Topic:Ultrastructure

Equine grass sickness is associated with major abnormalities in the ultrastructure of skeletal neuromuscular junctions.
Equine veterinary journal    February 1, 2024   doi: 10.1111/evj.14063
McGorum BC, Davey T, Dosi MCM, Keen JA, Morrison LR, Pirie RS, Shaw DJ, Harris JB.Equine grass sickness (EGS) is a frequently fatal multisystem neuropathy of equids. The aetiology is unknown; proposed causes include toxicoinfection with Clostridium botulinum and a mycotoxicosis. The effect of EGS on the organisation and structural integrity of the skeletal neuromuscular junction (NMJ), the target of botulinum neurotoxins (BoNTs), is unknown. Objective: To compare the organisation and structural integrity of skeletal NMJs from EGS horses, control horses and one horse with a presumptive diagnosis of botulism. Methods: Blinded, retrospective case control. Methods: NMJs in samp...
Mule embryos share identical morphological features to horse embryos.
Theriogenology    December 1, 2023   Volume 216 196-202 doi: 10.1016/j.theriogenology.2023.11.026
Podico G, Canisso IF.This study aimed to compare the morphometry of horse and mule embryos. The study's hypothesis was that the micronuclei and nuclear fragmentation indexes are higher in mule embryos than in horse embryos. Twenty-two mares were randomly assigned in a crossover design to receive semen from a horse and a donkey; thirteen horse and thirteen mule embryos were obtained. Embryos were recovered eight days post-ovulation and classified according to the stage of development and quality with a score from 1 (excellent) to 4 (degenerate). Embryos were stained with Hoechst33342, and images were acquired with ...
Erosion behaviour of human, bovine and equine dental hard tissues.
Scientific reports    November 10, 2023   Volume 13, Issue 1 19617 doi: 10.1038/s41598-023-46759-9
Hertel S, Basche S, Schmidt V, Staszyk C, Hannig C, Sterzenbach T, Hannig M.Dental hard tissues from different species are used in dental research, but little is known about their comparability. The aim of this study was to compare the erosive behaviour of dental hard tissues (enamel, dentin) obtained from human, bovine and equine teeth. In addition, the protective effect of the pellicle on each hard tissue under erosive conditions was determined. In situ pellicle formation was performed for 30 min on enamel and dentin samples from all species in four subjects. Calcium and phosphate release was assessed during 120 s of HCl incubation on both native and pellicle-cove...
Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue.
Reproductive biology    October 25, 2023   Volume 23, Issue 4 100814 doi: 10.1016/j.repbio.2023.100814
Borges MA, Curcio BR, Gastal GDA, Gheno L, Junior ASV, Corcini CD, Nogueira CEW, Aguiar FLN, Gastal EL.The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and o...
Identification of telocytes in the oviduct of the mare.
Theriogenology    April 14, 2023   Volume 205 18-26 doi: 10.1016/j.theriogenology.2023.04.012
Etcharren V, Mouguelar H, Aguilar Valenciano JJ.Telocytes (TCs), a recently discovered special type of stromal cells, have been identified in many organs of many species, including the female and male reproductive system, with proposed multiple potential bio-functions such as homeostasis, immunomodulation, tissue remodeling and regeneration, embryogenesis, angiogenesis and even tumorigenesis. The aim of this study was to investigate the existence, and characteristics of telocytes in normal equine oviduct. To identify them, we used routine light microscopy, non-conventional light microscopy (NCLM), transmission electron microscopy (TEM), and...
Implication of Cellular Senescence in Osteoarthritis: A Study on Equine Synovial Fluid Mesenchymal Stromal Cells.
International journal of molecular sciences    February 4, 2023   Volume 24, Issue 4 3109 doi: 10.3390/ijms24043109
Teti G, Mazzotti E, Gatta V, Chiarini F, Alfieri ML, Falconi M.Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we ai...
Larvae and adult flies of Rhinoestrus purpureus and R. usbekistanicus: morphology and pupation (Diptera: Oestridae).
