Monika Martiniaková

Differences Among Species in Compact Bone Tissue Microstructure of Mammalian Skeleton: Use of a Discriminant Function Analysis for Species Identification:

ABSTRACT: In order to develop an identification key for distinguishing between several mammalian species, bone structure of their compact bone tissue was analyzed using qualitative and quantitative characteristics. Ninety femora of adult male humans, pigs, cows, sheep, rabbits, and rats were studied. The average area, perimeter, minimum, and maximum diameter of 1863 Haversian canals and 1863 secondary osteons were measured using a digital image device. The observed data were first used to evaluate inter‐ and intraspecies diversity. After that, we applied a discriminant function analysis for differentiation of the species by these variables. Classification functions for investigated species give cross‐validated correct classification rates for 76.17% of cases. This percentage value can be increased by integrating conclusions from the qualitative analysis.

Heavy metal content in the femora of yellow-necked mouse (Apodemus flavicollis) and wood mouse (Apodemus sylvaticus) from different types of polluted environment in Slovakia

Heavy metal content in the femora of yellow-necked mouse (Apodemus flavicollis) and wood mouse (Apodemus sylvaticus) caught in different polluted biotopes of a low hill level in Slovakia (Nováky and Kolín̆any) was investigated in the present study. Length, weight and histological structure of mouse bones have also been analysed. According to our results, higher concentrations of Cd, Ni, Fe, Cu and Zn were detected in the femora of A. flavicollis from Kolín̆any area. Similarly, we observed higher concentrations of Ni, Fe, Cu and Zn in the bones of A. sylvaticus trapped at the same biotope. Significant differences were observed for concentrations of Ni and Zn in both species (P < 0.05). The measured values for bone length and bone weight were higher in yellow-necked mice and wood mice from Nováky locality (P < 0.01). Histological observation of thin sections from femora of A. flavicollis and A. sylvaticus revealed an outer and inner non-vascular lamellar layer around a poorly developed reticular layer. We did not identify demonstrable changes in qualitative histological characteristics of the femora between the mice (A. flavicollis and A. sylvaticus separately) from different types of polluted environment. Also, no statistically significant differences for all the measured variables of primary osteons’ vascular canals were observed. Correlation analysis in yellow-necked mouse showed high positive relation between bone weight and bone length (r = 0.66), area and perimeter (r = 0.87) and perimeter and maximum diameter (r = 0.87). In wood mouse, high positive correlation between bone weight and bone length (r = 0.80), area and perimeter (r = 0.72), area and maximum diameter (r = 0.66) and perimeter and maximum diameter (r = 0.74) was found. Our results demonstrate slightly elevated accumulation of some heavy metals in the femora of yellow-necked mouse and wood mouse from Kolín̆any biotope and thus give an evidence of a contamination of the environment.

Differences in Femoral Compact Bone Tissue Microscopic Structure between Adult Cows (Bos taurus) and Pigs (Sus scrofa domestica)

The purpose of this investigation was to study in detail compact bone tissue microscopic structure of adult cows and pigs with an emphasis to find an adequate key for the species identification. Altogether 18 femurs were analysed. Each of the bones was sectioned at the smallest breadth of their diaphysis. Specimens were prepared using standard histological equipment, producing thin sections approximately 80–100 μm thick. The qualitative differences between investigated species were examined in anterior, posterior, medial and lateral views of thin sections. The quantitative ones were counted using the specific computer software Scion Image. We measured the area, perimeter, minimal and maximal diameter of the Haversian canals, the Haversian systems and the vascular canals of primary osteons. After that a discriminant function analysis was used for the species identification. According to our results the basic structural pattern of femur diaphysis was primary vascular plexiform in both species. However, non‐vascular bone tissue was identified in cows and resorption lacunae were found between the secondary osteons in pigs. The measured variables of the Haversian canals, the Haversian systems and the primary osteons vascular canals were higher in most cases in cows. Classification functions for investigated species give a correct classification of 64.69% of cases. This percentage value can be increased by integrating conclusions from the qualitative analysis.

Effects of subchronic exposure to cadmium and diazinon on testis and epididymis in rats.

