The hepatocytes of the brown trout (Salmo trutta fario): A stereological study of some cytoplasmic components with the breeding cycle

Sex differences exist in fish hepatocytes, but studies for characterizing their cytology throughout the breeding cycle are still scarce; suggesting changes, but most lacking quantitative data. To address this limitation, to complement baseline data generated from the brown trout model, and to prove that sex‐specific seasonal changes exist, we made an unbiased stereological evaluation of the hepatocytic cytoplasm. Unprecedentedly for fish liver, the stereological design was exempt from model (biased) assumptions. Five (3 years old) animals per sex were studied in endogenous vitellogenesis, exogenous vitellogenesis, and spawning season end. Liver pieces for analysis were systematically sampled. Stereology was done in transmission electron microscopy (TEM) micrographs. Primary data generated relative volume estimates of the major cytoplasmic components. Such values were used for deriving absolute volumes (per cell and per liver). Lipid droplets did not show changes. As to other targets, trends at cell and liver levels were not always equal. If the hepatocyte was the reference space, the contents in mitochondria, dense bodies, glycogen, and cytosol changed seasonally, in both sexes. If taking the liver as the reference, changes attained the Golgi apparatus and rough endoplasmic reticulum (RER), besides dense bodies, glycogen (in females), and cytosol. The components volumes (namely per liver) were often positively (negatively for glycogen) correlated with the ovary weight, disclosing new associations and implications in fish. While also offering gold‐standard data for backing morphofunctional correlations and pathology, we revealed a new process by which females increase the amount of RER and Golgi throughout vitellogenesis, breaking from the idea on how this event happens in fish. Microsc. Res. Tech. 73:766–778, 2010. © 2010 Wiley‐Liss, Inc.     

Seasonal changes in hepatocytic lipid droplets,glycogen deposits,and rough endoplasmic reticulum along the natural breeding cycle of female ohrid trout (Salmo letnica Kar.)—A semiquantitative ultrastructural study

This study on wild female Ohrid trout was primarily designed to provide a general overview of the breeding cycle influence upon selected aspects of hepatocytes. According with a semiquantitatively evaluation, some of these cell's structural compartments change during the breeding cycle. Structural modifications were disclosed in the relative occurrence of lipid, glycogen, and RER content during breeding cycle. The relative amount of lipid deposits in the hepatocytes was much greater in previtellogenesis, and decreased postspawning. So, while the seasonal changes in RER were positively related with the ovary maturation status, those of the lipid droplets followed an opposite trend. The hepatocytic glycogen occurred rarely, mainly in late‐vitellogenesis and spawning, suggesting that in this species such kind of energy storage is comparatively unimportant. Lipid accumulation and later usage is, probably, the relevant biochemical pathway for Ohrid trout in the wild. While glycogen and RER contents were positively correlated with the gonadosomatic index, lipids were negatively correlated. Additionally, glycogen inclusions were positively correlated with the plasma estradiol levels. When comparing seasonal patterns from wild Ohrid trout with those from well‐studied rainbow and brown trout (specimens studied were from aquaculture), there are contradicting results as to lipid and glycogen reserves, and also as to RER loads. The differences among the mentioned trout can result from intrinsic interspecies differences or may be associated with natural feeding conditions versus feeding with commercially prepared diets, or other factors. This study offers new data useful as standard to access liver pathology in wild and aquacultured Ohrid trout. Microsc. Res. Tech. 79:700–706, 2016. © 2016 Wiley Periodicals, Inc.     

