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1.
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 mum 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 osteon's 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.  相似文献   

2.

Background

While gross morphological changes in the skeleton between males and females are well know, differences between sexes in the histomorphology are less known. It is important to have knowledge on the bone structure of rabbits, as this is a widely used species in biomedical research. A study was performed to evaluate the association between sex and the compact bone morphology of the femoral diaphysis in juvenile rabbits.

Methods

Seventeen clinically healthy 2–3 month-old rabbits (9 females, 8 males) were included in the study. The rabbits were euthanized and the right femur was sampled for analysis. 70–80 microns thick bone sections of the femoral diaphysis were prepared using standard histological equipment. The qualitative histological characteristics were determined according to internationally accepted classification systems while the quantitative parameters were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of primary osteons'' vascular canals, Haversian canals and secondary osteons were measured. Additionally, blood plasma concentrations of progesterone, corticosterone, IGF-I, testosterone and estradiol were analyzed.

Results

Qualitative histological characteristics were similar for both sexes. However, variations of certain quantitative histological characteristics were identified. Measured parameters of the primary osteons'' vascular canals were higher in males than for females. On the other hand, females had significant higher values of secondary osteons parameters. Differences in Haversian canals parameters were only significant for minimum diameter.

Conclusion

The study demonstrated that quantitative histological characteristics of compact bone tissue of the femoral diaphysis in juvenile rabbits were sex dependent. The variations may be associated with different growth and modeling of the femur through influence by sex-specific steroids, mechanical loads, genetic factors and a multitude of other sources. The results can be applied in experimental studies focusing on comparison of the skeletal biology of the sexes.  相似文献   

3.
Histological sections of femoral diaphysis from adult sheep were investigated in this study. The qualitative characteristics in microstructure of the compact bone were examined in anterior, posterior, medial and lateral views; the quantitative ones were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of the Haversian canals, the Haversian systems and the primary osteons' vascular canals were measured. Our results indicate that ovine diaphysis of the femur is occupied mainly with primary vascular plexiform (laminar, fibrolamellar) tissue. In addition, irregular and dense Haversian bone tissues were observed. All measured variables disposed lower values in comparison with other artiodactyls (cattle and pig).  相似文献   

4.
Apricot seeds due to the presence of cyanogenic glycoside amygdalin belong to the popular “alternative cancer cures", although anticancer effect of amygdalin remains controversial. This in vivo study points to the effect of long-term peroral administration of bitter apricot seeds on bone microstructure of rabbits since chronic amygdalin toxicity in relation to bone parameters has not been investigated yet. Rabbits (n = 16) were randomly divided into four experimental groups of 4 animals each. Three experimental groups S1, S2 and S3 received commercial feed for rabbits mixed with crushed bitter apricot seeds at doses 60, 300 and 420 mg/kg bw during five months, respectively. The control (C) group received no apricot seeds. The long-term consumption of apricot seeds had no impact on total body weight, femoral weight and femoral length of rabbits. Also, microcomputed tomography (3D analysis) of cortical and trabecular bone tissues did not reveal any significant impact of amygdalin toxicity on relative bone volume, BMD, cortical bone thickness, bone surface, trabecular number, thickness, and their separation. On the other hand, histological (2D) analysis demonstrated evident changes in cortical bone microstructure consistent with a decreased density of secondary osteons in the middle part of substantia compacta due to a replacement of Haversian bone tissue by plexiform bone tissue, vasoconstriction in the primary osteons' vascular canals, Haversian canals, and decreased sizes of secondary osteons in rabbits from S1, S2 and S3 groups. These negative changes are associated with different vascularization and biomechanical properties of cortical bones.  相似文献   

5.
A high number of differences exist in bone histological features depending on the species, breed, age and bone. Moreover, osteon distribution may vary in the different sides of a bone as a consequence of different biomechanical strains. The aim of this work was to study the distribution and morphology of osteons in different sides of the equine femoral diaphysis with the attempt to correlate them to the main strains operating on them. The following parameters of secondary osteons and Haversian canals were measured in the transverse sections of diaphyses: perimeter, area, minimum and maximum diameter, eccentricity and osteon population density. A typical Haversian tissue was observed with elliptic secondary osteons consisting in about 10 well‐defined lamellae surrounding a circular Haversian canal. Quantitative analysis displays a different population density of secondary osteons depending on the side. The caudal and medial sides, where compression strains are higher, have more secondary osteons in comparison with the cranial and lateral sides, where tension strains are prevalent. These data suggest that secondary osteon population density may depend on the predominant strains. Even the elliptical shape of secondary osteons may be related to biomechanical strains, as their major axes are oriented cranio‐caudally parallel to prevalent strains.  相似文献   

6.

