Quantifying free behaviour in an open field using k-motif approach.

Quantification and parametrisation of movement are widely used in animal behavioural paradigms. In particular, free movement in controlled conditions (e.g., open field paradigm) is used as a "proxy for indices of baseline and drug-induced behavioural changes. However, the analysis of this is often time- and labour-intensive and existing algorithms do not always classify the behaviour correctly. Here, we propose a new approach to quantify behaviour in an unconstrained environment: searching for frequent patterns (k-motifs) in the time series representing the position of the subject over time. Validation of this method was performed using subchronic quinpirole-induced changes in open field experiment behaviours in rodents. Analysis of this data was performed using k-motifs as features to better classify subjects into experimental groups on the basis of behaviour in the open field. Our classifier using k-motifs gives as high as 94% accuracy in classifying repetitive behaviour versus controls which is a substantial improvement compared to currently available methods including using standard feature definitions (depending on the choice of feature set and classification strategy, accuracy up to 88%). Furthermore, visualisation of the movement/time patterns is highly predictive of these behaviours. By using machine learning, this can be applied to behavioural analysis across experimental paradigms.

Lead poisoning and its in vivo biomarkers in Mallard and Coot from two hunting activity areas in Poland.

In this study, we aimed to present the status of lead (Pb) poisoning in birds from southern Poland and the evaluation of in vivo biomarkers which may be used in the diagnosis without killing animals. This included the activity of δ-aminolevulinic acid dehydratase (ALA-d), hemoglobin (Hb) concentration, hematocrit (Ht) level and concentrations of Pb in blood, feathers and excrements. The significance of this work is the use of hunted birds which allow us to compare the signals of the chosen biomarkers with the internal response. Birds collected in the area of lower hunting activity (the Milicz ponds) revealed statistically lower Pb concentrations than birds from the Zator area. Pb poisoning was diagnosed in almost 8% of birds (including specimens from both areas), but lead pellets were found in 3%. The highest tissue concentration found was noted in kidneys of Mallard from the Zator area (36.55 µg g(-1) d.w.). Significantly higher concentrations were noted in a few samples of gizzard content (up to 1047 µg g(-1) d.w.) and excrements (up to 82.95 µg g(-1) d.w.). Hb concentration, Ht level, concentrations in feathers and excrements seem not to be efficient biomarkers at noted Pb concentrations in internal tissues (brain, pectoral muscle, kidney, liver, spleen, bone). In contrast, we found a significant negative correlation between Pb concentration in blood and ALA-d activity which confirmed that this parameter can be used successfully as in vivo biomarker of lead poisoning also in low environmental pollution.

Cadmium concentrations and their implications in Mallard and Coot from fish pond areas.

During the studies in 2003, high concentrations of cadmium (Cd) were noted among Mallards and Coots in Poland. Further research, five years later, showed the histopathological alterations in birds from the same area which were probably stimulated by Cd. This paper presents the analysis of Cd in ten different materials collected from these species (n=180) in years 2006-2009 in two areas, including southern - Zator and western Poland - Milicz. Similarly high concentrations to those from 2003 were noted only in kidneys of Mallards from the Zator area (up to 56.49 µg g(-1) d.w.). Other comparable materials accumulated significantly lower concentration of Cd. Generally, lower concentrations occurred in birds from Milicz. Concentrations varied also between species and age groups. They correlated between liver and kidneys, brain and muscle and brain and spleen. Potential in vivo bioindicators (blood, feathers and excrements) did not seem to be useful as biomarkers in both areas.

Histopathology of liver and kidneys of wild living Mallards Anas platyrhynchos and Coots Fulica atra with considerable concentrations of lead and cadmium.

Concentrations of cadmium and lead were measured in liver and kidneys of Mallard (n=60) and Coot (n=50). Free living birds were collected by hunters in years 2006-2008 in the area of fishponds near Zator in southern Poland. Age group was determined according to the appearance of the plumage (Mallards) and iris color (Coot). Concentrations of metals were measured with ET-AA spectrometer. Among all birds specimens with negligible (n=5) and high concentrations (Mallards n=18 and Coots n=17) of cadmium and lead were chosen for further analysis. Histopathological alterations were observed, ranging from circulatory disturbances, retrogressive changes, inflammations to leukocytic infiltration in liver and kidney. They dominated among birds with the highest concentrations of metals. The control group of birds was characterized by a very small number of mentioned lesions. Probably the higher cadmium and lead concentrations in tissues are co-factors in the development of lesions.

