Volume X-Ray Micro-Computed Tomography Analysis of the Early Cephalized Central Nervous System in a Marine Flatworm, Stylochoplana pusilla.

Platyhelminthes are a phylum of simple bilaterian invertebrates with prototypic body systems. Compared with non-bilaterians such as cnidarians, the bilaterians are likely to exhibit integrated free-moving behaviors, which require a concentrated nervous system "brain" rather than the distributed nervous system of radiatans. Marine flatworms have an early cephalized 'central' nervous system compared not only with non-bilaterians but also with parasitic flatworms or freshwater planarians. In this study, we used the marine flatworm Stylochoplana pusilla as an excellent model organism in Platyhelminthes because of the early cephalized central nervous system. Here, we investigated the three-dimensional structures of the flatworm central nervous system by the use of X-ray micro-computed tomography (micro-CT) in a synchrotron radiation facility. We found that the obtained tomographic images were sufficient to discriminate some characteristic structures of the nervous system, including nerve cords around the cephalic ganglion, mushroom body-like structures, and putative optic nerves forming an optic commissure-like structure. Through the micro-CT imaging, we could obtain undistorted serial section images, permitting us to visualize precise spatial relationships of neuronal subpopulations and nerve tracts. 3-D micro-CT is very effective in the volume analysis of the nervous system at the cellular level; the methodology is straightforward and could be applied to many other non-model organisms.

Hydrops fetalis caused by a complex congenital heart defect with concurrent hypoplasia of pulmonary blood vessels and lungs visualized by micro-CT in a French Bulldog.

BACKGROUND: Hydrops fetalis (HF) is fluid accumulation in fetus body cavities and subcutaneous tissue. The condition has been described in various farm and companion animal species, including dogs. Most of cases result from a heart defect. Exact nature of this defect is rarely clarified. CASE PRESENTATION: A newborn, male French bulldog puppy with severe HF underwent a full anatomopathological examination to diagnose the primary cause of HF. Based on the anatomopathological examination, fetal ultrasound, and micro-computed tomography, transposition of the great arteries with hypoplasia of the ascending aorta, aortic arch interruption, ostium secundum atrial septal defect, severe tricuspid valve dysplasia, as well as hypoplasia of pulmonary vessels and lungs were diagnosed. CONCLUSIONS: This is the first report of HF caused by severe, complex congenital heart defects with concurrent pulmonary vessel and lung hypoplasia.

Different methionine to cysteine supplementation ratios altered bone quality of broilers with or without Eimeria challenge assessed by dual energy X-ray absorptiometry and microtomography.

Despite the acknowledged significance of nutrition in bone development, effects of methionine (Met) and cysteine (Cys) on bone quality remain under-researched, particularly during Eimeria challenge. We investigated the effects of different supplemental Met to Cys ratios (MCR) on bone quality of broilers under Eimeria challenge. A total of 720 fourteen-day old Cobb500 broilers were allocated into a 5 × 2 factorial arrangement. Five diets with Met and Cys supplemented at MCR of 100:0, 75:25, 50:50, 25:75, and 0:100 were fed to the birds with or without Eimeria challenge. Body composition was measured by dual energy x-ray absorptiometry, and the femur bone characteristics were assessed by microtomography. Data were analyzed by two-way ANOVA and orthogonal polynomial contrast. The results reaffirmed the detrimental effects of Eimeria challenge on bone quality. On 9 d post inoculation (DPI), significant interaction effects were found for whole body bone mineral content (BMC), lean tissue weight, and body weight (P < 0.05); in the nonchallenged group (NCG), these parameters linearly decreased as MCR decreased (P < 0.05). In the challenged group (CG), body weight and lean tissue weight were unaffected by MCR, and BMC linearly increased as MCR decreased (P < 0.05). For the cortical bone of femoral metaphysis on 6 DPI, bone mineral density (BMD) linearly increased as MCR decreased (P < 0.05). Bone volume to tissue volume ratio (BV/TV) in the CG linearly increased as MCR decreased (P < 0.05). On 9 DPI, BMC and TV linearly increased as MCR decreased (P < 0.05) in the NCG. BMD and BV/TV changed quadratically as MCR decreased (P < 0.05). For the trabecular bone of femoral metaphysis on 9 DPI, BV/TV, and trabecular number linearly increased as MCR decreased (P < 0.05) in the NCG. For the femoral diaphysis, BV, TV, BMC on 6 DPI, and BMD on 9 DPI linearly increased as MCR decreased (P < 0.05). In conclusion, this study showed that both Eimeria challenge and varying supplemental MCR could influence bone quality of broilers.