A qualitative and quantitative study of the hepatic pigmented macrophage aggregates during the breeding cycle of Ohrid trout, Salmo letnica Kar. (Teloestei, Salmonidae)

It is well known that factors such as aging and water quality may influence pigmented macrophages (PMacs) in fishes, but it is not established yet if PMacs undergo seasonal and breeding dependent variations. This study explored this caveat and reports qualitative histological and stereological data on liver PMacs from wild female Ohrid trout, Salmo letnica, during the annual breeding cycle. Data showed that a minority of PMacs contained melanin and that the vast majority of them contained only hemosiderin or hemosiderin and lipofuscin/ceroid pigment. It was suggested that this is the normal scenario for the species. One remarkable result was the demonstration of a striking increase, after spawning, of the relative and total volumes of the hepatic macrophages, both parenchyma and stroma located. Because the melano PMacs did not vary, those changes were due to fluctuations in the hemosiderin-laden PMacs. We concluded that Ohrid trout presented a pigment composition in liver macrophages that differed from other fish, including salmonids, where most liver phagocytes essentially display melanin. Our quantitative data support interspecies differences in the amount of liver macrophages and also that after spawning expansion of the macrophage pool is crucial and most likely connected with the needs of liver remodeling (leading to a decrease in hepatic mass). So, we suggested that the hormonal (sex steroidal) constellation influenced the liver macrophage pool. Additionally, we proved that the use of fish liver macrophages for biomonitoring should take into account the considerable natural breeding/seasonal dependent variations that are expected to occur.     

An electron microscopic analysis on the ultra structural abnormalities in sperm of the common carp Cyprinus carpio L. inhabiting a polluted lake, Umiam (Meghalaya, India)

The present communication reports the ultra structural abnormalities in sperm of a fish species Cyprinus carpio inhabiting a polluted lake, Umiam in North‐East India. Transmission electron microscopy (TEM) revealed absence of differentiation between head and midpiece (neck) of some sperm while scanning electron microscopy (SEM) showed some sperm tails with highly reduced length and some sperm with folded tail. Abnormal shape of some sperm head was also revealed by Scanning electron microscopy. Detachment of membrane from some parts of the sperm head and an outward expansion of the same was observed from Transmission electron micrographs of transverse section of sperm head. The well developed mitochondria surrounding the cytoplasmic channel in the sperm tail, as observed in control were found to be drastically disorganized in fish inhabiting the polluted lake. The study suggests that the fish C. carpio inhabiting the polluted lake Umiam is under severe stress as far as its male reproductive system is concerned. The study further suggests that Electron microscopic approach is extremely important in the assessment of adverse effects of environmental pollution on fish tissue. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.     

Effects of heavy metal pollution on pigmented macrophages in kidney of Vardar chub (Squalius vardarensis Karaman)

Pollution with heavy metals may influence the immune system of fish, leading to impairment of their health or even increase their mortality. The fish kidney is one of the first fish organs to be affected by water contamination. Amounts of kidney macrophages (MACs), which are involved in fish immune response, as well as the qualitative and quantitative changes in the pigmented MACs in fish kidney, are used as biomarkers of pollution. Therefore, in this study, we have evaluated relative and total volumes of trunk kidney pigmented MACs, and analyzed the pigments accumulated within them. Fish were sampled from two mining impacted rivers, Kriva and Zletovska, highly contaminated with heavy metals, and from one reference river, Bregalnica, in spring and autumn of 2012. We have observed that main pigments found in kidney MACs of Vardar chub were melanin and lipofuscin/ceroid, as well as that relative volumes of MACs ranged from 0.56 to 1.68%. Moreover, the results showed that relative volumes of pigmented MACs were higher in metal contaminated rivers, especially in autumn season in the Zletovska River, concurrently with extremely high metal exposure. In addition, condition factors and kidney somatic indices were found significantly lower in the Zletovska River in both seasons, autumn and spring, possibly also as a consequence of high water pollution. Our data confirm that increase in relative volumes of pigmented MACs may serve as warning sign of potential heavy metal pollution in aquatic environment.     