Veterinaria italiana    December 30, 2022   Volume 58, Issue 2 doi: 10.12834/VetIt.2085.12058.2
Ibrahim M.Rhinoestrus species larvae are considered obligatory parasites of the nasal cavities of equine. This type of myiasis is characterised by sneezing, coughing, olfactory nerve damage and encephalomyelitis. Also, it has a zoonotic importance as the larvae might cause ophthalmomyiasis and conjunctivitis in human. While few studies describing R. purpureus adult fly antennal sensillae are available, the R. usbekistanicus antennal sensillae have never been described. Also, scanty data are available on the adult flies of Rhinoestrus species morphology. For this reason, the current study aimed at identi...
Pulmonary bleeding in racehorses: A gross, histologic, and ultrastructural comparison of exercise-induced pulmonary hemorrhage and exercise-associated fatal pulmonary hemorrhage.
Veterinary pathology    August 16, 2022   Volume 59, Issue 6 973-982 doi: 10.1177/03009858221117859
Rocchigiani G, Verin R, Uzal FA, Singer ER, Pregel P, Ressel L, Ricci E.Exercise-induced pulmonary hemorrhage (EIPH) is a common condition of Thoroughbred racehorses that is usually responsible for reduced performance, while exercise-associated fatal pulmonary hemorrhage (EAFPH) is characterized by severe pulmonary bleeding of unknown pathogenesis resulting in sudden death during strenuous exercise. The aim of the study was to characterize and compare anamnestic data together with pulmonary gross, histologic, and ultrastructural findings in racehorses with EIPH (n = 10), EAFPH (n = 10), and control horses (n = 5). No differences in anamnesis were identified betwee...
Morphomolecular characterization of Strongylus vulgaris isolated from donkeys with special references to histopathological study on the affected organs.
Journal of parasitic diseases : official organ of the Indian Society for Parasitology    June 6, 2022   Volume 46, Issue 3 795-803 doi: 10.1007/s12639-022-01498-y
El-Gameel SM, Al-Mokaddem AK, Salaeh NMK, Attia MM.Equine gastrointestinal tract is infected with () which is highly pathogenic parasite for its harmful effect on cranial mesenteric artery during its migration. So, this study was applied for identification of in donkeys ultramorphologically and molecularly. In addition to, detection of the pathological effect of larval stage of on the mesenteric arterial system using histopathology and immunohistochemistry. During the period from September to December; 2019, 60 male and 20 female donkeys at the Giza Zoo was postmortem examined. adults and larvae were collected from the large intestine and ...
Morphological, Immunohistochemical, and Ultrastructural Studies of the Donkey’s Eye with Special Reference to the AFGF and ACE Expression. Attaai AH, Hussein MT, Aly KH, Abdel-Maksoud FM.The donkey is mainly used as a working animal for riding and pack transport, as well as for dairy and meat production. Eye afflictions are common in donkeys, thus requiring a detailed study. A few studies had focused on the donkey's eye, and most of them had considered it, merely, a horse's eye. This study aimed to investigate the anatomy, histology, ultrastructure, and immunohistochemical features of the donkey's eye. The results were recorded and compared to those of horses in certain dimensions. Unlike horses, the donkey's eye is more circular in the contour of the cornea, has smaller lenti...
Morphological study of equine amniotic compartment.
Theriogenology    October 19, 2021   Volume 177 165-171 doi: 10.1016/j.theriogenology.2021.10.019
Lanci A, Ingru00e0 L, Dondi F, Tomasello F, Teti G, Mariella J, Falconi M, Castagnetti C.Exfoliative cytology of human amniotic fluid (AF) has been extensively studied since 1940s, but no data exist in equine species. The AF compartment represents the environment in which the foetus grows and matures, and its composition changes, reflecting foetal well-being and development. The aim of this study was to describe for the first time the morphology of equine AF cells and amniotic membrane (AM) with light microscopy (LM) and transmission electron microscopy (TEM). AF was collected at parturition within 5 min after the appearance of the AM with a 60 mL syringe from 34 mares and sampl...
