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Topic:Ultrastructure

Ultrastructure refers to the detailed architecture of biological cells as observed through electron microscopy. In horses, the study of cellular ultrastructure provides insights into the organization and function of various tissues and organs at a microscopic level. This includes the examination of cellular components such as mitochondria, endoplasmic reticulum, and lysosomes. Understanding the ultrastructure of equine cells aids in identifying cellular changes associated with disease processes, developmental stages, and physiological adaptations. This page compiles peer-reviewed research studies and scholarly articles that explore the ultrastructural characteristics of equine cells and tissues, contributing to a deeper understanding of equine biology and pathology.
Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation.
Theriogenology    July 23, 2007   Volume 68, Issue 5 804-812 doi: 10.1016/j.theriogenology.2007.06.009
Morris GJ, Faszer K, Green JE, Draper D, Grout BW, Fonseca F.The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. An alternative mechanism has been proposed by Morris (2006) that cell damage is a result of an osmotic imbalance encountered during thawing. This paper examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. Horse spermatozoa were cooled at a range of cooling rates from 0.3 to 3,000 degrees C/min in the presence of a cryoprotectant. The ultra...
Immunocytochemical localization of lysozyme and beta-defensin in the apocrine glands of the equine scrotum.
Archives of dermatological research    July 17, 2007   Volume 299, Issue 8 393-397 doi: 10.1007/s00403-007-0766-5
Yasui T, Fukui K, Nara T, Habata I, Meyer W, Tsukise A.The present study revealed in detail the subcellular localization of lysozyme and beta-defensin in the apocrine glands of the equine scrotal skin, a specific body region. The apocrine glandular cells were equipped with a varying number of secretory granules, a well-developed Golgi apparatus and abundant cisternae of the rough endoplasmic reticulum within their cytoplasm. In these cells, reactive gold particles representing lysozyme were detectable in the secretory granules as well as the Golgi apparatus and elements of the rough endoplasmic reticulum. Additionally, the antimicrobial peptide gr...
Multiple masses in a horse’s tongue resulting from an atypical perineurial cell proliferative disorder.
Veterinary pathology    May 11, 2007   Volume 44, Issue 3 398-402 doi: 10.1354/vp.44-3-398
Vashisht K, Rock RW, Summers BA.A 5-year-old National Show horse mare presented with a soft mass on the left dorsolateral aspect of the tongue. Over the next 2 years, the mare developed numerous, similar, coalescing masses that extended along the left dorsolateral aspect to the tip of the tongue. Microscopically, the bases for these masses were slender, fusiform, mesenchymal cells that formed compact whorls around myelinated and unmyelinated nerves. These cells were labeled by antibodies directed against vimentin but not by S-100. Ultrastructurally, multiple, concentrically arranged, long, slender cell processes, with discon...
Equine stomach worm, Drashia megastoma (Spirurida: Habronematidae): first SEM report.
Parasitology research    May 10, 2007   Volume 101, Issue 4 913-918 doi: 10.1007/s00436-007-0558-z
Naem S.Drashia megastoma (Spirurida: Habronematidae) occurs in nodules in the stomach wall and rarely free in the stomach of the horse, mule and zebra throughout the world. D. megastoma develops in the housefly Musca domestica and causes gastric haemorrhage or even perforation of the stomach. Larvae of this nematode may be found in the lung, skin and eye of the host. Scanning electron microscopy (SEM) was used to study the surface ultrastructure of adult worms of this nematode. In both sexes, the head end was separated from the rest of the body by a constriction. The stoma was infundibuliform with a ...
Morphological alterations in oxidative muscles and mitochondrial structure associated with equine atypical myopathy.
Equine veterinary journal    January 19, 2007   Volume 39, Issue 1 26-32 doi: 10.2746/042516407x157765
Cassart D, Baise E, Cherel Y, Delguste C, Antoine N, Votion D, Amory H, Rollin F, Linden A, Coignoul F, Desmecht D.There is a lack of well documented studies about muscular lesions in equine atypical myopathy (EAM). Objective: To characterise morphopathological changes of striated muscles and myocardium, to progress understanding of this disease. Methods: Thirty-two horses age 0.5-7 years kept on pasture were referred for a sudden ataxia/myoglobinuria syndrome. Clinical examination (stiffness, muscle pain, muscle fasciculations, abnormal gait, recumbency, myoglobinuria, tachycardia, sweating) and plasma CPK, LDH and AST levels were consistent with extensive myonecrosis and, together with anamnestic data, w...