Ultrastructure and distribution of superficial neuromasts of blind cavefish,Phreatichthys andruzzii,juveniles

Transmission and scanning electron microscopy (TEM, SEM) were used to study the ultrastructure of superficial neuromasts in 15 six-month old blind cavefish juveniles, Phreatichthys andruzzii (Cyprinidae). In five specimens examined with SEM, the number of superficial neuromasts over the fish body (480–538) was recorded. They were localized mainly on the head (362–410), including the dorsal surface, the mentomandibular region, and laterally from the mouth to the posterior edge of the operculum. Neuromasts were also present laterally on the trunk and near the caudal fin (116–140). A significantly higher number of neuromasts were present on the head compared to the trunk (t-test, P < 0.05). Superficial neuromasts of the head and those along the trunk were similar in ultrastructure. Each neuromast comprised sensory hair cells surrounded by nonsensory support cells (mantle cells and supporting basal cells) with the whole covered by a cupula. Each hair cell was pear-shaped, 15–21 μm high and 4–6 μm in diameter, with a single long kinocilium and several short stereocilia. Most support cells were elongated, with nuclei occupying a large portion of the cytoplasm. In the margin of the neuromast, mantle cells were particularly narrow. Both types of support cells had well-developed Golgi apparatus and rough endoplasmic reticulum. The number of hair cells and nonsensory support cells of the anterior lateral line (head) did not differ significantly from those of the posterior lateral line (trunk) (t-test, P > 0.05). Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

Characterization of Albanian water frog,Pelophylax shqipericus,sperm traits and morphology,by using phase contrast microscopy

Mature spermatozoa traits and morphology of endangered Albanian water frog, Pelophylax shqipericus, have been characterized for the first time through phase contrast microscopy, as part of successful implementation of in vitro fertilization technique for this species. The basic morphology of P. shqipericus spermatozoa consists of an elongated, thick, smooth‐edged, and solid‐staining head, continuing with a thin and long tail which usually extends 2.48 times the head length. The acrosome was not clearly discernible so the measurements were done on the head as a whole, while the middle section was better visible. Average length of head, including the acrosome and midsection was estimated to be 11.78 μm ± 0.32, while the tail length resulted 29.24 ± 1.75 μm. The average thickness of the head was shown to be 3.45 μm. The total sperm length resulted to be 41.02 ± 1.83 μm. The average sperm concentration was estimated of 25.5 × 10⁶/ml. Sperm amount, survival rate and motility were also measured. The sperm survival rate was maximal immediately after preparation of the suspension and tended to decrease over time of storage, reaching 50% after 72 hr. Decreased sperm motility seemed to follow the same trend as sperm viability. Sperm traits resulted to be very similar both in size and in shape with those of “Lessonae” frog group, one of the lineages of Western Palearctic species complex, suggesting a strong phylogenetic relationship among these species.     

基于RecurDyn的带式输送机起动阶段的动态分析

应用动力学分析软件RecurDyn建立了带式输送机的样机模型,通过仿真研究,得出在起动初始阶段,尾部滚筒速度要滞后于头部滚筒速度一定时间,这主要是由于弹性波在输送带中进行传播到达尾部需要一定的时间;并且在起动阶段,输送带的张力比稳定运行时变化大,起动时间越长,输送带出现的最大张力越小。     

基于伴随方法的高速列车头型气动优化

为改善高速列车明线运行时的气动性能,基于伴随方法和径向基函数网格变形技术,开展高速列车头型气动优化设计。采用径向基函数网格变形技术,避免列车头型优化过程中的网格重复生成,提高头型优化的效率。通过伴随方法求解目标函数对列车头型的敏感度,无须定义任何的头型设计变量,避免人为指定设计变量对优化结果的影响。将网格变形技术、伴随方法及计算流体动力学（Computational fluid dynamic,CFD）方法相结合,构建高速列车头型优化设计流程,选取整车气动阻力和尾车气动升力为优化目标,对高速列车头型进行多目标气动优化设计。结果表明：伴随方法可以有效地应用于高速列车的头型优化;优化后,在满足约束条件的情况下,列车的整车气动阻力减小2.83%,尾车气动升力减小25.86%;气动阻力减小主要位于头尾车流线型部位,中间车和头尾车车体气动阻力基本保持不变;尾车气动升力减小主要位于流线型部位,尾车车体向下的升力绝对值也有所减小。     