Steroid concentrations in plasma, whole blood and brain: effects of saline perfusion to remove blood contamination from brain.

The brain and other organs locally synthesize steroids. Local synthesis is suggested when steroid levels are higher in tissue than in the circulation. However, measurement of both circulating and tissue steroid levels are subject to methodological considerations. For example, plasma samples are commonly used to estimate circulating steroid levels in whole blood, but steroid levels in plasma and whole blood could differ. In addition, tissue steroid measurements might be affected by blood contamination, which can be addressed experimentally by using saline perfusion to remove blood. In Study 1, we measured corticosterone and testosterone (T) levels in zebra finch (Taeniopygia guttata) plasma, whole blood, and red blood cells (RBC). We also compared corticosterone in plasma, whole blood, and RBC at baseline and after 60 min restraint stress. In Study 2, we quantified corticosterone, dehydroepiandrosterone (DHEA), T, and 17ß-estradiol (E2) levels in the brains of sham-perfused or saline-perfused subjects. In Study 1, corticosterone and T concentrations were highest in plasma, significantly lower in whole blood, and lowest in RBC. In Study 2, saline perfusion unexpectedly increased corticosterone levels in the rostral telencephalon but not other regions. In contrast, saline perfusion decreased DHEA levels in caudal telencephalon and diencephalon. Saline perfusion also increased E2 levels in caudal telencephalon. In summary, when comparing local and systemic steroid levels, the inclusion of whole blood samples should prove useful. Moreover, blood contamination has little or no effect on measurement of brain steroid levels, suggesting that saline perfusion is not necessary prior to brain collection. Indeed, saline perfusion itself may elevate and lower steroid concentrations in a rapid, region-specific manner.

The effect of long term exposure to copper on physiological condition and reproduction of sheep.

The influence of copper upon some physiological parameters and reproduction in ewes was studied. Four groups of animals were investigated: 1/ control ewes (untreated); 2) ewes receiving copper as a supplement over the recommended amount of copper in food (10, 25 or 50 mg Cu/ewe/day); 3/ control, superovulated ewes; and 4) ewes treated with 50 mg copper during one month and then superovulated. After 10 months of daily exposure to 10 mg of copper/ewe/day no signs of toxicity on physiological condition and reproduction were found. In ewes exposed to 25 or 50 mg of copper a decrease in blood parameters and increase in concentration of Cu in blood and liver were noticed. The wavy pattern of follicles was disturbed and disorders in fecundity, prolificacy and pregnancy occurred. Significant differences between the number of corpora lutea in superovulated control animals and experimental (Cu 50 mg) ewes were observed.

Histopathological changes in the liver, kidneys, and testes of bank voles environmentally exposed to heavy metal emissions from the steelworks and zinc smelter in Poland.

Bank voles were trapped in the neighborhood of the Sendzimir steelworks in Krakow and the ZGH Boleslaw zinc smelter in Bukowno. The Borecka forest in the north of Poland served as a control area. Lead, cadmium, zinc, and iron concentrations were analyzed in the liver, kidneys, testes, and femur bones of the bank voles. Typically, high levels of lead and cadmium were found in the bones and kidneys, respectively. In the femurs of the rodents from Bukowno, 109.26 microg g(-1) dry weight of lead was detected. The kidneys of these animals had accumulated 32.98 microg g(-1) cadmium. Concentrations of zinc and iron in the tissues were at physiological levels. No damage was found in the tissues of the bank voles from the Borecka forest or in the testes of animals from other areas. Histopathological changes in the kidneys of the rodents from Krakow as well as changes to the liver and kidneys of the animals from Bukowno were demonstrated.

Further investigation of the heavy metal content of the teeth of the bank vole as an exposure indicator of environmental pollution in Poland.