Physiological death of hypertrophic chondrocytes.
Osteoarthritis and cartilage    December 13, 2006   Volume 15, Issue 5 575-586 doi: 10.1016/j.joca.2006.10.016
Ahmed YA, Tatarczuch L, Pagel CN, Davies HM, Mirams M, Mackie EJ.Post-proliferative chondrocytes in growth cartilage are present in two forms, light and dark cells. These cells undergo hypertrophy and die by a mechanism that is morphologically distinct from apoptosis, but has not been characterized. The aims of the current study were to document the ultrastructural appearance of dying hypertrophic chondrocytes, and to establish a culture system in which the mechanism of their death can be examined. Methods: Growth cartilage from fetal and growing postnatal horses was examined by electron microscopy. Chondrocytes were isolated from epiphyseal cartilage from ...
Sperm morphology in stallions: ultrastructure as a functional and diagnostic tool.
The Veterinary clinics of North America. Equine practice    November 30, 2006   Volume 22, Issue 3 683-692 doi: 10.1016/j.cveq.2006.08.001
Veeramachaneni DN, Moeller CL, Sawyer HR.Conventional light microscopic evaluation of a seminal ejaculate does not fully avail potential indicators of functional impairment in spermatozoal organelles. The technique of critical quantitative evaluation of morphologic features of individual structural components of spermatozoa at a light microscopic level in conjunction with critical qualitative evaluation of spermatozoal organelles at an ultrastructural level, as described in this article, is a valuable clinical tool. Compared with a battery of sperm function assays used in human andrology clinics, this relatively less expensive and si...
Post-testicular changes in the density and distribution of intramembrane particles of stallion sperm surface domains.
Animal reproduction science    October 2, 2006   Volume 100, Issue 1-2 204-210 doi: 10.1016/j.anireprosci.2006.08.027
López ML, Olea N, Retamal CA.Freeze-fracture replicas of stallion spermatozoa, collected from the proximal caput, corpus and cauda epididymides regions, were analyzed by electron microscopy to explore the distribution and density of intramembrane particles (IMP). Conspicuous differences in density and arrangement of the IMP were observed in the different topographical domains of mature and immature spermatozoa. A reduction of IMP, especially remarkable in the post-acrosomal domain, was observed in mature epididymal spermatozoa when compared with samples collected from ductuli efferentes. Some structural species-specific d...
Myopathy in horses with pituitary pars intermedia dysfunction (Cushing’s disease).
Neuromuscular disorders : NMD    September 26, 2006   Volume 16, Issue 11 737-744 doi: 10.1016/j.nmd.2006.07.019
Aleman M, Watson JL, Williams DC, LeCouteur RA, Nieto JE, Shelton GD.Fifteen horses with pituitary pars intermedia dysfunction were studied. The horses were of various breeds and between 15 and 28 years of age. Control horses matched for breed and age were studied for comparison. Evaluations included complete blood cell count and serum biochemical analysis, electromyography, and gluteus medius muscle biopsies for histochemical, morphometric, and ultrastructural analysis. No differences were found between groups of horses on routine laboratory analysis or electromyography. We demonstrated that muscle wasting in diseased horses was the result of atrophy of types ...
[Depiction of the structure of the vitreous body in horses without ocular diseases and in horses with equine recurrent uveitis (ERU) using transmission electron microscopy].
DTW. Deutsche tierarztliche Wochenschrift    July 22, 2006   Volume 113, Issue 6 211-217 
Niedermaier G, Wollanke B, Hoffmann R, Matiasek K, Gerhards H.Neither the ultrastructure of the vitreous body from horses without ocular diseases, nor the pathomorphological changes in the vitreous body associated with equine recurrent uveitis (ERU) have been described. However, the vitreous body plays an important role in the pathogenesis of ERU. Ten vitreous body samples obtained from 5 horses without ocular disease, and 38 vitreous body samples from horses with ERU (collected during vitrectomy) were examined by transmission electron microscopy. The vitreous body samples of horses without ocular diseases were characterized by a loose network of unbranc...