Background

Chronic exposure to cadmium (Cd), even at low concentrations, has an adverse impact on the skeletal system. Histologically, primary and secondary osteons as basic structural elements of compact bone can also be affected by several toxicants leading to changes in bone vascularization and mechanical properties of the bone. The current study was designed to investigate the effect of subchronic peroral exposure to Cd on femoral bone structure including histomorphometry of the osteons in adult male rats.In our study, 20 one-month-old male Wistar rats were randomly divided into two experimental groups. In the first group, young males received a drinking water containing 30 mg of CdCl2/L, for 90 days. Ten one-month-old males without Cd intoxication served as a control group. After 90 days of daily peroral exposure, body weight, femoral weight, femoral length, cortical bone thickness and histological structure of the femora were analysed.

Results

We found that subchronic peroral application of Cd had no significant effect on body weight, femoral length and cortical bone thickness in adult rats. On the other hand, femoral weight was significantly increased (P < 0.05) in Cd-intoxicated rats. These rats also displayed different microstructure in the middle part of the compact bone where vascular canals expanded into central area of substantia compacta and supplied primary and secondary osteons. Additionally, a few resorption lacunae which are connected with an early stage of osteoporosis were identified in these individuals. Histomorphometrical evaluations showed that all variables (area, perimeter, maximum and minimum diameter) of the primary osteons’ vascular canals, Haversian canals and secondary osteons were significantly decreased (P < 0.05) in the Cd group rats. This fact points to alterations in bone vascularization.

Conclusions

Subchronic peroral exposure to Cd significantly influences femoral weight and histological structure of compact bone in adult male rats. It induces an early stage of osteoporosis and causes reduced bone vascularization. Histomorphometrical changes of primary and secondary osteons allow for the conclusion that the bone mechanical properties could be weakened in the Cd group rats. The current study significantly expands the knowledge on damaging action of Cd on the bone.  相似文献   

7.

Background

The 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.

Methods

Twenty 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.

Results

The 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.

Conclusions

Se 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.  相似文献   

8.
Bone microstructure of domestic herbivores is still not completely understood. Indeed, works focused on the bone histology of numerous Mammalian species frequently led to misunderstandings because of the high number of variations such as the kind of bone, section orientation, species, breed and age. Moreover, attempts to identify the species in archaeozoological studies by a mere qualitative approach have not been encouraging and in recent years quantitative methods, based on image processing and statistical analysis, have appeared. The present study was undertaken to determine whether morphometrical and morphological differences exist in the compact bone structure of the femur and humerus between horses and cows. Measurements such as area, perimeter, minimum and maximum diameter of osteons and Haversian canals as well as the osteonal density were carried out on cross sections of eight humeri and eight femurs of the two herbivores investigated. In agreement with other authors, the qualitative investigation confirmed that the compact bone of horses and cows can be classified as dense Haversian tissue. Osteons of the horse were more numerous and composed of a higher number of well-defined lamellae when compared with the cow. Diameter, perimeter and area of osteons and Haversian canals were always higher in horses than in cows and this pattern could be related to the different locomotor behaviour of these animals.  相似文献   

9.
The aim of our study was to compare the hFVIII mRNA expression in different organs, pathological changes and selected haematological and biochemical blood parameters between transgenic and non-transgenic rabbits from F3 generation. Selected physiological parameters of 3- to 4-month-old transgenic rabbits from F3 generation carrying human factor VIII gene (hFVIII) were analysed and compared with those of non-transgenic ones. Before slaughtering, the blood for haematological and biochemical analysis was taken from the central ear artery. Pathological and histological examination of vital organs and RT-PCR analysis of several tissue organs of transgenic and non-transgenic animals were performed after slaughtering. Except for the mammary gland tissue, slight hfVIII mRNA expression in the spleen, lung and brain and none expression in the liver, kidney, skeletal muscle and heart of rabbits were recorded. pathological examination of vital organs showed some pathological changes in both transgenic and non-transgenic rabbits which were confirmed by histological qualitative evaluations. Statistically significant lower values of blood haemoglobin in blood of transgenic (11.86+/-0.86) animals compared with non-transgenic (12.41+/-1.02, P<0.05) ones and lower parameters of HCT (39.22+/-2.44 versus 40.89+/-2.26, P<0.01) in blood of transgenic rabbits were observed. Parameters of WBC, RBC and PLT showed no significant differences between the analysed groups. All biochemical serum parameters of transgenic rabbits were higher in comparison with non-transgenic ones. Significant differences were found in the concentration of the urea, AST and GMT between transgenic and non-transgenic animals (P<0.001) and in the total protein content, the difference was significant at P<0.05. In conclusion, our results showed that no considerable impact on the general health was found in transgenic rabbits.  相似文献   