Isolation and morphological characterization of equine mesenchymal stem cells from harvested adipose tissue and bone marrow and stably transfected with green fluorescent protein.
American journal of veterinary research    August 26, 2021   Volume 82, Issue 9 770-776 doi: 10.2460/ajvr.82.9.770
Pu00e9rez-Castrillo S, Gonzu00e1lez-Fernu00e1ndez ML, Gutiu00e9rrez-Velasco L, Villar-Suu00e1rez V.To characterize the ultrastructure of mesenchymal stem cells (MSCs) that were harvested from the adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) of horses and transfected with green fluorescent protein. Methods: MSCs from adipose tissue and bone marrow of 6 adult female Hispano-Bretón horses. Methods: Harvested equine MSCs were cultivated and transfected with green fluorescent protein, and the immunophenotypes of the MSCs were characterized by use of anti-CD90 and anti-CD105 monoclonal antibodies. When stable transfection of MSCs was achieved, the morphological and ultrastructural characte...
Evaluation of the Filum Terminale in Hereditary Equine Regional Dermal Asthenia.
Veterinary pathology    May 31, 2021   Volume 58, Issue 6 1100-1106 doi: 10.1177/03009858211018660
McElroy A, Klinge PM, Sledge D, Donahue JE, Glabman RA, Rashmir A.The objectives of this study were to describe the anatomy, histology, and ultrastructure of the equine filum terminale (FT) and to describe the FT in hereditary equine regional dermal asthenia (HERDA), a model of human Ehlers-Danlos syndromes (EDS). Those humans suffer from tethered cord syndrome (TCS) caused by an abnormally structured FT wherein its attachment at the base of the vertebral column leads to long-term stretch-induced injury to the spinal cord. The pathophysiology of TCS in EDS is poorly understood, and there is a need for an animal model of the condition. Histopathologic and ult...
Systemic Toxoplasmosis in a Horse.
Journal of comparative pathology    December 9, 2020   Volume 182 27-31 doi: 10.1016/j.jcpa.2020.11.004
Kimble KM, Gomez G, Szule JA, Dubey JP, Buchanan B, Porter BF.An adult American Quarter Horse gelding with a history of weight loss presented with an acute onset of colic, fever, soft faeces and elevated liver enzymes. At necropsy, there were gastric mucosal masses and evidence of caecal necrosis. Histologically, the masses were lymph nodes with granulomatous inflammation and areas of liquefactive necrosis. Within and surrounding necrotic areas were free and intrahistiocytic clusters of protozoal tachyzoites. Similar but milder inflammation was evident in the spleen, lungs and liver. Necrotizing typhlitis was also evident. Immunolabelling for Toxoplasma ...
Ultrastructural evidence for telocytes in equine tendon.
Journal of anatomy    October 18, 2020   Volume 238, Issue 3 527-535 doi: 10.1111/joa.13335
Luesma MJ, Cantarero I, Su00e1nchez-Cano AI, Rodellar C, Junquera C.The three-dimensional ultrastructure of the tendon is complex. Two main cell types are classically supported: elongated tenocytes and ovoid tenoblasts. The existence of resident stem/progenitor cells in human and equine tendons has been demonstrated, but their location and relationship to tenoblasts and tenocytes remain unclear. Hence, in this work, we carried out an ultrastructural study of the equine superficial digital flexor tendon. Although the fine structure of tendons has been previously studied using electron microscopy, the presence of telocytes, a specific type of interstitial cell, ...
Histomorphometric and vascular changes in equine endometrium after the infusion of conceptus fragments.