Detection of nephrocalcinosis using ultrasonography, micro-computed tomography, and histopathology in cats.

BACKGROUND: Identification of nephrocalcinosis in cats with chronic kidney disease (CKD) is of clinical interest but the ability of ultrasonography to detect nephrocalcinosis is uncertain. OBJECTIVES: To compare ultrasonography, micro-computed tomography (µCT) and histopathology for identification of nephrocalcinosis. ANIMALS: Twelve kidneys from 7 euthyroid client-owned cats with CKD. METHODS: Descriptive study. Renal ultrasonography was performed ante-mortem for nephrocalcinosis detection. Kidneys were grouped based on nephrocalcinosis: present, suspected, or absent. When cats died, necropsy was performed. Renal tissue was evaluated using µCT for macroscopic nephrocalcinosis, and nephrocalcinosis volume-to-kidney tissue ratio (macro-VN:KT) and sagittal nephrocalcinosis area-to-kidney tissue ratio (macro-AN:KT) were calculated. Each kidney subsequently was bisected longitudinally, formalin-fixed, and paraffin-embedded for microscopic nephrocalcinosis assessment using von Kossa and Alizarin red staining with AN:KT (VK-micro-AN:KT and AR-micro-AN:KT) quantified using ImageJ. Data are presented as median (range). Relationships between macroscopic and microscopic AN:KT were assessed using Spearman's correlation. RESULTS: Nephrocalcinosis by ultrasonography was considered to be absent in 3, suspected in 3, and present in 5 kidneys; 1 kidney had nephrolithiasis with nephrocalcinosis. The macro-VN:KT was 0.001%, 0.001%, and 0.019%, and the macro-AN:KT was 0.08%, 0.30%, and 1.47%, respectively. Histologically, VK-micro-AN:KT was 0.21%, 2.85%, and 4.56%, and AR-micro-AN:KT was 1.73%, 5.82%, and 8.90% for kidneys where ultrasonographic macro-nephrocalcinosis was absent, suspected, or present, respectively. A strong correlation was identified between macroscopic (macro-AN:KT) and microscopic (VK-micro-AN:KT) nephrocalcinosis (rs = 0.76; P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Ultrasonographically diagnosed nephrocalcinosis correlates well with macroscopic and microscopic nephrocalcinosis at necropsy despite their separation in time.

Microcomputed tomographic analysis of the ulnar trochlear notch in medium- and large-breed canine cadavers with and without medial coronoid disease.

Medial coronoid disease (MCD) is a common disease often associated with thoracic limb lameness in medium- and large-breed dogs. The term MCD includes subchondral changes of the medial coronoid process (MCP), as well as pathologies of both cartilaginous surfaces. As there are only a few comprehensive and detailed studies on the trabecular structure of the medial coronoid, the goal of this study was to compare the trabecular structure of the ulnar trochlear notch of canine cadavers with and without MCD using different micro-computed tomographic (micro-CT) parameters. Fifty-eight elbow joints from 29 canine cadavers of MCD-predisposed and non-predisposed breeds (control group) were examined radiographically, macroscopically and by microcomputed tomography. The study included elbow joints of eight Labrador Retrievers (21.6-37 kg), seven Golden Retrievers (26.3-42 kg), seven Bernese Mountain dogs (31-47 kg) and seven dogs of non-predisposed breeds (19.7-52 kg) (control group). The final diagnosis of MCD was based on necropsy and micro-computed tomographic examinations. Micro-CT examinations were performed using XtremeCT II (Scanco Medical, Zurich, Switzerland) and the following parameters were examined: bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), connectivity density (Conn.D) and degree of anisotropy (DA). Twenty-four elbows of 44 elbows of the predisposed breeds (Labrador Retrievers, Golden Retrievers Bernese Mountain dogs) showed subchondral changes and lesions of the cartilage surfaces. The result of this study is a higher density (BV/TV) of the trabecular bone of the ulnar trochlear notch in elbows affected by MCD compared to the control group. The increased density due to trabecular reconstruction in the ulnar trochlear notch is likely the result of selectively increased loading during life.