The content of heavy metals in mineralised dental tissues is an indicator of the exposure of their mineral phase to heavy metals during the time of tooth development and dental tissue formation. Therefore, teeth have been used as bio-indicators which accurately reflect the environmental or dietary exposure of animals and humans to heavy metals. This study follows from the earlier work in which it was demonstrated that the teeth from bank voles inhabiting various environmentally polluted and non-polluted forests in Southern Poland were reliable indicators of exposure to heavy metals. Using analytical techniques employed in the earlier study heavy metal concentrations were obtained in the teeth of bank voles trapped in 1998-2000 to determine if efforts to clean up the environment could be detected in changes in heavy metal concentrations in the teeth. The results show that these efforts are reflected in lower concentrations of heavy metals in the teeth but that cross border contamination remains a problem.

Input of heavy metals to the forest floor as a result of Cracow urban pollution.

The influence of Cracow's urban pollution on small forest ecosystems was studied during 1998-2000. Seven sites located at different distances from the Cracow conurbation and from busy road were selected. Two sites were situated 26 and 31 km from Cracow town along an eastern transect, the other five-3, 8, 14, 26 and 35 km respectively, from Cracow-along a southern transect. At the beginning of September, five litter traps were set at each site. The quantity of litter fall, along with percentage composition of each species, were estimated for an area of 1 m(2). The concentration of Cd, Pb, Cu, Zn and Fe in the litter fall and the input of these metals to the forest floor were determined. The input of cadmium ranged from 84 microg m(-2) at Kornatka in 1998 to 382 microg m(-2) at Bonarka in 2000. The input of lead was about 10 times higher than cadmium and varied from 406 microg m(-2) at Weglówka in 1998 to 3717 microg m(-2) at Bonarka in 2000. Input of Cu, Zn and Fe to the forest floor showed the similar tendency. During 1998-2000, the highest heavy metal concentrations in the litter fall and their impact on the forest floor were always found at sites close to the Cracow conurbation and, in the eastern transect, close to a steel works. Input of metals to the forest floor tended to decrease with increasing distance from the centre of Cracow.

Damage to the liver, kidney, and testis with reference to burden of heavy metals in yellow-necked mice from areas around steelworks and zinc smelters in Poland.

The influence of the steelworks in Warsaw and Krakow as well as the zinc smelters in Bukowno and Miasteczko Slaskie on lead, cadmium, zinc and iron concentrations and the structure of selected tissues of yellow-necked mice were analysed. The Borecka Forest was chosen as a control area. The highest concentrations of lead, 172.36 g/g dry weight, and cadmium, 23.58 g/g, were detected in the femurs and kidneys, respectively, of rodents caught in Bukowno. Zinc and iron concentrations ranged over physiological values. No histopathological changes were observed in analysed tissues of all rodents in the control area. Damage occurred in the liver and kidneys of animals from all other sites and in the testes of rodents from Bukowno. Decreased glycogen content, interstitial fibrosis, and increased number of pyknotic nuclei as well as necrosis were seen in hepatocytes. In the kidneys hyperplasia of the tubules, atrophy of glomeruli, interstitial fibrosis and necrosis were observed. Degenerate cells were present in the lumen of seminiferous tubules of animals from the Bukowno area. Even relatively low concentrations of lead and cadmium, like those found in the liver and kidneys of rodents from the neighbourhood of the steelworks, caused histopathological changes.

The long-term effect of cadmium exposure through food on the postnatal development of the bank vole (Clethrionomys glareolus Schreber, 1780).

Cadmium is well known for its toxicity to the animal body. However, its effect on pregnancy and the development of young animals is still not well understood. This study examined such effects, using bank voles captured from the wild to make the results closer to those which could be expected in the natural environment. One group of animals was fed 7 microg g(-1) cadmium in the food, a second 35 microg g(-1), and a third no cadmium, as a control. The concentrations of cadmium in the whole bodies of young bank voles were determined on the 3rd, 5th, or 10th day of life. The cadmium level in the bodies of animals exposed to 35 microg g(-1) of cadmium was significantly higher than in those from either the control group or the group receiving 7 microg g(-1) of cadmium, which did not differ from each other. The cadmium level did not change with animal age in any of the study groups. Concentrations of Zn, Cu, and Fe were also determined in the whole body of young animals, as cadmium is known to disturb the metabolism of these essential metals through antagonistic activity. Both Cu and Fe levels were negatively correlated with cadmium concentrations, while a positive correlation was found between zinc and cadmium in the young animal bodies. Also found was higher offspring mortality in the group receiving 35 microg g(-1) of cadmium in food. There was no difference in young animal body weight between the study groups.