Animal reproduction    June 29, 2020   Volume 17, Issue 2 e20200006 doi: 10.1590/1984-3143-AR2020-0006
Camacho CA, Estradu00e9 MJ, Cazales N, Caballeros JE, Fiala-Rechsteiner SM, Neves AP, Mattos RC.This experiment aimed to verify if the proteins present in a 13 day conceptus induce changes in the equine endometrial ultra-structure, histology, and vascularization, two days after its infusion. Ten healthy cyclic mares were used. Once estrus was confirmed, mares were examined daily to detect ovulation (day 0). After ovulation, mares were examined daily until day seven by transrectal palpation and B-mode and Doppler ultrasonography. In this first cycle, intrauterine biopsies were collected at day seven after ovulation, constituting the Cyclic group (n = 10). In the second cycle, the same mar...
Hanoverian F/W-line contributes to segregation of Warmblood fragile foal syndrome type 1 variant PLOD1:c.2032G>A in Warmblood horses.
Equine veterinary journal    May 19, 2020   Volume 53, Issue 1 51-59 doi: 10.1111/evj.13271
Metzger J, Kreft O, Sieme H, Martinsson G, Reineking W, Hewicker-Trautwein M, Distl O.Warmblood fragile foal syndrome (WFFS) is a lethal condition detected in Warmblood horses. Its origin and association with performance traits and fertility among horse populations is unknown. Objective: To validate the previously identified WFFS type 1 (WFFST1)-associated missense variant PLOD1:c.2032G>A and to investigate its distribution among various horses with particular focus on Hanoverian breed, as well as its pathomorphological picture. The study aimed at identifying the origin of the mutant allele and its correlation with performance and fertility traits in Warmblood horses. Method...
Quantitative Assessment of Tendon Hierarchical Structure by Combined Second Harmonic Generation and Immunofluorescence Microscopy.
Tissue engineering. Part C, Methods    May 5, 2020   Volume 26, Issue 5 253-262 doi: 10.1089/ten.TEC.2020.0032
Durgam S, Singh B, Cole SL, Brokken MT, Stewart M.Histological evaluation of healing tendons is primarily focused on monitoring restoration of longitudinal collagen alignment, although the elastic property of energy-storing flexor tendons is largely attributed to interfascicular sliding facilitated by the interfascicular matrix (IFM). The objectives of this study were to explore the utility of second harmonic generation (SHG) imaging to objectively assess cross-sectional tendon fascicle architecture, to combine SHG microscopy with elastin immunofluorescence to assess the ultrastructure of collagen and elastin in longitudinal and transverse se...
Age-Related Alterations Affecting the Chondrogenic Differentiation of Synovial Fluid Mesenchymal Stromal Cells in an Equine Model.
Cells    September 20, 2019   Volume 8, Issue 10 1116 doi: 10.3390/cells8101116
Osteoarthritis is a degenerative disease that strongly correlates with age and promotes the breakdown of joint cartilage and subchondral bone. There has been a surge of interest in developing cell-based therapies, focused particularly on the use of mesenchymal stromal cells (MSCs) isolated from adult tissues. It seems that MSCs derived from synovial joint tissues exhibit superior chondrogenic ability, but their unclear distribution and low frequency actually limit their clinical application. To date, the influence of aging on synovial joint derived MSCs' biological characteristics and differen...
Changes on the Structural Architecture and Growth Factor Release, and Degradation in Equine Platelet-Rich Fibrin Clots Cultured Over Time.
Journal of equine veterinary science    August 9, 2019   Volume 82 102789 doi: 10.1016/j.jevs.2019.102789
Jimu00e9nez-Aristazu00e1bal RF, Carmona JU, Prades M.The aims of the present study were (1) to describe the microscopic and ultrastructural appearance of equine platelet-rich fibrin (PRF) clots and (2) to determine the release and degradation of transforming growth factor beta 1 (TGF-β) and insulin-like growth factor type I (IGF-I) from PRF clots incubated over 14 days. Whole blood from six horses was collected into plain tubes and centrifuged at 240 g for 8 minutes. Clots were evaluated by histology and by both transmission and scanning electronic microscopy (TEM and SEM). Growth factor concentrations were measured by ELISA at 48-hour inter...
Comparison between adult and foetal adnexa derived equine post-natal mesenchymal stem cells.
