmium and lead were modestly higher in males (55.78, 0.346 and 0.424mg/kg, respectively) compared to females (45.25, 0.305 and 0.341mg/kg). Our results indicate that, although gender did not significantly affect heavy metal uptake and toxicokinetics of contaminants in wild boars, these effects could vary between species, populations, organs, and elements. It is therefore essential to investigate gender-related differences for each species.

Use of computed tomography as a non-invasive method for diagnosing cephenemyiosis in roe deer (Capreolus capreolus).

This study was conducted to assess the reliability of computed tomography (CT) for diagnosing bot fly infestations by Cephenemyia stimulator (Clark) (Diptera: Oestridae) in roe deer (Capreolus capreolus L.) (Artiodactyla: Cervidae). For this purpose, the heads of 30 animals were analysed, firstly by CT and then by necropsy, which was used as the reference standard method. The prevalence values obtained by both methods were identical; the prevalence of infestation was 40.0% overall, and was higher in males (45.5%) than in females (25.0%). These results highlight the usefulness of CT as an alternative or non-invasive method for diagnosing cephenemyiosis in live-captured roe deer and in hunting trophies or museum collections that cannot be destroyed or damaged.

Quantification of pericaudal adipocyte diameter in dairy cattle during peripartum: a complementary method to study energetic status using conventional histology.

The energetic status of high-yielding Holstein-Friesian dairy cattle was studied during peripartum under field conditions using body condition score (BCS), glycemia, seric ß-hydroxybutyrate and adipose tissue cellularity. This last method was tested as a complementary tool for energetic status assessment. Biopsies of pericaudal subcutaneous adipose tissue were obtained from 25 multiparous animals at 28 days before and 21 days after parturition. Samples were routinely processed for histological examination and stained with haematoxylin and eosin (H&E). The mean diameter of adipocytes (MDA) was measured with the aid of a digital image processor. During the same period, blood samples were collected weekly for metabolite determinations. The MDA at 28 days pre-partum and 21 days post-partum were 72.1 vs. 66.2 µm respectively (p = 0.055), and the corresponding BCS at these moments was 3.32 vs. 3.19 (p = 0.068). At -28 days pre-partum, the BCS was positively correlated with MDA (Pearson's r = 0.521, p = 0.016) and with glycemia (Pearson's r = 0.404, p = 0.056). Correlations between BCS and MDA, and between BCS and glycemia, with ß-hydroxybutyrate although not significant, suggest that routine histological preparations of biopsies from subcutaneous adipose tissue could be included as an easy and valuable tool for research purposes to evaluate metabolic adaptation of dairy cows to peripartum, as well as the incidence of metabolic disorders and productive performance.

Calbindin D28k expression and the absence of apoptosis in the cerebellum of Solanum bonariense L-intoxicated bovines.

Solanum bonariense intoxication is characterized by cerebellar neuronal vacuolation, degeneration, and necrosis. Cerebellar Purkinje cells seem especially susceptible, but more research is needed to determine the pathogenesis of neuronal necrosis and the mechanism of Purkinje cell susceptibility. Calbindin D28k (CbD28k) is highly expressed in Purkinje cells and has been used as a marker for normal and degenerative Purkinje cells. The goal of this study was to describe S bonariense-induced disease by ascertaining Purkinje cell-specific degenerative changes using CbD28k expression and to correlate this with apoptosis in Purkinje cells, as determined using TUNEL (transferase-mediated dUTP-biotin nick end-labeling) and ultrastructural changes. In all cases, an increase in both dose and duration of S bonariense intoxication resulted in a decrease in the number of Purkinje cells. CbD28k immunohistochemistry was an excellent marker for Purkinje cells because immunoreactivity did not change in normal or degenerative tissues. This finding suggests that excessive calcium excitatory stimulation does not induce rapid neuronal degeneration and death. As found in previous studies, TUNEL tests and electron microscopy suggest that Purkinje cell degeneration and death are not occurring via an apoptotic process. These findings suggest that S bonariense poisoning induces progressive Purkinje cell death that is not mediated by excitotoxicity or apoptotic activation.

Meningoencephalitis associated with Staphylococcus warneri in a dog.

A case of meningoencephalitis in a dog caused by Staphylococcus warneri is reported here. The history and clinical signs were suggestive of possible central nervous system infection. Analysis of cerebrospinal fluid documented a neutrophilic pleocytosis (890 cells/mul) and the presence of occasional intracellular cocci. Staphylococcus warneri was isolated from the microbiological culture of the cerebrospinal fluid. Treatment consisted of intravenous antibiotics, supportive care and anticonvulsants for the generalised seizures that developed after admission. Histological assessment confirmed the location and extension of bacterial meningoencephalitis. Thrombotic meningoencephalitis associated with Staphylococcus warneri infection has not, to the authors' knowledge, been previously reported in dogs.

