Evaluation of point-of-care analysers for blood gas and clinical chemistry in Hermann's tortoises (Testudo hermanni).

OBJECTIVE: To assess the agreement between point-of-care and laboratory analysers in measuring biochemical and blood gas analytes in venous samples from tortoises and to define preliminary reference intervals for venous blood gas analysis in Hermann's tortoises (Testudo hermanni). MATERIALS AND METHODS: Jugular venous blood samples from 47 Hermann's tortoises underwent paired analysis with a portable gas analyser (i-STAT 1, Abaxis), a portable chemical analyser (VetScan VS2, Abaxis), and with the respective reference analysers. Agarose gel electrophoresis was used to determine albumin concentrations on 12 specimens. Agreement was evaluated with Bland-Altman plots and regression analysis using the Passing-Bablok method. RESULTS: Point-of-care analysers had variable agreement with the reference analysers, presenting constant or proportional bias depending on the analyte. Relevant analytes in reptiles, such as ionised and total calcium, had acceptable agreement. The method for determining albumin concentration currently available in both point-of-care and laboratory analysers significantly overestimated albumin concentrations as compared to protein electrophoresis. CLINICAL SIGNIFICANCE: While the use of POC analysers is extremely advantageous in small animal primary care facilities, agreement between point-of-care and laboratory analysers varies depending on the analyte. For certain analytes, interchangeability of results is limited and specific reference intervals for point-of-care analysers are required. Veterinarians should be aware of the size and the direction of the bias of each analyte.

Total plasma magnesium in healthy and critically ill foals.

Abnormalities in total Mg (tMg) concentration in plasma and/or serum are common in critically ill humans, and the association with increased mortality has been documented in several clinical studies in adults and newborns with hypoxic-ischemic encephalopathy. Abnormalities in tMg were studied in hospitalized dogs, cats, and adult horses. Newborn foals were scarcely studied with regard to Mg concentration. The aims of the present study were: (1) to compare two analytical methods for the determination of tMg in plasma: the automated colorimetric method and the atomic absorption spectrometry; (2) to measure plasma tMg in healthy foals during the first 72 hours after birth and in sick foals during the first 72 hours of hospitalization; (3) to compare total plasma Mg concentration among healthy foals, foals affected by perinatal asphyxia syndrome (PAS), prematurity and/or dismaturity, and sepsis; (4) to evaluate tMg plasma concentration in surviving and non-surviving foals. One hundred seventeen foals were included in the study: 20 healthy and 97 sick foals. The automated method used in clinical practice probably overestimates plasma tMg. Due to its higher sensitivity and specificity, the atomic absorption spectrometry should be considered the method of choice from an analytical point of view, but requires an instrumentation not easily available in any laboratory and specific technical skills and competencies. Plasma tMg in healthy foals were included in the range 0.52 to 1.01 mmol/L and did not show any time-dependent change during the first 72 hours of life. In sick foals, tMg evaluated at T0 was statistically higher than tMg measured at subsequent times. Foals affected by PAS had a tMg at T0 significantly higher (P < 0.01) than healthy, septic, and premature and/or dysmature foals. The t test found significantly higher (P < 0.01) plasma tMg measured at T0 in non-surviving than in surviving foals. Plasma tMg could be a useful parameter for the diagnosis of PAS and the formulation of the prognosis in critically ill foals.

The effect of chronic kidney disease on the urine proteome in the domestic cat (Felis catus).

Chronic kidney disease (CKD) is a major cause of mortality in cats, but sensitive and specific biomarkers for early prediction and monitoring of CKD are currently lacking. The present study aimed to apply proteomic techniques to map the urine proteome of the healthy cat and compare it with the proteome of cats with CKD. Urine samples were collected by cystocentesis from 23 healthy young cats and 17 cats with CKD. One-dimensional sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) was conducted on 4-12% gels. Two-dimensional electrophoresis (2DE) was applied to pooled urine samples from healthy cats (n = 4) and cats with CKD (n = 4), respectively. Sixteen protein bands and 36 spots were cut, trypsin-digested and identified by mass spectrometry. 1D-SDS-PAGE yielded an overall view of the protein profile and the separation of 32 ± 6 protein bands in the urine of healthy cats, while CKD cats showed significantly fewer bands (P < 0.01). 2-DE was essential in fractionation of the complex urine proteome, producing a reference map that included 20 proteins. Cauxin was the most abundant protein in urine of healthy cats. Several protease inhibitors and transport proteins that derive from plasma were also identified, including alpha-2-macroglobulin, albumin, transferrin, haemopexin and haptoglobin. There was differential expression of 27 spots between healthy and CKD samples (P < 0.05) and 13 proteins were unambiguously identified. In particular, increased expression of retinol-binding protein, cystatin M and apolipoprotein-H associated with decreased expression of uromodulin and cauxin confirmed tubular damage in CKD cats suggesting that these proteins are candidate biomarkers.