Myopathy with central cores in a foal.
Veterinary pathology    July 19, 2006   Volume 43, Issue 4 579-583 doi: 10.1354/vp.43-4-579
Paciello O, Pasolini MP, Navas L, Russo V, Papparella S.Central core disease is a nonprogressive or slowly progressive congenital myopathy with a variable degree of hypotonia and axial and proximal muscle weakness that is histologically characterized by areas devoid of oxidative enzyme activity, resulting from an absence or low numbers of mitochondria in these regions (central core). A 10-month-old, male, pony foal was examined because of stiff gait, marked contractures of the distal portion of the limbs, flexion deformities of the hooves, and moderate hypotonia that had been present from birth. The foal had increased creatine kinase (282 U/liter; ...
Assessment of platelet function in horses: ultrastructure, flow cytometry, and perfusion techniques.
Journal of veterinary internal medicine    June 1, 2006   Volume 20, Issue 3 581-588 doi: 10.1892/0891-6640(2006)20[581:aopfih]2.0.co;2
Segura D, Monreal L, Pérez-Pujol S, Pino M, Ordinas A, Brugués R, White JG, Escolar G.We studied equine platelet function and activation using ultrastructural examination, flow cytometry, and perfusion. The main aim of the study was to evaluate hemostatic mechanisms in horses using these techniques. Ultrastructural observations were done on resting and activated platelets. Flow cytometry was used to evaluate binding of antibodies to major platelet glycoproteins (GPIIb-IIIa, GPIV, and GPIb) and activation-dependent antigens (P-selectin and lysosomal integral membrane protein [LIMP]). Perfusion techniques were used to evaluate the interaction between platelets and damaged subendo...
Histology, immunohistochemistry and ultrastructure of the tonsil of the soft palate of the horse.
Anatomia, histologia, embryologia    January 26, 2006   Volume 35, Issue 1 1-6 doi: 10.1111/j.1439-0264.2005.00622.x
Kumar P, Timoney JF.The tonsil of the soft palate was an oval, flat structure located centro-rostrally on the oral surface of the soft palate. Its stratified squamous non-keratinized epithelium was perforated by holes or small crypts the deeper parts of which were loosely spongiform inter-digitated with lymphoid tissue. These unusual features have not previously been reported in tonsils of any species. Crypts and reticulated epithelium as found in the lingual and palatine tonsils were not observed. Lectins showed varying affinities for specific layers of the epithelium. M cells were not observed. A few Langerhans...
Scanning electron microscopy of the choroid plexus of the lateral ventricle of the horse.
Anatomia, histologia, embryologia    November 18, 2005   Volume 34, Issue 6 379-382 doi: 10.1111/j.1439-0264.2005.00628.x
Adami M, Faria MM, Almeida AE, Pinto MG, Prada IL.The present study examined the ultrastructure of the choroid plexus of the lateral ventricle of the horse. The material was fixed in 2.5% glutaraldehyde in 0.1 m sodium phosphate buffer, pH 7.3, processed and analysed by scanning electron microscopy. The choroid plexus was characterized by regions with a predominance of villi, which resembled finger-like projections or bunches of grapes, and others where straight and uniform folds predominated. Epithelial cells projected into the ventricle and large amounts of cilia and microvilli were observed on their surface. The choroid glomus corresponded...
Co-infection between influenza virus and flagellated bacteria.
Revista do Instituto de Medicina Tropical de Sao Paulo    November 16, 2005   Volume 47, Issue 5 275-280 doi: 10.1590/s0036-46652005000500007
Mancini DA, Mendonça RM, Dias AL, Mendonça RZ, Pinto JR.Trypsin is required in the hemagglutinin (HA) cleavage to in vitro influenza viruses activation. This HA cleavage is necessary for virus cell entry by receptor-mediated endocytosis. Bacteria in the respiratory tract are potential sources of proteases that could contribute to the cleavage of influenza virus in vivo. From 47 samples collected from horses, pigs, and from humans, influenza presence was confirmed in 13 and these samples demonstrated co-infection of influenza with flagellated bacteria, Stenotrophomonas maltophilia from the beginning of the experiments. Despite treatment with antibio...