10.

Background

Exposure to cadmium (Cd) is considered a risk factor for various bone diseases in humans and experimental animals. This study investigated the acute effects of Cd on femoral bone structure of adult male rats after a single intraperitoneal administration.

Methods

Ten 4-month-old male Wistar rats were injected intraperitoneally with a single dose of 2 mg CdCl2/kg body weight and killed 36 h after the Cd had been injected. Ten 4-month-old males served as a control group. Differences in body weight, femoral weight, femoral length and histological structure of the femur were evaluated between the two groups of rats. The unpaired Student''s t-test was used for establishment of statistical significance.

Results

A single intraperitoneal administration of Cd had no significant effect on the body weight, femoral weight or femoral length. On the other hand, histological changes were significant. Rats exposed to Cd had significantly higher values of area, perimeter, maximum and minimum diameters of the primary osteons'' vascular canals and Haversian canals. In contrast, a significant decrease in all variables of the secondary osteons was observed in these rats.

Conclusions

The results indicate that, as expected, a single intraperitoneal administration of 2 mg CdCl2/kg body weight had no impact on macroscopic structure of rat''s femora; however, it affected the size of vascular canals of primary osteons, Haversian canals, and secondary osteons.  相似文献   

11.
The mammary gland of transgenic animals has been used for the production of recombinant proteins of therapeutic and nutraceutical use. The objective of this study was to compare the ultrastructure of transgenic and non-transgenic rabbit mammary gland tissue. New Zealand White transgenic rabbits were obtained by breeding non-transgenic rabbits with transgenic founder rabbits containing a whey acidic protein-human factor VIII (WAP-hFVIII) transgene integrated into their genome. Samples of mammary gland tissue from lactating rabbit females were isolated by surgical procedures. These samples were examined by optical and electron microscopy and photographs were taken. Measurements of ultrastructural organelles were made from digital images of the mammary cells. No differences were found in the cellular structure of mammary tissue, but significant differences t((0.001)) in the relative volume of mitochondria and vacuoles between transgenic and non-transgenic mammary gland epithelium were observed.  相似文献   

12.
The aim of this study was to compare morphological characteristics of testes from transgenic (the WAP-hFVIII gene) and non-transgenic rabbits with emphasis on the histological and ultrastructural aspects. Samples of testes from both groups were fixed and embedded into Durcupan ACM for transmission electron microscopy. For histological analysis, semi-thin toluidine blue-stained sections were evaluated under a Jenaval light microscope. Male fertility was tested based on egg fecundity and blastocyst yield; transgene transmission was proved using PCR assay. Spermatogenesis in rabbit testes had not been destroyed both in transgenic and non-transgenic rabbits. No significant differences were found in the occurrence of individual cell organelles of the Sertoli cells in transgenic and non-transgenic rabbits. The ultrastructure of Leydig cells in testes of transgenic and non-transgenic rabbits was rather similar. No differences in the occurrence of individual organelles of Leydig cells between transgenic and non-transgenic males were found. These results were in concert with fertilizing capacity of transgenic spermatozoa. The presented status of organelles in this study indicates functional activity of the analysed cells.  相似文献   

13.

Background

Osteoporosis and its main health outcome, fragility fractures, are large and escalating health problems. Skeletal damage may be the critical result of low-level prolonged exposure to several xenobiotics in the general population, but the mechanisms of their adverse effects are not clearly understood. The current study was aimed to investigate the possible ability of simultaneous subchronic peroral administration of selenium (Se) and diazinon (DZN) to induce changes in bone of adult male rats.In our study, twenty 1-month-old male Wistar rats were randomly divided into two experimental groups. In the first group, young males were exposed to 5 mg Na2SeO3/L and 40 mg of DZN/L in drinking water, for 90 days. Ten 1-month-old males without Se and DZN intoxication 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.