Alkaline phosphatase in boar sperm function.

Alkaline phosphatase (AP) catalyses the detachment of phosphate residues from different substrates. Its activity has been demonstrated in seminal plasma and spermatozoa from porcine and other mammalian species; anyway, the role of AP in male reproduction has not been clarified yet and the aim of this study was to determine AP function in boar sperm capacitation and in vitro fertilization (IVF). AP activity was assayed in seminal plasma and in uncapacitated and in vitro capacitated (IVC) spermatozoa; in addition, capacitation was studied in presence of different doses of AP (1.2 and 2.5 IU/mL). The effect of different doses of AP (1.2 and 2.5 IU/mL) on several sperm parameters after IVC (viability, acrosome integrity with FITC-PSA, capacitation status with CTC staining, tyrosine phosphorylation) and on fertilizing ability during IVF were also evaluated. High AP activity was detected in seminal plasma, in particular in sperm-rich fraction; a lower activity was detected in uncapacitated spermatozoa while a significant decrease was evidenced after IVC. Viability was not changed by AP supplementation of the capacitating medium, whereas acrosome integrity and capacitation status were significantly affected by 1.2 and 2.5 doses, with a dose-dependent decrease in acrosome-reacted cells as well as in CTC B pattern displaying cells. As for sperm head protein phosphorylation, a decrease in relative fluorescence was detected in AP 2.5 group, if compared with capacitated one. After IVF, a dose-dependent decrease in penetrated oocytes was recorded, with an increase in monospermic zygote rate. In conclusion, we demonstrated that AP activity decreases under capacitating condition and that addition of AP to spermatozoa during capacitation results in a depression of the capacitating process and IVF. We can infer that AP plays a role in keeping spermatozoa quiescent until they are ejaculated and in modulating the acquisition of the fertilizing ability.

Cadmium accumulation and antioxidant responses in Sparus aurata exposed to waterborne cadmium.

Cadmium (Cd), a nonessential trace element, is rapidly accumulated by most living organisms and subsequently exerts its toxicity at different molecular levels. This study exposed gilthead sea bream (Sparus aurata) to waterborne 0.1 mg/l Cd for 11 days and investigated the Cd accumulation pattern, lipid oxidation, and response of antioxidant defences. At the end of the experiment, mean Cd concentrations in gills and liver, the organs most prone to metal accumulation, were 209.4 and 371.7 ng/g ww, respectively. Muscle did not show any Cd retention during the 11 days of exposure. In liver, the cytosolic fraction of the metal was chelated into the nontoxic form by metallothionein (MT), a specific Cd-inducible protein. Zn and Cu concentrations were not influenced by Cd exposure. Glutathione (GSH) concentrations and the antioxidant enzyme activities of GSH reductase and GSH peroxidase showed an overall decreasing trend. In addition, lipid and aqueous hydroperoxide levels did not show any significant variation. Oxidative stress indirectly generated by Cd seems to be compensated for by the different biochemical systems tailored to decrease cellular damage. In particular, the negative effects of Cd accumulation in tissues were probably counteracted by the induction of MT.

Detection and localization of GLUTs 1, 2, 3 and 5 in donkey spermatozoa.

GLUTs are a family of proteins that facilitate the transport of glucose and other hexoses through the plasma membrane of the cells. GLUTs are present in mammalian spermatozoon's membrane in different isoforms and they supply metabolic substrates for all the cell's activities such as motility, homoeostasis and fertilization. As studies about donkey spermatozoa and their metabolism are lacking, this study was aimed at detecting GLUTs 1, 2, 3 and 5 presence by western blotting technique and at determining their localization on the plasma membrane by indirect immunofluorescence. Each protein showed a typical localization on the sperm cells' plasma membrane, differencing the one to the other on the basis of the hexose they transport. We also highlighted some differences between GLUTs distribution and molecular weight in donkey spermatozoa and its nearest relative, the horse.