Control of the collagen fibril diameter in the equine superficial digital flexor tendon in horses by decorin.
The Journal of veterinary medical science    October 8, 2005   Volume 67, Issue 9 855-860 doi: 10.1292/jvms.67.855
Watanabe T, Hosaka Y, Yamamoto E, Ueda H, Sugawara K, Takahashi H, Takehana K.The distribution pattern of collagen fibril diameter in the equine superficial digital flexor tendon (SDFT) is known to differ in central and peripheral areas of some regions. This study reports the essence of collagen fibril differences among different regions of the equine SDFT by transmission electron microscopic (TEM) and high-voltage electron microscopic observations and biochemical analysis. The distribution of large collagen fibrils increased but the density of collagen fibrils decreased from the proximal metacarpal region to the distal metacarpal region. Large collagen fibrils with an ...
Membrane-coating granules and the intercellular cementing substance (membrane-coating material) in the epidermis in different regions of the equine hoof.
Anatomia, histologia, embryologia    September 15, 2005   Volume 34, Issue 5 298-306 doi: 10.1111/j.1439-0264.2005.00616.x
Anthauer K, Mülling C, Budras KD.This study aimed to carry out a comprehensive morphological examination of the structure and ultrastructure of the membrane-coating granules (MCG) and membrane-coating material (MCM) common to all hoof segments as well as segment-specific features in the MCG and MCM regarding their synthesis, structure and biochemical composition. Tissue samples from the hooves of the left front and right hind limb of 20 adult riding horses were used. Samples were preserved and embedded for histological routine methods as well as for histochemical techniques and tracer techniques for light and electron microsc...
Comparative morphological study on the tongue and lingual papillae of horses (Perissodactyla) and selected ruminantia (Artiodactyla).
Italian journal of anatomy and embryology = Archivio italiano di anatomia ed embriologia    August 17, 2005   Volume 110, Issue 2 Suppl 1 55-63 
Kobayashi K, Jackowiak H, Frackowiak H, Yoshimura K, Kumakura M, Kobayashi K.A common characteristic of horses, Rocky Mountain goats, and cattle is that they all have a well developed lingual prominence on the dorsal surface of the posterior area of the tongue. Foliate papillae were found in the horse studied but not in the goat or in cattle. The horse filiform papillae had a long and slender external form with a thin and slender CTC, while in the goat and cattle the external form consisted of a large thick main process and the CTC consisted of a bundle of numerous rod-shaped protrusions. The special papilla found on the lingual prominence resembled larger filiform-lik...
Unilateral basement membrane zone alteration of the regenerated laminar region in equine chronic laminitis.
The Journal of veterinary medical science    August 6, 2005   Volume 67, Issue 7 685-691 doi: 10.1292/jvms.67.685
Kuwano A, Ueno T, Katayama Y, Nishiyama T, Arai K.Between the laminar epidermis and the laminar dermis of laminar region (LR) in equine foot, it can be observed the basement membrane zone (BMZ), which is composed of a basement membrane and its accompaniments like the hemidesmosome and anchoring fibril. Alteration in the BMZ in equine laminitis is possibly related with not only development but also recovery outcome and recurrence of this disease. However, there is little known about the structure of the BMZ during the recovery phase of this disease. To assess the condition of the BMZ of LR affected by chronic laminitis, the tissue was examined...
Ultrastructural immunolocalization of cartilage oligomeric matrix protein (COMP) in relation to collagen fibrils in the equine tendon.
Matrix biology : journal of the International Society for Matrix Biology    July 12, 2005   Volume 24, Issue 5 376-385 doi: 10.1016/j.matbio.2005.06.003
Södersten F, Ekman S, Eloranta ML, Heinegård D, Dudhia J, Hultenby K.The structure and organisation of the extracellular matrix, and in particular the axial alignment of type I collagen fibrils, are essential for the tensile strength of tendons. The resident tenocytes synthesize and maintain the composition of the extracellular matrix, which changes with age and maturation. Other components of the extracellular matrix include less abundant collagen types II, III, V, VI, XII, proteoglycans and glycoproteins. Cartilage oligomeric matrix protein (COMP) is an abundant non-collagenous pentameric glycoprotein in the tendon, which can bind to collagen types I and II. ...