Research Note: Three-dimensional microstructural basis for chicken eggshell translucency.

Eggshell translucency, an externally observable feature, can influence consumer purchase intentions. The eggshell quality often deteriorates in aging laying hens, leading to increased eggshell translucency. Eggs exchange water vapor through the pores. It is commonly believed that the moisture remaining in the mastoid space contributes to translucent egg formation. However, the precise mechanisms underlying eggshell translucency require further structural investigations. This study aimed to explore the 3-dimensional microstructure of eggshells using micro-computed tomography (micro-CT) to provide insights into the microstructural basis of eggshell translucency. The results revealed that the ratio of pore volume to mastoid space volume was significantly higher in the high-translucency (TH) group than that in the low-translucency (TL) group (P < 0.01); however, the ratio of mastoid space to eggshell volume showed no significant difference. Additionally, measurements of the individual pore morphological features showed that the roundness of the pores in the TH group was significantly higher than that in the TL group, whereas the flatness of the pores in the TH group was significantly lower (P = 0.01). Eggshell porosity directly reflects the water vapor exchange capacity of eggshells, with the TL group exhibiting significantly higher eggshell porosity compared to the TH group (P = 0.02). In conclusion, based on observations of the 3-dimensional microstructure of eggshells, this study elucidated how the volume and morphological characteristics of pores influence the water vapor exchange capacity of eggshells, resulting in eggshell translucency.

Grey parrot (Psittacus erithacus) beak papillae and nerves identified using novel 2-D and 3-D imaging modalities.

OBJECTIVE: The avian beak is a complex organ containing bone, neurovascular tissue, and keratinized covering (rhamphotheca). Nerve-rich papillae extend through bone into rhamphotheca providing sensory input from the beak tip. Beak trimming is a common procedure in avian species and is used for corrective, cosmetic, and behavioral modification purposes. Yet, practitioners are not well versed in complete beak anatomy, and therefore, beak trimming often disregards neurovasculature, injuring the patient and hampering recovery. Here, using comprehensive anatomical description, we aim to provide recommendations on how to safely perform beak trimming without damaging underlying sensory papillae. ANIMALS: Here, we evaluated beaks of 2 deceased grey parrots (Psittacus erithacus). PROCEDURES: In one, we used a novel stain and microcomputed tomography to visualize papillae in the upper and lower beaks. In a second, we hand isolated the upper and lower beak dermal papillae and used high-resolution photography plus traditional paraffin histology. RESULTS: Papillae and their nerves were easily identified in these 2- and 3-dimensional approaches. This allowed us to determine the approximate lengths of papillae within the upper and lower beak. CLINICAL RELEVANCE: Based on these findings, the authors recommend lateral radiographs of the bird's head and beak to identify the location of the underlying bone relative to the overlying rhamphotheca before performing beak trims. Specifically in grey parrots, the authors recommend the upper and lower beak should not be trimmed closer than 8 to 10 mm from the underlying bone. Further work is needed to support these recommendations and provide guidelines for other species.

Histomorphometry and µCT scan analysis of osteoporosis in spayed female dogs.