Comparative immunolocalization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa.

Spermatozoa, as other eukaryotic cells, need hexoses to produce energy to maintain membrane homeostasis, to move along the female genital tract and to carry the male genome to the female gamete. GLUTs are a family of proteins that permit and improve the passive transport of hexoses inside cells. This study was aimed at investigating the presence and localization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa by both immunofluorescence and western blotting. GLUTs exhibited a peculiar distribution along the sperm cell depending on the isoforms considered, the hexose they transport and the different species. The localization of GLUTs after capacitation and acrosome reaction highlighted the possible changes in their distribution because of the different functional moment. Only in dog spermatozoa changes in GLUTs distribution were demonstrated; these changes could be related to the different metabolic needs and modifications occurring in the sperm cell.

Cadmium accumulation and biochemical responses in Sparus aurata following sub-lethal Cd exposure.

Cadmium (Cd), a heavy metal with limited biological function, is widely distributed in the aquatic environment as a result of natural and anthropogenic activities. The effect of 4 and 11 days exposure of gilthead sea bream Sparus aurata to sub-lethal concentrations of Cd was evaluated as levels of Cd content and Cd-metallothionein (MT) presence in different organs. The possible genotoxic effect was also evaluated in erythrocytes by using the "comet assay", a promising tool for estimating DNA damage at the single-cell level. The results obtained show that in the controls, Cd content was significantly higher in gills compared to in liver, but the treatment of fish with 0.1mg/l Cd induced a stronger accumulation of metal in liver depending on the length of the exposure period. Cd traces were found in plasma, muscle and kidney. Cd forms complexes in the cytosol with MT only in the liver but Cd-MT content significantly increased after 11 days of exposure to the metal, while after 4 days of treatment the protein level was similar to the control. The "comet assay" performed on S. aurata eryhtrocytes isolated from fish treated for 4 and 11 days with 0.1mg/l Cd, showed that there was no DNA damage at both exposure periods.

Trace metals in arcid clam Scapharca inaequivalvis: effects of molluscan extracts on bioluminescent bacteria.

The relationship between a supposed effect of molluscan extracts on bioluminescent bacteria and metal concentrations in the extracts was investigated. For this purpose a biotoxicological assay based on bioluminescent bacteria (BLB) and extracts from metal exposed molluscs, Scapharca inaequivalvis, was optimized to monitor Cd and Cu marine pollution. Cu and Cd concentrations increased in tissues of experimentally exposed molluscs. Molluscan extracts inhibited the bacterial luminescence, the inhibition decreasing as the time of mollusc exposure to metals increased, suggesting a reduction of the "bioactive" metals. In regard to the use of BLB test in environmental monitoring, the analysis of Cu, Cd, and metallothionein (MT) was first performed in tissues from molluscs collected in three different areas of Northern Adriatic Sea. Metal concentrations reached maximum values in the gills, while Cd was mostly bound to MT in the kidney. Significant differences in metals and MT concentrations were found depending on the sampling sites. The biotoxicological assay resulted slightly correlated with the biochemical parameters.

Leptin does not seem to influence glucose uptake by bovine mammary explants.

Leptin, a protein produced and secreted by adipocytes, is know to regulate food intake and whole-body energy metabolism, but knowledge about its possible effect in bovine mammary gland is scarce. Leptin may be involved in the regulation of glucose transport even though this effect at the tissue level remains controversial. Once uptaken by the mammary gland, glucose is utilised in several ways but the majority, about 60-70%, is drained for lactose synthesis. This study was aimed at investigating the effect of leptin on glucose regulation in bovine mammary gland. We have examined the effects of leptin on the expression of GLUT1 mRNA, pyruvate kinase (PK) as well as glucose-6-phosphate dehydrogenase (G6PDH) activity. Treatment of mammary gland explants with recombinant leptin did not influence glucose assimilation, pathway transport (GLUT1 mRNA) and glucose metabolism (PK and G6PDH) in this tissue. The results from this study seem to exclude an involvement of leptin in glucose uptake and metabolism in bovine mammary gland.

Zinc-bound metallothioneins as potential biological markers of ageing.