Ultrastructural and immunocytochemical evaluation of the effects of extracorporeal shock wave treatment in the hind limbs of horses with experimentally induced suspensory ligament desmitis.
American journal of veterinary research    June 7, 2005   Volume 66, Issue 5 892-896 doi: 10.2460/ajvr.2005.66.892
Caminoto EH, Alves AL, Amorim RL, Thomassian A, Hussni CA, Nicoletti JL.To evaluate the effects of extracorporeal shock wave therapy (ESWT) on affected ligaments in the hind limbs of horses with experimentally induced suspensory ligament desmitis by use of ultrasonographic, ultrastructural, and immunocytochemical techniques. Methods: 10 horses. Methods: Suspensory ligament desmitis was induced in both hind limbs of each horse by use of 2 collagenase injections (administered 2 weeks apart) in each suspensory ligament. Two weeks after the second injection, the right hind limb of each horse was treated with ESWT (3 treatments at 3-week intervals); the left hind limb ...
Histology, immunohistochemistry and ultrastructure of the equine tubal tonsil.
Anatomia, histologia, embryologia    June 3, 2005   Volume 34, Issue 3 141-148 doi: 10.1111/j.1439-0264.2005.00582.x
Kumar P, Timoney JF.The tubal tonsil of the horse surrounds the pharyngeal opening of the eustachian tube and is lined by pseudostratified columnar ciliated epithelium interspersed with areas of follicle-associated epithelium (FAE) heavily infiltrated by lymphocytes but devoid of goblet and ciliated cells. Scanning and transmission electron microscopy revealed microvillous cells and cells with features characteristic of M cells such as reduced microvilli or depressed bare surface, more numerous mitochondria, small vesicles and lysosomes, as well as vimentin filaments and epitopes specific for GS 1-B4 as previousl...
Histology, immunohistochemistry and ultrastructure of the equine palatine tonsil.
Anatomia, histologia, embryologia    June 3, 2005   Volume 34, Issue 3 192-198 doi: 10.1111/j.1439-0264.2005.00594.x
Kumar P, Timoney JF.The palatine tonsils of five young horses formed 10-12 cm elongated follicular structures extending from the root of the tongue on either side to the base of the epiglottis and lateral to the glossoepiglottic fold. The stratified squamous non-keratinized epithelium of the outer surface was modified into crypts as reticular epithelium by heavy infiltration of lymphoid cells from underlying lymphoid follicles. In places, lymphoid tissue reaching almost to the surface and with only one to two cell layers intact was identified as the lymphoepithelium. Langerhans cells with Birbeck granules were in...
Advanced fertility diagnosis in stallion semen using transmission electron microscopy.
Animal reproduction science    May 31, 2005   Volume 91, Issue 3-4 285-298 doi: 10.1016/j.anireprosci.2005.04.004
Pesch S, Bostedt H, Failing K, Bergmann M.Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3,...
In vivo interaction of pulmonary intravascular macrophages with activated platelets in microvessels of equine lung after multiple exposures to halothane, isoflurane, and thiamylal: a comparative ultrastructural and cytochemical study.
The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology    May 10, 2005   Volume 284, Issue 2 574-584 doi: 10.1002/ar.a.20196
Atwal OS, McDonell W.The pulmonary intravascular macrophages (PIMs) of equines contain a unique electron-dense surface coat that is predominantly composed of lipoproteins. A single exposure of inhalatory halothane causes mobilization of the surface coat into the endocytotic system of the PIMs, followed by expansion of the Golgi apparatus and its enrichment with acid phosphatase. Simultaneously, the cells of the lymphocytic series show hyperplasia in the form of mitotic changes inside the microvascular compartment of the lung. Halothane is known to cause acute and chronic hepatotoxicity because of its biotransforma...