Background: Both humans and small animals suffer from similar metabolic and structural diseases that impact the musculoskeletal system; however, instead of studying animal disease in its own right, animals are more often used as models for research into various human ailments, such as osteoporosis. There are few studies indicating that animals may suffer from osteoporosis, which raises the question of why small animals, which we believe to be equally susceptible, receive so little attention. With this research, we hope to draw the attention of researchers to the fact that the examination of animals for this disease is just as important as the examination of humans; human osteoporosis research receives a great deal of attention, while animals and their health are neglected. Aims: We aimed to analyze the bone volume fraction (BV/TV) and thickness of first (L1) and second (L2) lumbar vertebrae samples from five cadavers using histomorphometric analysis. In addition, we aimed to investigate one cadaver using microcomputed tomography (µCT) imaging. Methods: The L1 and L2 vertebrae from five dog carcasses were used to evaluate the BV/TV and the trabecular thicknesses. We used precise sampling criteria, and also developed a methodological approach to the study of the vertebrae. Using semi-automated methods, we performed histomorphometric analysis and µCT data analysis. Results: We used five dog cadavers in this research. During the histomorphometry study, we observed that the lowest L1 BV/TV ratio was 7.88% and the highest was 23.08%. The L2 vertebrae BV/TV ranged from 11.58% to 23.7%. The L1 and L2 lumbar trabeculae thicknesses were also measured. L1's smallest trabecula was 17.34 microns and its largest was 31.88. The L2 vertebrae trabecula thickness was 18.76-30.75 microns. BV/TV and trabecular thickness were positively correlated (and vice versa). The two-tailed p value was less than 0.00001. This difference is statistically significant. After µCT analysis, we discovered regions in the vertebral body with low porosity; these cavities are usually filled with connective tissue. The bone tissue in these areas is more vulnerable, meaning fracture risk has increased. Conclusions: Animals should not just be considered as models for osteoporosis in humans, but also as potential patients. A single test, such as histomorphometry, may not be sufficient; more advanced technology, such as µCT, is required, since it reveals the pores that make the vertebral column more brittle and susceptible to fracture.

Morphology and volume of Meibomian glands ex vivo pre and post partial tarsal plate excision, cryotherapy and laser therapy in the dog using microCT.

OBJECTIVE: To determine the morphology and volume of Meibomian glands (MG) of dogs with microCT before and after partial tarsal plate excision (PTPE), cryotherapy, and laser therapy. PROCEDURE: MicroCT scans were made of 12 upper lids (ULs) and lower lids (LLs) of 12 dogs. After undergoing PTPE, 10 ULs and LLs were scanned again, and one UL and one LL was scanned after laser therapy and one UL and one LL after cryotherapy. RESULTS: The length of the area containing MGs did not change pre- and post-PTPE, and cryo- or laser therapy. The mean number of MGs in the ULs and LLs was 30.50 and 29.42, respectively, and did not change during the procedures. The average length of one individual MG was 2.60 mm. The mean volume of MGs in the 10 ULs and LLs pre-PTPE was 21.45 and 17.2 mm3 , respectively, and 12.84 and 11.25 mm3 in the UL and LL after PTPE, respectively. The mean volume of MGs decreased from 29.78 mm3 precryotherapy to 28.91 mm3 post-treatment and in the lower eyelid from 22.87 to 22.4 mm3 after cryotherapy. The mean volume of MGs in the UL and LL before laser therapy was 8.95 and 6.78 mm3 , respectively, and after 9.25 and 6.38 mm3 , respectively. CONCLUSION: MicroCT is a valuable tool to determine the morphology and the volume of MGs and to demonstrate changes that occur after PTPE, laser-, and cryotherapy. There is no need for additional preparation, such as staining, of the specimen prior to scanning.

Dual-contrast micro-CT enables cartilage lesion detection and tissue condition evaluation ex vivo.

BACKGROUND: Post-traumatic osteoarthritis is a frequent joint disease in the horse. Currently, equine medicine lacks effective methods to diagnose the severity of chondral defects after an injury. OBJECTIVES: To investigate the capability of dual-contrast-enhanced computed tomography (dual-CECT) for detection of chondral lesions and evaluation of the severity of articular cartilage degeneration in the equine carpus ex vivo. STUDY DESIGN: Pre-clinical experimental study. METHODS: In nine Shetland ponies, blunt and sharp grooves were randomly created (in vivo) in the cartilage of radiocarpal and middle carpal joints. The contralateral joint served as control. The ponies were subjected to an 8-week exercise protocol and euthanised 39 weeks after surgery. CECT scanning (ex vivo) of the joints was performed using a micro-CT scanner 1 hour after an intra-articular injection of a dual-contrast agent. The dual-contrast agent consisted of ioxaglate (negatively charged, q = -1) and bismuth nanoparticles (BiNPs, q = 0, diameter ≈ 0.2 µm). CECT results were compared to histological cartilage proteoglycan content maps acquired using digital densitometry. RESULTS: BiNPs enabled prolonged visual detection of both groove types as they are too large to diffuse into the cartilage. Furthermore, proportional ioxaglate diffusion inside the tissue allowed differentiation between the lesion and ungrooved articular cartilage (3 mm from the lesion and contralateral joint). The mean ioxaglate partition in the lesion was 19 percentage points higher (P < 0.001) when compared with the contralateral joint. The digital densitometry and the dual-contrast CECT findings showed good subjective visual agreement. MAIN LIMITATIONS: Ex vivo study protocol and a low number of investigated joints. CONCLUSIONS: The dual-CECT methodology, used in this study for the first time to image whole equine joints, is capable of effective lesion detection and simultaneous evaluation of the condition of the articular cartilage.