Melanization stimulating factor (MSF) and melanization inhibiting factor (MIF) in the integument of fish

The present studies were directed to demonstrate that adult fish skin contains putative factors that affect chromatophore and/or chromatoblast function. This hypothesis is based upon the possibility that hypo and/or hyperpigmented areas of the skin are so pigmented because of the localized expression of intrinsic factors that are either stimulatory or inhibitory to the differentiation of specific pigment cell types. In all the morphological and biochemical experiments carried out, we used culture media conditioned by dorsal (DCM) or ventral (VCM) skin from different species of fish. Both DCM and VCM were capable of stimulating differentiation of melanophores in neural crest explants. While the stimulation of melanization is an activity present in both dorsal and ventral skin, an inhibitory activity is also present in ventral skin at such a concentration that it overrides the stimulatory activity afforded by DCM. With biochemical assays, we demonstrated that the three important sequential enzymatic steps in melanogenesis are all stimulated by the conditioned media in a dose-dependent manner and this results in an increase in the amount of melanin present in cultured cells. The results of our investigations provide strong evidence that there are intrinsic pigment cell regulatory factors in the integument of fish, the inhibitory activity being stronger in the ventrum, and that those factors strongly influence, perhaps even determine, the pigment patterns of fish.     

Protein detection in spermatids and spermatozoa of the butterfly Euptoieta hegesia (Lepidoptera).

This study was undertaken to detect protein components in both sperm types of the butterfly Euptoieta hegesia. These spermatozoa possess complex extracellular structures for which the composition and functional significance are still unclear. In the apyrene sperm head, the proteic cap presented an external ring and an internal dense content; basic proteins were detected only in external portions. In the tail, the paracrystalline core of mitochondrial derivatives and the axoneme are rich in proteins. The extratesticular spermatozoa are covered by a proteic coat, which presented two distinct layers. In eupyrene spermatozoa, acrosome and nucleus were negatively stained, probably because of their high compaction. In the tail, there is no paracrystalline core and the axoneme presented a very specific reaction for basic proteins. The lacinate and reticular appendages are composed of cylindrical sub-units and presented a light reaction to E-PTA and a strong reaction to tannic acid. A complex proteic coat also covers the extratesticular spermatozoa. We found similarities between both extratesticular coats, indicating a possible common origin. Both spermatozoon types are rich in proteins, especially the eupyrene appendages and the extratesticular coats. We believe that both coats are related to the sperm maturation and capacitation processes.     

Comparative repeatability of guide-pin axis positioning in computer-assisted and manual femoral head resurfacing arthroplasty

The orientation of the femoral component in hip resurfacing arthroplasty affects the likelihood of loosening and fracture. Computer-assisted surgery has been shown to improve significantly the surgeon's ability to achieve a desired position and orientation; nevertheless, both bias and variability in positioning remain and can potentially be improved. The authors recently developed a computer-assisted surgical (CAS) technique to guide the placement of the pin used in femoral head resurfacing arthroplasty and showed that it produced significantly less variation than a typical manual technique in varus/valgus placement relative to a preoperatively determined surgical plan while taking a comparable amount of time. In the present study, the repeatability of both the CAS and manual techniques is evaluated in order to estimate the relative contributions to overall variability of surgical technique (CAS versus manual), surgeon experience (novice versus experienced), and other sources of variability (e.g. across specimens and across surgeons). This will enable further improvements in the accuracy of CAS techniques. Three residents/fellows new to femoral head resurfacing and three experienced hip arthroplasty surgeons performed 20-30 repetitions of each of the CAS and manual techniques on at least one of four cadaveric femur specimens. The CAS system had markedly better repeatability (1.2 degrees) in varus/valgus placement relative to the manual technique (2.8 degrees), slightly worse repeatability in version (4.4 degrees versus 3.2 degrees), markedly better repeatability in mid-neck placement (0.7 mm versus 2.5 mm), no significant dependence on surgeon skill level (in contrast to the manual technique), and took significantly less time (50 s versus 123 s). Proposed improvements to the version measurement process showed potential for reducing the standard deviation by almost two thirds. This study supports the use of CAS for femoral head resurfacing as it is quicker than the manual technique, independent of surgeon experience, and demonstrates improved repeatability.     