Results

The body weight, femoral length and cortical bone thickness were significantly decreased in rats simultaneously exposed to Se and DZN (P < 0.05). These rats also displayed different microstructure in the middle part of the compact bone where vascular canals expanded into central area of substantia compacta. The canals occurred only near endosteal surfaces in rats from the control group. Additionally, a smaller number of primary and secondary osteons, as well as a few resorption lacunae were observed near endosteal surfaces in rats simultaneously administered to Se and DZN. The resorption lacunae as typical structures of bone resorption manifestation are connected with an early stage of osteoporosis. Histomorphometric analysis revealed that area, perimeter, maximum and minimum diameters of primary osteons’ vascular canals were significantly increased (P < 0.05) in the Se-DZN-exposed rats. On the other hand, all measured variables of Haversian canals and secondary osteons were considerable reduced (P < 0.05) in these rats.

Conclusions

Simultaneous subchronic peroral exposure to Se and DZN induces changes in macroscopic and microscopic structures of the femurs in adult male rats, and also it can be considered as possible risk factor for osteoporosis. The current study contributes to the knowledge on damaging impact of several xenobiotics on the bone.  相似文献   

14.

Background

Patulin, a toxic mold metabolite, has been found as natural contaminant of processed fruits, most notably apples, apple juices and other apple-based products. A number of adverse health effects in humans and animals are associated with patulin intoxication. The current study was performed to analyse possible toxic effects of patulin on femoral bone microstructure in adult rabbits in detail. Fourteen clinically healthy four-month-old rabbits of both sexes (6 males and 8 females) were included in the study. Animals from the experimental groups (group E♂, n = 3; group E♀, n = 4) were injected intramuscularly with patulin at dose 10 μg/kg body weight two times a week for 28 days. The dose of patulin was estimated based on the maximum permitted level of patulin for apple products intended for infants and young children. Three males and four females without patulin administration served as controls (groups C♂ and C♀). Cortical bone thickness and qualitative and quantitative histological characteristics of compact bone tissue were investigated.

Results

Intramuscular applications of patulin significantly increased the thickness of cortical bone in both sexes of rabbits. In patulin-exposed males, an absence of primary vascular longitudinal bone tissue near the endosteal border was observed, which could be associated with intensive bone remodeling. Femoral diaphyses of females displayed a lower number of secondary osteons in the middle part of the substantia compacta, and occurrence of the osteons near the periosteum. This could indicate alterations in bone turnover. Histomorphometrical evaluations showed significantly increased sizes of the primary osteons’ vascular canals (P < 0.05) in males exposed to patulin possibly due to mycotoxin-induced increased levels of testosterone.

Conclusions

This study demonstrates significant impact of intramuscular application of patulin on bone microstructure in adult rabbits. Moreover, we have found that the effects of patulin on qualitative and quantitative histological characteristics of compact bone are sex-dependent.  相似文献   

15.
This study was aimed to compare structures of the thyroid tissue of transgenic rabbits expressing the human clotting factor VIII under the murine whey acidic protein promoter (mWAP-hFVIII rabbits) with the non-transgenic controls. Thyroid tissue samples were taken from transgenic and non-transgenic New Zealand White rabbits, examined by optical microscopy and analysed morphometrically. The analysis revealed no significant differences (P > 0.05) in the relative volume of basic thyroid structures. Furthermore, no significant differences (P > 0.05) were observed when measuring the epithelial height and nuclear diameter of the follicular cells. Altogether, this study demonstrates no negative effect of the mWAP-hFVIII transgenesis on the rabbit thyroid gland structure.  相似文献   

16.

Background

Nickel (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.

Methods

One 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.

Results

The 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.