An optimized workflow for microCT imaging of formalin-fixed and paraffin-embedded (FFPE) early equine embryos.

Here, we describe a workflow for high-detail microCT imaging of formalin-fixed and paraffin-embedded (FFPE) equine embryos recovered on Day 34 of pregnancy (E34), a period just before placenta formation. The presented imaging methods are suitable for large animals' embryos with intention to study morphological and developmental aspects, but more generally can be adopted for all kinds of FFPE tissue specimens. Microscopic 3D imaging techniques such as microCT are important tools for detecting and studying normal embryogenesis and developmental disorders. To date, microCT imaging of vertebrate embryos was mostly done on embryos that have been stained with an X-ray dense contrast agent. Here, we describe an alternative imaging procedure that allows to visualize embryo morphology and organ development in unstained FFPE embryos. Two aspects are critical for high-quality data acquisition: (i) a proper sample mounting leaving as little as possible paraffin around the sample and (ii) an image filtering pipeline that improves signal-to-noise ratio in these inherently low-contrast data sets. The presented workflow allows overview imaging of the whole embryo proper and can be used for determination of organ volumes and development. Furthermore, we show that high-resolution interior tomographies can provide virtual histology information from selected regions of interest. In addition, we demonstrate that microCT scanned embryos remain intact during the scanning procedure allowing for a subsequent investigation by routine histology and/or immunohistochemistry. This makes the presented workflow applicable also to archival paraffin-embedded material.

Proximal sesamoid bone microdamage is localized to articular subchondral regions in Thoroughbred racehorses, with similar fracture toughness between fracture and controls.

OBJECTIVE: To determine whether proximal sesamoid bone (PSB) microdamage and fracture toughness differ between Thoroughbred racehorses sustaining PSB fracture and controls. STUDY DESIGN: Cadaveric case-control. ANIMALS: Twenty-four Thoroughbred racehorses (n = 12 PSB fracture, n = 12 control). METHODS: Proximal sesamoid bones were dissected, and gross pathological changes and morphological measurements were documented. High-speed exercise history data were evaluated. Microdamage was assessed in fracture, fracture-contralateral limb (FXCL) and control PSBs using whole bone lead uranyl acetate (LUA) staining with micro-CT imaging or basic fuchsin histological analysis. Fracture toughness mechanical testing was carried out in 3-point-bending of microbeams created from PSB flexor cortices. Data were analyzed using ordinal logistic and linear regression models. RESULTS: Microdamage was detected most commonly in the articular subchondral region of PSBs via LUA micro-CT and basic fuchsin histology. There were no differences in microdamage between FXCL and control PSBs. Fracture toughness values were similar for FXCL (1.31 MPa√m) and control (1.35 MPa√m) PSBs. Exercise histories were similar except that horses sustaining fracture spent a greater percentage of their careers in rest weeks. CONCLUSION: Microdamage was detected in the articular region of PSBs but was not greater in horses sustaining catastrophic PSB fracture. Fracture toughness of PSB flexor cortices did not differ between FXCL and control PSBs. CLINICAL SIGNIFICANCE: Although uncommon, microdamage is localized to the articular region of Thoroughbred racehorse PSBs. Catastrophic PSB failure is not associated with lower PSB flexor cortex fracture toughness.

Clinical insights into the three-dimensional anatomy of cheek teeth in alpacas based on micro-computed tomography. Part 1: mandibular cheek teeth.