Metallothioneins (MTs) (I+II) play pivotal roles in metal-related cell homeostasis because of their high affinity for metals forming clusters. The main functional role of MTs is to sequester and/or dispense zinc participating in zinc homeostasis. Consistent with this role, MT gene expression is transcriptionally induced by a variety of stressing agents to protect cells from reactive oxygen species. In order to accomplish this task, MTs induce the secretion of pro-inflammatory cytokines by immune and brain cells, such as astrocytes, for a prompt response against oxidative stress. These cytokines are in turn involved in new synthesis of MTs in the liver and brain. Such protective mechanism occurs in the young-adult age, when stresses are transient. Stress-like condition is instead constant in the old age, and this causes continuous stealing of intracellular zinc by MTs and consequent low bioavailability of zinc ions for immune, endocrine, and cerebral functions. Therefore, a protective role of zinc-bound MTs (I+II) during ageing can be questioned. Because free zinc ions are required for optimal efficiency of the immune-endocrine-nervous network, zinc-bound MTs (I+II) may play a different role during ageing, switching from a protective to a deleterious one in immune, endocrine, and cerebral activities. Physiological zinc supply, performed cautiously, can correct deficiencies in the immune-neuroendocrine network and can improve cognitive performances during ageing and accelerated ageing. Altogether these data indicate that zinc-bound MTs (I+II) can be considered as novel potential markers of ageing.

Metallothioneins (MTs) in marine molluscs.

The presence of MTs in marine molluscs was firstly hypothesized in oyster and in mussel during the seventies, however mussel's and oysters' MTs were completely purified and sequenced rather later. Already from the first studies it was evident that the purification of molluscan MTs was more difficult than in mammals. Mussel's MTs are characterized by the presence of a monomeric and a dimeric form. Several physiological and biochemical parameters can influence the concentration and the isolation of MT from molluscan tissues. Remarkable variations in MT isolation and quantification might depend on the purification and storage protocol. Because of possible artefacts due to the isolation procedure the establishment of a standard protocol for MT quantification in marine mollusc is still an important goal. In a few species the presence of very low molecular weight metal binding ligands has also been reported, in these cases it cannot be excluded that the native MT has been cleaved by the action of proteases. This review aims to report: 1) importance of a standard method for MT purification and quantification in molluscs; 2) distribution of MT among molluscan species; 3) data concerning oyster's and mussel's MTs which are the two more deeply investigated marine molluscs; 4) biotic and abiotic factors influencing MT concentration, and 5) biological role of MT and use of MT as a biochemical marker of heavy metal pollution.

Seasonal dependence of cadmium accumulation and Cd-binding proteins in Mytilus galloprovincialis exposed to cadmium.

At different periods of the year specimens of Mytilus galloprovincialis were exposed to 0.5 microg Cd/ml seawater for 7 days. Concentrations of trace elements (Cd, Zn, Cu and Fe) and Cd-binding proteins in gills, viscera, muscle and mantle were determined after 1 weeks exposure. Cadmium accumulation was higher in May and June and was tissue dependent; it was highest in the gills and decreased in the order: gills > viscera > mantle and adductor muscle. Significant seasonal variations of zinc, copper and iron, were also found, in both exposed and control molluscs. The percentage of Cd distribution between cytosol and pellet changed during the year; a clear shift from the particulate fractions to the cytosolic fractions was measured during May and June, especially in the gills. Metallothionein (MT) was the main ligand responsible for Cd accumulation, and this protein reached a maximum between May and June. Inclusion of mercaptoethanol during the purification procedure was found to improve MT isolation by gel chromatography. In the absence of mercaptoethanol, MT showed polymerization patterns which were season dependent and temperature independent, whereas its concentration was increased in mussels kept at higher temperature.

Seasonal changes of zinc, copper, and iron in gilthead sea bream (Sparus aurata) fed fortified diets.

Four groups of gilthead sea bream (Sparus aurata) were fed diets with additional metal contents: a basal diet (diet A) contained Zn at 60.9 +/- 1.9 mg/kg diet, Cu at 3.9 +/- 0.9 mg/kg diet, and Fe at 138.3 +/- 6.8 mg/kg diet; the other diets were supplemented with copper (20 mg/kg, diet B), iron (100 mg/kg, diet C), or zinc (300 mg/kg, diet D). Two consecutive year-classes (0+ and 1+ age fish) from the same parent stock were examined. Several fish tissues were analyzed for metal contents in five different periods of each year in order to determine (1) the sensitivity of certain tissues as indicators of trace element metabolism and (2) possible seasonal variations. Growth data were similar for gilthead sea bream fed the basal diet and the metal-fortified diets. Mineral concentrations in tissues were found to be little affected by the dietary supplementation of trace elements, suggesting an efficient homeostatic control of these three metal concentrations. Tissues involved in metal metabolism (e.g., liver, kidney, gills) presented greater variations between minimum and maximum values with respect to other tissues (e.g., brain, muscle, eye). Seasonal variations were observed during the 2 yr of this study and were especially evident for zinc and copper concentrations in the liver. The overall pattern of metal variations showed a decreasing trend during the 2 yr. Results from this study indicate that (1) trace element concentrations in fish tissues vary with age and life cycle and (2) trace element requirements may vary in function of age and life cycle.