车间纵向减振器失效对列车动力学性能影响

为了研究车间纵向减振器失效对动车组动力学性能的影响,基于SIMPACK建立某高速动车组动力学模型;分析车间纵向减振器在失效工况下对整车动力学性能影响,有助于解决高速动车组车间纵向减振器异响及异常问题。研究结果表明:车间纵向减振器失效对稳定性影响不大,对垂向平稳性影响也较小,横向平稳性和舒适性有明显恶化;中间车横向平稳性和舒适性恶化情况比两端车严重,两端车横向平稳性指标最大增加率为5.4%,舒适性指标最大增加率约25%,中间车横向平稳性指标最大增加率约10%,舒适性指标最大增加率约45%;轮轴横向力、轮轨垂向力、脱轨系数及轮重减载率等安全性指标受其影响较小。     

Ultrastructural neurobiology of the olfactory mucosa of the brown trout, Salmo trutta.

This paper describes four investigations of the olfactory mucosa of the brown trout: 1) the ultrastructure of the olfactory mucosa as revealed by scanning (SEM), conventional transmission (TEM), and high voltage (HVEM) electron microscopy; 2) light and electron-microscopic investigations of retrograde transport of the tracer macromolecule horseradish peroxidase (HRP) when applied to the cut olfactory nerve; 3) SEM and TEM investigations of the effects of olfactory nerve transection on cell populations within the olfactory epithelium; and 4) ultrastructural investigations of reversible degeneration of olfactory receptors caused by elevated copper concentrations. The trout olfactory epithelium contains five cell types: ciliated epithelial cells, ciliated olfactory receptor cells, microvillar olfactory receptor cells, supporting cells, and basal cells. The ciliated and microvillar olfactory receptor cells and a small number of basal cells are backfilled by HRP when the tracer is applied to the cut olfactory nerve. When the olfactory nerve is cut, both ciliated and microvillar olfactory receptor cells degenerate within 2 days and are morphologically intact again within 8 days. When wild trout are taken from their native stream and placed in tanks with elevated copper concentrations, ciliated and microvillar cells degenerate. Replacement of these trout into their stream of origin is followed by morphologic restoration of both types of olfactory receptor cells. Ciliated and microvillar receptor cells are primary sensory bipolar neurons whose dendrites make contact with the environment; their axons travel directly to the brain. Consequently, substances can be transported directly from the environment into the brain via these "naked neurons." Since fish cannot escape from the water in which they swim, and since that water may occasionally contain brain-toxic substances, the ability to close off--and later reopen--this anatomic gateway to the brain would confer a tremendous selective advantage upon animals that evolved the "brain-sparing" capacity to do so. Consequently, the unique regenerative powers of vertebrate olfactory receptor neurons may have their evolutionary origin in fishes.     