BACKGROUND: Despite dental disease being a common health concern in alpacas, important dental pathology including apical infection, remains poorly understood. Treatment options are limited compared to veterinary dentistry techniques in other species. The primary goal of this study was to increase understanding of the external and internal anatomy of mandibular cheek teeth to enable the development of tooth sparing techniques in this species. Also, an objective evaluation of the sub-occlusal dentinal thickness in normal mandibular cheek teeth is warranted to understand the risks associated with reduction of overgrown teeth. RESULTS: Overall pulp anatomy was variably characterized by the presence of a common pulp chamber in younger teeth, and segmentation of pulp cavities into multiple separate pulp entities within the same tooth with increasing age. A common pulp chamber was identified in 55.3% (26/47) of teeth with a mean dental age of 1 year and 11 months (± 1 year and 8 months). Columnar segmentation was recorded in the remaining teeth with a mean dental age of 6 years and 5 months (± 3 years and 11 months). Age of segmentation of the common pulp chamber into multiple separate pulp entities shows wide variation and is dependent of the specific Triadan position. The present study illustrates the presence of disto-mesial root contacts between adjacent tooth roots, often leading to morphological adaptations, most frequently observed between Triadan 09-10s (80%) and 10-11s (67%). The measured sub-occlusal dentinal thickness was as low as 1.11 mm over some pulp horns. The sub-occlusal dentinal thickness was lower than 2, 3, and 4 mm in 13.1, 38.1 and 61.4% of performed measurements, respectively. CONCLUSION: This study provides detailed information on age-dependent mandibular cheek teeth anatomy in alpacas, which may support the use and development of advanced dental treatments in this species such as endodontics and tooth sectioning techniques. Apical morphological adaptations caused by disto-mesial root contact between adjacent mandibular cheek teeth are clearly illustrated. The limited amount of sub-occlusal secondary dentin warrants a cautious approach with regards to dental floating in alpacas.

Ultrastructural studies of developing egg tooth in grass snake Natrix natrix (Squamata, Serpentes) embryos, supported by X-ray microtomography analysis.

The egg tooth development is similar to the development of all the other vertebrate teeth except earliest developmental stages because the egg tooth develops directly from the oral epithelium instead of the dental lamina similarly to null generation teeth. The developing egg tooth of Natrix natrix changes its curvature differently than the egg tooth of the other investigated unidentates due to the presence of the rostral groove. The developing grass snake egg tooth comprises dental pulp and the enamel organ. The fully differentiated enamel organ consists of outer enamel epithelium, stellate reticulum, and ameloblasts in its inner layer. The enamel organ directly in contact with the oral cavity is covered with periderm instead of outer enamel epithelium. Stellate reticulum cells in the grass snake egg tooth share intercellular spaces with the basal part of ameloblasts and are responsible for their nutrition. Ameloblasts during egg tooth differentiation pass through the following stages: presecretory, secretory, and mature. The ameloblasts from the grass snake egg tooth show the same cellular changes as reported during mammalian amelogenesis but are devoid of Tomes' processes. Odontoblasts of the developing grass snake egg tooth pass through the following classes: pre-odontoblasts, secretory odontoblasts, and ageing odontoblasts. They have highly differentiated secretory apparatus and in the course of their activity accumulate lipofuscin. Grass snake odontoblasts possess processes which are poor in organelles. In developing egg tooth cilia have been identified in odontoblasts, ameloblasts and cells of the stellate reticulum. Dental pulp cells remodel collagen matrix during growth of the grass snake egg tooth. They degenerate in a way previously not described in other teeth.

Comparative microcomputed tomographic structural analysis of the trabecular and cortical bone architecture of radius and ulna in toy dog breeds.