Effects of different levels of dietary zinc on the gilthead, Sparus aurata during the growing season.

Gilthead were fed three diets. Diet A was the control diet, whereas diets B and C were supplemented with 300 and 900 mg Zn/kg, respectively. Fish fed with diet C, at the end of the experiment, showed the lowest weight. Zinc concentrations presented the higher values in gills, liver, and kidney. Muscle and brain had the lower mean values and showed a tight control of zinc levels. These results reinforce the hypothesis that zinc in the CNS should be strictly controlled in order to maintain the functional role of the metal. Significant differences in tissue zinc concentrations were obtained between fish fed different amounts of zinc, the metal concentrations being higher in tissues of fish fed diet C. The tissue decrease of zinc, found at the end of the experiment, may depend on a lower feed consumption or on different zinc requirements during the cold season. These changes, even if not univocal among the three diets, may be associated with the life cycle of fish. Furthermore, copper concentrations were little affected by the different concentrations of zinc in the three diets; liver and kidney presented the highest concentrations; liver showed a significant decrease in copper content at the end of the experiment. We conclude that: zinc concentrations of the diet may affect the gilthead weights and the tissual metal content; and zinc concentrations in the diets, depending on the growth rate, may be varied depending on the season.

Heavy metals in some species of waterfowl of northern Italy.

Concentrations of heavy metals (zinc, copper, cadmium, and iron) were measured in several tissues (brain, gizzard, leg-muscle, heart, breast-muscle, intestine, liver and kidney) of moorhens (Gallinula chloropus), black-headed gulls (Larus ridibundus), and coots (Fulica atra) collected between autumn 1985 and spring 1989 in northern Italy. Cadmium concentrations in the liver and kidney of water-rails (Rallus aquaticus) and in five species of Anatidae collected also were measured. High mean (+/- SD) copper levels were detected in aerobic muscles such as heart (38 +/- 5 micrograms/g dry weight (DW)) and pectoral muscles (35 +/- 7 micrograms/g DW). Compared to other tissues, the iron content of brain was rather low and constant, with a mean value of 160 +/- 17 micrograms/g DW in moorhens, 157 +/- 60 micrograms/g DW in black-headed gulls, and 157 +/- 25 micrograms/g DW in coots. Iron concentrations in tissues of moorhens from the Reno River were significantly higher than those from the Sile River. Cadmium was detectable only in the liver and kidney; there was a linear relationship between cadmium levels in these two organs. The highest mean (+/- SD) cadmium concentrations were present in the kidney of black-headed gull (30 +/- 20 micrograms/g DW).

Cd-metallothionein in liver and kidney of goldfish (Carassius auratus): effects of temperature and salinity.

Treatment of goldfish with Cd, by intraperitoneal injection, resulted in Cd-metallothionein (Cd-MT) synthesis mainly in liver and kidney. The relative amount of Cd sequestered by liver metallothionein was always greater in fish maintained at 20 degrees C compared to those reared at 10 degrees C, indicating a temperature dependence of metallothionein biosynthesis; in the kidney this dependence was not so clearly evident. Changes in MT levels induced by adapting fish to different salinities did not correlate with the salinity change.

Cd-metallothionein and metal-enzymes interactions in the goldfish Carassius auratus.

Goldfish injected with cadmium chloride synthesized metallothionein. Ten days after the first injection, cadmium reached a maximum in the metallothionein peak (2 micrograms/ml) obtained after gel filtration of liver cytosol. Pyruvate kinase activity was inhibited from the beginning of the experiment; after the fourth day, the enzyme activity again started to increase but did not reach the control level. Alkaline phosphatase and fructose biphosphatase did not show any apparent inhibition. From the results here reported, a detoxifying role of metallothionein could be suggested.