The present study aimed to elucidate the structural changes in testis and epididymis of adult rats following subchronic peroral administration of cadmium at 30 mg/L, diazinon at 40 mg/L, cadmium at 30 mg/L, and diazinon at 40 mg/L, respectively. At the end of 90-day experiment, the samples of the testes and epididymis were assayed by qualitative and quantitative histological methods. The testis and epididymis weights increased following exposure to cadmium and simultaneous exposure to cadmium and diazinon. Testicular damage following cadmium and diazinon coexposure was significantly less expressive than in groups with individual administration of these compounds. Cadmium caused a significant thickening of seminiferous epithelium, cellular degeneration, and necrosis. Desquamation of immature germ cells resulted in a significant increase of intraepithelial spaces and reduced tubule volume in all experimental groups. Vascular dilation and congestion were detected in the interstitial tissue. The changes in epididymal histology in the group exposed to cadmium and group exposed simultaneously included a reduction of epithelium, necrotic epithelial cells, vasoconstriction, and interstitial edema together with mononuclear cell infiltration. Results did not indicate a synergistic or any additional effect from the simultaneous administration of both toxicants. Further research is needed to determine the significance and the mechanism of the adverse effects.

Quantitative histological analysis of the mouse testis after the long-term administration of nickel in feed

In this study, the effects of nickel chloride (NiCl2) applied per os on testis histopathology and morphometry of mice were investigated. The metal was applied in pellets at a dose of 10 mg NiCl2/kg bw to male mice 4 weeks of age. After 3, 6, 9 and 12 weeks of nickel administration, the relative volume of whole seminiferous tubule, germinal epithelium, tubule lumen, interstitium and blood vessels as well as the diameter of seminiferous tubules were determined in the experimental and corresponding control groups. Microscopic examination of testis showed significant changes in all nickel-exposed groups. The degeneration of germinal epithelium, with released germ cells into the lumen of the tubules, and occurrence of empty spaces in the seminiferous epithelium were found in all experimental groups. The changes in the testes were time-dependent. The relative volume of empty spaces in the seminiferous epithelium significantly increased (P < 0.001) in all experimental groups when compared with the corresponding control. A significant decrease in the relative volume of seminiferous epithelium was observed after 6 and 12 weeks of Ni-exposure. The increased luminization of the tubules was found after 6 (P < 0.001), 9 (P < 0.01) and 12 (P < 0.001) weeks. Interstitial tissue significantly decreased after 6 and 9 weeks of Ni exposure and increased after 12 weeks of Ni intake. The seminiferous tubule diameter significantly (P < 0.001) decreased after 12 weeks. Results of this study report a serious, time-dependent changes in the testes, mainly in the germinal epithelium, after a peroral intake of nickel.

Yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) as zoomonitors of environmental contamination at a polluted area in Slovakia.

BackgroundFree-living wild rodents are often used as zoomonitors of environmental contamination. In the present study, accumulation of cadmium (Cd), copper (Cu), iron (Fe), and zinc (Zn) in critical organs of yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) trapped in a polluted area in Nováky, Slovakia was investigated.MethodsYellow-necked mice (n = 8) and bank voles (n = 10) were collected using standard theriological methods for wood ecosystems. All animals were adult males in good physical condition. The concentrations of Cd, Cu, Fe, and Zn in the liver, kidney, and bone were determined by atomic absorption spectrophotometry.ResultsThe highest concentrations of Cd and Zn were found in the bone of both species while Cu and Fe accumulated mainly in kidney or liver. Significant higher concentrations of Cd and Cu were detected in the liver of bank voles than in yellow-necked mice. Similar significant higher levels of Cd and Zn were found in the bone of bank voles. In contrast, significant higher concentrations of Cu and Fe were present in the kidney of yellow-necked mice.ConclusionsIn the yellow-necked mouse and bank vole, bone seems to accumulate Cd and Zn following prolonged exposure. On the contrary, kidney and liver store Cu and Fe after a long-term environmental exposure. In the present study, bank voles seemed to be more heavy metal loaded zoomonitors than yellow-necked mice.

Effects of dietary supplementation of nickel and nickel-zinc on femoral bone structure in rabbits

BackgroundNickel (Ni) and zinc (Zn) are trace elements present at low concentrations in agroecosystems. Nickel, however, may have toxic effects on living organisms and is often considered as a contaminant. This study reports the effect of peroral administrated Ni or a combination of Ni and Zn on femoral bone structure in rabbits.MethodsOne month-old female rabbits were divided into three groups of five animals each. Group 1 rabbits were fed a granular feed mixture with addition of 35 g NiCl2 per 100 kg of mixture for 90 days. In group 2, animals were fed a mixture containing 35 g NiCl2 and 30 g ZnCl2 per 100 kg of mixture. Group 3 without administration of additional Ni or Zn served as control. After the 90-day experimental period, femoral length, femoral weight and histological structure of the femur were analyzed and compared.ResultsThe results did not indicate a statistically significant difference in either femoral length or weight between the two experimental groups and the control group. Also, differences in qualitative histological characteristics of the femora among rabbits from the three groups were absent, except for a fewer number of secondary osteons found in the animals of groups 1 and 2. However, values for vascular canal parameters of primary osteons were significantly lower in group 1 than in the control one. Peroral administration of a combination of Ni and Zn (group 2) led to a significant decreased size of the secondary osteons.ConclusionsThe study indicates that dietary supplementation of Ni (35 g NiCl2 per 100 kg of feed mixture) and Ni-Zn combination (35 g NiCl2 and 30 g ZnCl2 per 100 kg of the mixture) affects the microstructure of compact bone tissue in young rabbits.