Radius and ulna fractures can be detected in dogs with an incidence of 18.0% and occur more frequently in toy breeds with increased complication rates. The reasons for the predisposition and the increased complication rate of antebrachial fractures in toy breeds are not yet fully understood. The aim of this study was to research the associated mechanisms by investigating the trabecular structure parameters (BV/TV (%), Tb.N (mm-1), Tb.Th (mm), Tb.Sp (mm), Conn. D (mm-3), DA) and the cortical bone density (Mean Density (mg HA/ccm)) for toy breeds and small dogs of other breeds using microcomputed tomography and to identify the differences between both groups. A total of 70 forearms, originating from toy breeds (n = 36) and small dogs of other breeds (n = 34) were evaluated by microcomputed tomography. The results of this study showed a lower bone volume fraction (BV/TV), more (Tb.N) but thinner trabeculae (Tb.Th) with greater separation (Tb.Sp) and decreased diaphyseal cortical density (Mean Density) in toy breed dogs. This could thus contribute to the increased risk of antebrachial fractures in toy breeds. Within the group of toy breeds inhomogeneous results were observed, whereby the values of the Chihuahuas deviated. This breed had significantly thinner trabeculae (Tb.Th) with a greater trabecular separation (Tb.Sp), lower bone volume fraction (BV/TV) and significantly lower diaphyseal cortical density (Mean Density). These parameters could therefore indicate an increased antebrachial fracture and complication risk, especially in Chihuahuas.

Connections between the internal and the external capsules and the globus pallidus in the sheep: A dichromate stain X-ray microtomographic study.

Sheep are recognized as useful species for translational neurodegeneration research, in particular for the study of Huntington disease. There is a lack of information regarding the detailed anatomy and connections of the basal ganglia of sheep, in normal myeloarchitectonics and in tract-tracing studies. In this work, the organization of the corticostriatal projections at the level of the putamen and globus pallidus (GP) are explored. For the first time, the myeloarchitectonic pattern of connections between the internal (IC) and the external (EC) capsules with the GP have been investigated in the sheep. Formaldehyde-fixed blocks of the striatum were treated with a metallic stain containing potassium dichromate and visualized using micro-CT (µ-CT). The trivalent chromium (Cr3+), attached to myelin phospholipids, imparts a differential contrast to the grey and white matter compartments, which allows the visualization of myelinated fascicles in µ-CT images. The fascicles were classified according to their topographical location in dorsal supreme fascicles (X, Y, apex) arising from the IC and EC; pre-commissurally, basal fascicles connecting the ventral part of the EC with the lateral zone of the ventral pallidum (VP) and, post-commissurally, superior (Z1 ), middle (Z2 ) and lower (Z3 ) fascicles, connecting at different levels the EC with the GP. The results suggest that the presumptive cortical efferent and afferent fibres to the pallidum could be organized according to a dorsal to ventrolateral topography in the sheep, similar to that seen in other mammals. The proposed methodology has the potential to delineate the myeloarchitectonic patterns of nervous systems and tracts.

Synchrotron microtomography applied to the volumetric analysis of internal structures of Thoropa miliaris tadpoles.

Amphibians are models for studying applied ecological issues such as habitat loss, pollution, disease, and global climate change due to their sensitivity and vulnerability to changes in the environment. Developmental series of amphibians are informative about their biology, and X-ray based 3D reconstruction holds promise for quantifying morphological changes during growth-some with a direct impact on the possibility of an experimental investigation on several of the ecological topics listed above. However, 3D resolution and discrimination of their soft tissues have been difficult with traditional X-ray computed tomography, without time-consuming contrast staining. Tomographic data were initially performed (pre-processing and reconstruction) using the open-source software tool SYRMEP Tomo Project. Data processing and analysis of the reconstructed tomography volumes were conducted using the segmentation semi-automatic settings of the software Avizo Fire 8, which provide information about each investigated tissues, organs or bone elements. Hence, volumetric analyses were carried out to quantify the development of structures in different tadpole developmental stages. Our work shows that synchrotron X-ray microtomography using phase-contrast mode resolves the edges of the internal tissues (as well as overall tadpole morphology), facilitating the segmentation of the investigated tissues. Reconstruction algorithms and segmentation software played an important role in the qualitative and quantitative analysis of each target structure of the Thoropa miliaris tadpole at different stages of development, providing information on volume, shape and length. The use of the synchrotron X-ray microtomography setup of the SYRMEP beamline of Elettra Synchrotron, in phase-contrast mode, allows access to volumetric data for bone formation, eye development, nervous system and notochordal changes during the development (ontogeny) of tadpoles of a cycloramphid frog Thoropa miliaris. As key elements in the normal development of these and any other frog tadpole, the application of such a comparative ontogenetic study, may hold interest to researchers in experimental and environmental disciplines.