Immunohistochemical and ultrastructural detection of intestinal spirochetes in Thoroughbred horses. Shibahara T, Kuwano A, Ueno T, Katayama Y, Ohya T, Taharaguchi S, Yamamoto S, Umemura T, Ishikawa Y, Kadota K.Studies of equine intestinal spirochetes have long focused on intestinal contents alone, but intestinal spirochetosis has been reported recently in a 21-month-old Thoroughbred colt in Japan. To define the clinical and pathological significances of intestinal spirochetosis in several horses, an epizootiologic survey with histologic, immunohistochemical, and ultrastructural methods was conducted for Brachyspira antigen-containing intestinal spirochetes in 12 diseased or injured Thoroughbred horses, aged from 35 days to 17 years. Brachyspira antigen-containing spirochetes were found in 7 of 12 ho...
Histology and ultrastructure of the equine lingual tonsil. II. Lymphoid tissue and associated high endothelial venules.
Anatomia, histologia, embryologia    March 18, 2005   Volume 34, Issue 2 98-104 doi: 10.1111/j.1439-0264.2004.00579.x
Kumar P, Timoney JF.The stratified squamous epithelium of the lingual tonsil of five young horses was infiltrated with CD4 and CD8 positive cells, which were very numerous in the crypt reticular epithelium along with macrophages and IgGb and IgA positive cells. Lymphoid follicles of the lamina propria mucosae consisted of a parafollicular area, corona and germinal centre. The parafollicular area was populated by large numbers of CD4 and CD8 positive lymphocytes as well as macrophages, inter-digitating cells, and a few B-lymphocytes. The germinal centre contained mainly IgGb and IgG(T) positive cells, plasma cells...
Species-specific ultrastructure of neuronal lipofuscin in hippocampus and neocortex of subhuman mammals and humans.
Ultrastructural pathology    March 15, 2005   Volume 28, Issue 5-6 341-351 doi: 10.1080/019131290882330
Boellaard JW, Schlote W, Hofer W.Lipofuscin represents an integral part of neurons and glial cells in mammals and in submammalian species. It is a special lysosomal organelle, takes part of cellular metabolism, and is a structural expression of catabolic pathways. Species-specific differences of lipofuscin indicate metabolic differences of the relevant neurons. The authors have studied the ultrastructure of neuronal lipofuscin in the hippocampus and cerebral neocortex of dogs, horses, cows, elephants, rats, mice, apes, and humans to answer the question of species-specific differences of this organelle. Paraffin sections of fo...
Experimental infection of ponies with Sarcocystis fayeri and differentiation from Sarcocystis neurona infections in horses.
The Journal of parasitology    February 18, 2005   Volume 90, Issue 6 1487-1491 doi: 10.1645/GE-313
Saville WJ, Dubey JP, Oglesbee MJ, Sofaly CD, Marsh AE, Elitsur E, Vianna MC, Lindsay DS, Reed SM.Sarcocystis neurona and Sarcocystis fayeri infections are common in horses in the Americas. Their antemortem diagnosis is important because the former causes a neurological disorder in horses, whereas the latter is considered nonpathogenic. There is a concern that equine antibodies to S. fayeri might react with S. neurona antigens in diagnostic tests. In this study, 4 ponies without demonstrable serum antibodies to S. neurona by Western immunoblot were used. Three ponies were fed 1 x 10(5) to 1 x 10(7) sporocysts of S. fayeri obtained from dogs that were fed naturally infected horse muscles. A...
Histology and ultrastructure of the equine lingual tonsil. I. Crypt epithelium and associated structures.
Anatomia, histologia, embryologia    January 15, 2005   Volume 34, Issue 1 27-33 doi: 10.1111/j.1439-0264.2004.00560.x
Kumar P, Timoney JF.The microstructural and ultrastructural features of the equine lingual tonsil were studied in five young horses. Located at the root of the tongue it presented an irregular surface with rounded elevations, numerous folds and crypts. Stratified squamous non-keratinized epithelium lining its outer surface was modified by heavy infiltration of lymphoid cells to form reticular epithelium within the crypt. The latter implies a role in initiating and maintaining immune responses to incoming infectious agents and antigens. Lamellated structures resembling Hassall's corpuscle were observed towards the...