Conclusions

The 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.  相似文献   

17.
The objective of this research was to compare (i) the content of milk protein and recombinant human factor VIII (rhFVIII) in the milk of transgenic and non-transgenic rabbit females at three lactations and (ii) histological structure, ultrastructural morphology and occurrence of apoptosis in rabbit transgenic and non-transgenic mammary gland during third lactation and involution. Significant differences (t0.05) in milk protein content were found between transgenic and non-transgenic at all three lactations. The percentage of apoptotic cells was significantly higher (t0.01) in non-transgenic ones compared with transgenic mammary gland tissues (6.5% versus 2.4%) taken at the involution stage. Morphometrical analysis of histological preparations at the involution stage detected a significantly higher (t0.05) relative volume of lumen in transgenic animals compared with non-transgenic ones (60.00 versus 46.51%). Ultrastructural morphology of the transgenic mammary gland epithelium at the involution stage revealed an increased relative volume of protein globules (t0.05); at the lactation stage, a significantly higher volume of mitochondria (13.8%) compared with the non-transgenic (9.8%) ones was observed. These results, although revealing differences in some parameters of ultrastructure and histology, indicate no harmful effect of the mouse whey acid protein-hFVIII transgene expression on the state of mammary gland of transgenic rabbit females.  相似文献   

18.
The osteonal pattern of cortical bone is gradually built around the intracortical vessels by the progression of the cutting cones (secondary remodelling); therefore, the central canal size can be used as index of the remodelling activity. An experimental model in the rabbit femur was used to investigate, through central canal morphometry and frequency distribution analysis, the remodelling activity, comparing the middle of the diaphysis (mid-shaft) with the extremity (distal-shaft) and at the same level sectors and layers of the cortex in transversal sections. The study documented a higher density of canals in the mid-shaft than in the distal-shaft and a higher remodelling in the distal-shaft. There were no significant differences between dorsal, ventral, medial and lateral sectors at both mid-shaft and distal-shaft levels, while the number of canals was higher in the sub-periosteal layers than in the sub-endosteal. A lower threshold of 40 μm2 was observed in the central canal area. Sealed osteons in the midshaft were 22.43% of the total number of osteons of the central canal area between 40 and 200 μm2 and 0.44% of those of the distal-shaft. Micro-CT allowed a 3D reconstruction of the vascular canal system, which confirmed the branched network pattern rather than the trim architecture of the traditional representation. Some aspects like the lower threshold of the central canal size and the sealed osteons documented the plasticity of the system and its capacity for adaptation to changes in the haemodynamic conditions.  相似文献   

19.
本研究对转基因牛以及非转基因牛精液经流式细胞仪分离冷冻后精子活力、分离准确率进行了比较,同时对分离的性控冷冻精液进行了人工输精,对受体牛的情期受胎率进行了统计分析。结果表明,转基因牛与非转基因牛精液在冷冻解冻后活力以及分离准确率方面差异不显著(P>0.05);转基因牛与非转基因牛的性控冷冻精液的情期受胎率分别为57.4%、59.3%,二者之间差异不显著(P>0.05)。  相似文献   

20.
Some young large farm animals show a laminar bone formation in the long-bone cortex. Such a laminar bone is gradually replaced by Haversian bone with osteons during their growth periods. In this preliminary study, we observed the transverse ground samples of tibia cortex in young calves, pigs, and sheep by backscattered electron imaging. The cortex bones of all the newborn (NB) animals were basically formed with laminar bone structures. The NB and 1-month-old (1-M) calves had a typical concentric structure of laminar bone, whereas the NB and 1-M pigs showed a wire-netting bone with laminar-bone units. The NB sheep was similar to the calf rather than the pig. In the growth rate of bone volume, sheep was similar to calf up to 6 months after birth (6-M). Such calf and sheep showed a more rapid ratio of bone volume than pig. A few osteons had initially appeared in the innermost layer of the 6-M calf. A 1-year-old (1-Y) calf showed scattered osteons in the bone cortex, but many laminar-bone units were still retained in the outer layer. A 6-M pig had many osteons in the entire cortex but only a few osteons in the outermost layer. In the 6-M sheep, no osteons were observed, whereas a 1-Y sheep showed a relatively small number of osteons mainly in the middle layer but a higher osteon-volume than the 1-Y calf. In the 1-Y sheep, the more widely absorbed areas by bone-remodeling with osteons were observed as compared with the 1-Y calf, and the bone volume was decreased from the 6-M into the 1-Y sheep because of the remarkable bone-absorption. Thus, calf kept on possessing many laminar-bone units for a longer time in the growth period than sheep, while pig showed the earliest bone-remodeling with osteons. These results may be caused by their different body size and withers height in calf and sheep after growing and the difference of the dependence upon mother's body during juvenile period between pig and calf with sheep. The initial region of osteon formation may be distinguishable among their animals, respectively. However, further detailed investigations of their young animals at successive stages will be necessary.  相似文献   

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