Structural changes in femoral bone tissue of rats after subchronic peroral exposure to selenium

BackgroundThe role of selenium (Se) on bone microarchitecture is still poorly understood. The present study aims to investigate the macroscopic and microscopic structures of femoral bone tissue in adult male rats after subchronic peroral administration of Se.MethodsTwenty one-month-old male Wistar rats were randomly divided into two experimental groups. In the first group (Se group) young males were exposed to 5 mg Na2SeO3/L in drinking water, for 90 days. Ten one-month-old males without Se administration served as a control group. At the end of the experiment, macroscopic and microscopic structures of the femurs were analysed using analytical scales, sliding instrument, and polarized light microscopy.ResultsThe body weight, femoral length and cortical bone thickness were significantly decreased in Se group rats. These rats also displayed different microstructure in the middle part of the femur, both in medial and lateral views, where vascular canals expanded into the central area of the bone while, in control rats, these canals occurred only near the endosteal surfaces. Additionally, a smaller number of primary and secondary osteons was identified in Se group rats. Histomorphometric analyses revealed significant increases for area, perimeter, maximum and minimum diameters of primary osteons’ vascular canals but significant reductions for all measured variables of Haversian canals and secondary osteons.ConclusionsSe negatively affected the macroscopic and microscopic structures of femoral bone tissue in adult male rats. The results contribute to the knowledge on damaging impact of Se on bone.

Accumulation of risk elements in kidney, liver, testis, uterus and bone of free-living wild rodents from a polluted area in Slovakia

Free-living wild rodents are usually used as indicators of pollution, with elements being determined in either whole body or in specific organs. In the present study, the accumulation of cadmium (Cd), copper (Cu), iron (Fe), and zinc (Zn) in kidney, liver, testis, uterus and bone of yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) trapped in a polluted area of Nováky, Slovakia was investigated. Yellow-necked mice and bank voles were collected using standard theriological methods for wood ecosystems. All animals were adults in good physical condition. Concentrations of Cd, Cu, Fe, and Zn in all analyzed organs were determined by atomic absorption spectrophotometry. The highest concentrations of Cd and Zn were found in the bone of both species while Cu and Fe accumulated most in the uterus. Significantly higher concentrations of Cd and Cu were detected in the liver of the bank vole in comparison with the yellow-necked mouse (P<0.05). Similar significantly higher levels of Cd and Zn were found in the bone of the bank vole (P<0.05) than in the yellow-necked mouse, while these rodents had significantly higher Cu and Fe concentrations (P<0.05) in the kidney. Significantly higher levels of Fe and Zn were detected in the testis and uterus of bank voles, respectively. On the other hand, significantly higher concentration of Cu was found in the testis of yellow-necked mice. Results of this study suggest that bank voles are more sensitive heavy metal loaded bioindicators than yellow-necked mice.

Comparative Study of Compact Bone Tissue Microstructure between Non-transgenic and Transgenic Rabbits with WAP-hFVIII Gene Construct

The aim of this study was to investigate differences in compact bone tissue microstructure between New Zealand White transgenic rabbits with human blood clotting factor VIII gene and the non‐transgenic ones. The transgene was under the transcriptional control of the whey acidic protein (WAP) promoter. Altogether, 42 femora were analysed. Specimens were prepared using standard histological equipment, producing thin sections approximately 80–100 μm. Areas, perimeters, minimum and maximum diameters of vascular canals of primary osteons, Haversian canals and secondary osteons were measured. Our results indicate the basic structural pattern of the bone tissue was primary vascular longitudinal in both groups of rabbits. However, a fibrolamellar bone tissue was identified only in the transgenic rabbits. The measured variables of the vascular canals of the primary osteons, the Haversian canals and the secondary osteons were higher in transgenic rabbits in comparison with non‐transgenic ones in most cases and the differences were often statistically significant (P < 0.01; P < 0.001). More significant differences were noticed in males (transgenic versus non‐transgenic). However, it is not easy to find the real cause of the changes. Despite the fact that no hFVIII mRNA expression was found in the analysed bone of transgenic rabbits, we presume that the observed differences could be associated with transgenesis. In this article, we discuss possible associations between transgene integration as well as aneuploidy and/or observed microstructural changes in compact bone tissue.