Effects of dietary cation-anion difference on blood cortisol and ACTH levels in reproducing ewes.

Formulation of rations to induce a compensated metabolic acidosis in the post-partum cow has proved a useful strategy for prevention of milk fever. Such acidification improves the ability of the animal to maintain calcium homeostasis by promoting the absorption of calcium from the intestine and mobilization of calcium from the bone. In humans, an acidogenic diet results in mild metabolic acidosis in association with a state of cortisol excess and this increase in plasma cortisol may increase bone catabolism. The objective of our experiment was to induce acidification by anionic salt supplementation and to study the effects of anionic supplementation on plasma cortisol and adrenocorticotropic hormone (ACTH) levels in sheep. Twenty-seven twin-bearing sheep were assigned to two experimental groups and a control group, depending on dietary cation-anion difference (DCAD) (+272.6, -88.9 and +164.5 mEq/kg DM respectively). Sheep assigned to each dietary treatment received their respective rations beginning 6 weeks pre-partum and continuing until 12 days post-partum. Anionic diet induced a non-respiratory systemic acidosis in association with a mild increase in plasma cortisol concentration without changes in plasma ACTH levels. Our data suggest that the mild hypercortisolism observed in sheep fed the anionic diet may not be an effector for bone resorption induced by anionic salts. A mild hypercortisolism of this magnitude may lead to osteoporosis but this might require many years of adrenal hypersecretion while anionic salts are only used during the last weeks of pregnancy.

Suspected lasalocid poisoning in three dogs.

The ionophore lasalocid has been used as a feed additive for broilers chickens and for improving feed efficiency in ruminants. Although dogs appear to be more sensitive to lasalocid intoxication than other species, there is only 1 report in the veterinary literature about lasalocid poisoning in dogs. We describe the clinical signs, treatment and resolution of 3 hunting dogs that developed acute neurological signs consistent with lasalocid poisoning after the consumption of several broilers that had died on a nearby farm.

Long-term effects of dietary anion-cation balance on acid-base status and bone morphology in reproducing ewes.

The beneficial effects of anionic salts on calcium metabolism have been shown by supplementing rations with such salts during the last 3 weeks of pre-partum. However, there are few reports on the effects of anionic salts supplementation for periods of 4 weeks or longer on acid-base status, mineral metabolism and bone morphology. This study was conducted to evaluate the effects of the long-term dietary supplementation of anionic salts on the acid-base status, plasma minerals concentrations and bone morphology in sheep. Twenty-seven twin-bearing sheep were assigned to two experimental groups and a control group, depending on dietary cation-anion difference (DCAD) (+272.6, -88.9 and + 164.5 mEq/kg DM, respectively). Sheep assigned to each dietary treatment received their respective rations beginning 6 weeks prepartum and continuing until 12 days post-partum. Diets containing anionic salts induced a mild metabolic hyperchloraemic acidosis from 1 week pre-partum to 2 days post-partum that was completely compensated by non-respiratory mechanisms. These changes on acid-base status were accompanied by an increase of plasma ionized calcium levels. Plasma total calcium, phosphorus and magnesium concentrations were not affected by dietary treatment. Parathyroid hormone concentrations were related to the concentration of ionized calcium of plasma and were higher in sheep fed the cationic diet. Plasma osteocalcin levels were increased in sheep fed the anionic diet and cortical bone remodelling occurred in all the animals during late pregnancy in light and electron microscopy observation, but was particularly evident in the sheep fed the anionic diet. Bone turnover might be stimulated because of the role of the bone in buffering systemic acidosis. The data suggest that anionic salts ameliorated calcium metabolism around parturition by increasing bone resorption and the concentration of ionised calcium in plasma, possibly mediated by a mild hyperchloraemic metabolic acidosis induced by the salts.

Zinc poisoning associated with separation anxiety in an Argentinean bulldog.

Separation anxiety in dogs is a complex behavioral syndrome produced by many causes. A hunter 4y-old male Argentinean Bulldog had dental disturbances and gastrointestinal and cutaneous symptoms due to high plasma zinc and low copper and calcium plasma concentration. His behavior made him bite galvanized wire fences in his cage and bunk that contained his food, both of a Zn-containing material. Specific treatment resulted in complete recovery.