Internal morphological changes during metamorphosis in the sheep nasal bot fly, Oestrus ovis.

During the larval stage, oestrid flies (Diptera: Oestridae) are obligate parasites, whereas during the adult stage they are free-living and do not feed. Like other cyclorrhaphous flies, oestrids undergo metamorphosis inside an opaque puparium, formed by the contracted and hardened cuticle of the third-instar larva. The present study documents the internal morphological changes taking place during metamorphosis of the sheep nasal bot fly, Oestrus ovis L., using non-invasive, micro-CT-based virtual histology and provides quantitative data of volumetric changes in specific organs. Virtual histological sections allowed visualisation of the progression and completion of the apolyses, which delimit the different intra-puparial stages, and the connection to the tracheal system of a large gas bubble, which plays an essential role during early metamorphosis. Overall, our results show that the sequence of morphological and volumetric changes in tissues and organs is similar to those found in other cyclorrhaphous flies, but they also reveal developmental differences that result in an adult vestigial digestive tract. Future studies could develop non-invasive, reliable methods for aging the intra-puparial forms of different oestrid species of veterinary importance, based on both qualitative and quantitative markers, thus improving our knowledge of their development and the efficiency of control strategies.

A salamander that chews using complex, three-dimensional mandible movements.

Most non-mammal tetrapods have a hinge-like jaw operation restricted to vertical opening and closing movements. Many mammal jaw joints, by contrast, operate in more complex, three-dimensional (3D) ways, involving not only vertical but also propalinal (rostro-caudal) and transverse (lateral) movements. Data on intraoral food processing in lissamphibians and sauropsids has prompted a generally accepted view that these groups mostly swallow food unreduced, and that in those cases where lissamphibians and sauropsids chew, they mostly use simple vertical jaw movements for food processing. The exception to this generally accepted view is the occurrence of some propalinal chewing in sauropsids. We combined 3D kinematics and morphological analyses from biplanar high-speed video fluoroscopy and micro-computed tomography to determine how the paedomorphic salamander Siren intermedia treats captured food. We discovered not only that S. intermedia uses intraoral food processing but also that the elaborated morphology of its jaw joint facilitates mandibular motions in all three planes, resulting in complex 3D chewing. Thus, our data challenge the commonly held view that complex 3D chewing movements are exclusive to mammals, by suggesting that such mechanisms might have evolved early in the tetrapod evolution.

A Novel Method for Assessing Enamel Thickness Distribution in the Anterior Dentition as a Signal for Gouging and Other Extractive Foraging Behaviors in Gummivorous Mammals.

Gummivory poses unique challenges to the dentition as gum acquisition may often require that the anterior teeth be adapted to retain a sharp edge and to resist loading because they sometimes must penetrate a highly obdurate substrate during gum extraction by means of gouging or scraping. It has been observed previously that the enamel on the labial surface of the teeth used for extraction is thicker relative to that on the lingual surface in taxa that extract gums, while enamel is more evenly distributed in the anterior teeth of taxa that do not regularly engage in extractive behaviors. This study presents a quantitative methodology for measuring the distribution of labial versus lingual enamel thickness among primate and marsupial taxa in the context of gummivory. Computed microtomography scans of 15 specimens representing 14 taxa were analyzed. Ten measurements were taken at 20% intervals starting from the base of the crown of the extractive tooth to the tip of the cutting edge across the lingual and labial enamel. A method for including worn or broken teeth is also presented. Mann-Whitney U tests, canonical variates analysis, and between-group principal components analysis were used to examine variation in enamel thickness across taxa. Our results suggest that the differential distribution of enamel thickness in the anterior dentition can serve as a signal for gouging behavior; this methodology distinguishes between gougers, scrapers, and nonextractive gummivores. Gouging taxa are characterized by significantly thicker labial enamel relative to the lingual enamel, particularly towards the crown tip. Examination of enamel thickness patterning in these taxa permits a better understanding of the adaptations for the extraction of gums in extant taxa and offers the potential to test hypotheses concerning the dietary adaptations of fossil taxa.