Ovary of Matsucoccus pini (Insecta,Hemiptera, Coccinea: Matsucoccidae): Morphology,ultrastructure, and phylogenetic implications

The structure of ovary in a representative of the scale insect family Matsucoccidae, Matsucoccus pini, is described at the ultrastructural level. The paired ovaries of M. pini are composed of about 50 ovarioles of telotrophic type that develop asynchronously. An individual ovariole consists of an anterior tropharium (trophic chamber) and posterior vitellarium. The tropharium encloses trophocytes (nurse cells) and early previtellogenic oocytes termed arrested oocytes. In the vitellarium from 1 to 6, linearly arranged oocytes may develop. Analysis of serial sections has shown that each ovariole contains 32 germ cells (trophocytes, arrested oocytes, and developing oocytes). In the cytoplasm of all these cells, small rod‐shaped bacteria are present. In the early vitellogenic oocytes, accessory nuclei arise. As vitellogenesis progresses, these nuclei migrate toward the cortical ooplasm. The obtained results are discussed in a phylogenetic context. Microsc. Res. Tech. 77:327–334, 2014. © 2014 Wiley Periodicals, Inc.     

Microscopy of the Drosophila facet eye: vademecum for standardized fixation, embedding, and sectioning

We describe here a standardized method for histological processing of the Drosophila compound eye. Primary fixation with 2.5% glutaraldehyde, obligatorily supplemented with 0.1% household detergent regularly yielded the best structural preservation, as compared with that of other, more complicated fixation protocols tested. Notably, it proved indispensable not only to cut off the fly's head to facilitate the penetration of the reagents but also to open the chitinous head capsule. For this, we locally pierced the cuticle between the eyes, leaving the head structurally almost intact, a prerequisite for precisely aligning the head for microtomy. We developed a two-step re-embedding procedure allowing for exact and reproducible orientation of the fly heads. Thus, highly comparable series of cross sections through a representative number of ommatidia were obtained. The feasibility of our embedding and sectioning approach is finally demonstrated by three-dimensional reconstructions of the middle segments of the R1, R7, and R8 photoreceptor cells. We present reconstructions from structurally modified ommatidia, as seen after RNAi-mediated depletion of the endosomal adaptor protein p14, and from normal ommatidia corresponding to the wildtype.     

A study on the influence of the seat and head restraint foam stiffnesses on neck injury in low speed offset rear impacts

Soft tissue neck injury (STNI) is thought to be related to automotive seat characteristics, and it is important to know how the stiffness of the seatback and head restraint foam affect the occupant’s motions in low speed rear impacts. The combined MADYMO(MAthematical DYnamic MOdel, TNO) 50th percentile facet male human body model with a detailed neck model was selected for this analysis. In order to determine the relationship between the occupant’s motions and seatback and head restraint foam stiffnesses, 40% offset rear impact simulations were performed by changing the stiffness of seatback and head restraint. Simulated results show that the maximum acceleration of head and trunk would tend to increase with those stiffnesses. Increased stiffness of the seatback and head restraint resulted in increased ligament and disc forces. On the other hand a proper combination of seatback and head restraint stiffness can reduce a possible neck injury. It is clear that an appropriate combination of stiffnesses between seatback and head restraint allows to minimize a neck injury.     

热轧带钢尾部鱼尾形状与控制研究

采用刚塑性有限元法建立立轧／平轧三维热应力耦合有限元模型，并对宝钢2050热轧带钢粗轧机组轧制过程进行了全过程模拟。模拟所得的带钢尾部形状与现场图片吻合良好，有效地验证了计算模型。通过有限元仿真计算，研究带钢尾部鱼尾形状形成机理，得到了粗轧各道次轧后带钢尾部鱼尾切损的分布规律。提出了立轧／平轧负张力控制轧制带钢尾部的方法，通过计算表明该方法能够有效减少带钢尾部鱼尾切损量。建立了E】机架新的短行程控制模型，通过对比分析可知新模型比原模型能更好地改善尾部形状。     

柔性机器鱼的仿生运动拟合控制研究

基于一种新型弹性钢片尾部的仿生机器鱼机构,结合鱼类的运动学模型以及弹性钢片受力变形特性,得出机器鱼尾部的运动控制模型。通过实验验证了该运动控制模型能逼真地模仿鱼类的游动姿态。