BMC veterinary research    August 2, 2019   Volume 15, Issue 1 277 doi: 10.1186/s12917-019-2023-5
Merlo B, Teti G, Lanci A, Burk J, Mazzotti E, Falconi M, Iacono E.Little is known about the differences among adult and foetal equine mesenchymal stem cells (MSCs), and no data exist about their comparative ultrastructural morphology. The aim of this study was to describe and compare characteristics, immune properties, and ultrastructural morphology of equine adult (bone marrow: BM, and adipose tissue: AT) and foetal adnexa derived (umbilical cord blood: UCB, and Wharton's jelly: WJ) MSCs. Results: No differences were observed in proliferation during the first 3 passages. While migration ability was similar among cells, foetal MSCs showed a higher adhesion a...
Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105+K14+ progenitor cells.
In vitro cellular & developmental biology. Animal    July 11, 2019   Volume 55, Issue 8 641-655 doi: 10.1007/s11626-019-00380-1
Yang Q, Lopez MJ.The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning e...
Evaluation of the Structure of Myodural Bridges in an Equine Model of Ehlers-Danlos Syndromes.
Scientific reports    July 10, 2019   Volume 9, Issue 1 9978 doi: 10.1038/s41598-019-46444-w
McElroy A, Rashmir A, Manfredi J, Sledge D, Carr E, Stopa E, Klinge P.Myodural bridges have been described in various species as connective tissue structures "bridging" small cranio-cervical muscles to the dura. Myodural bridges are thought to stabilize the dural sac during head and neck movements and promote cerebrospinal fluid motion; however, their role in neurological diseases has not yet been established. We report ultrasonographic visualization, necropsy, histopathologic and ultrastructural findings of myodural bridges in horses with hereditary equine regional dermal asthenia (HERDA), an equine model of Ehlers-Danlos syndromes. Five HERDA and 5 control hor...
A histological and ultrastructural study of equine peripheral caries.
Equine veterinary journal    May 30, 2019   Volume 52, Issue 1 104-111 doi: 10.1111/evj.13134
Borkent D, Smith S, Dixon PM.Peripheral caries (PC) has recently been shown to affect over half of UK and Australian horses. Objective: To examine affected cheek teeth histologically and ultrastructurally, to assess the possible route(s) of carious infection into the dental tissues and to further describe the pathological changes in affected teeth. Methods: Descriptive pathological study. Methods: Four control and 16 cheek teeth with different grades of PC were examined grossly, histologically and by electron microscopy. Results: Cariogenic bacteria from plaque entered peripheral cementum perpendicular to the sides of tee...
Histologically, immunohistochemically, ultrastructurally, and molecularly confirmed neosporosis abortion in an aborted equine fetus.
Veterinary parasitology    May 3, 2019   Volume 270 20-24 doi: 10.1016/j.vetpar.2019.04.009
Anderson JA, Alves DA, Cerqueira-Cu00e9zar CK, da Silva AF, Murata FHA, Norris JK, Howe DK, Dubey JP.Neosporosis is a common cause of abortion in cattle worldwide but is rare in horses. Here, the first case of histologically, ultrastructurally, immunohistochemically, and molecularly confirmed equine abortion caused by neosporosis is reported. Samples of lung, heart, liver, skeletal muscle, tongue, brain, and the placenta from a female fetus aborted at 280 days of gestation were fixed in formalin and submitted for diagnosis. Histologically, there was disseminated neosporosis with severe lesions in lungs, liver and the heart. Protozoal tachyzoites in all tissues reacted with polyclonal anti-Neo...
Ultrastructural and histological characteristics of the equine endometrium at day 5 post ovulation.
Theriogenology    April 14, 2019   Volume 132 106-112 doi: 10.1016/j.theriogenology.2019.04.006
Caballeros JE, Camacho C, Cazales N, Estradu00e9 MJ, Fiala-Rechsteiner S, Jobim MIM, Mattos RC.Rapid endometrial adaptations occur with the embryo entering the uterus to create a receptive uterine environment, which is essential for the conceptus' development. The aim of this study was to demonstrate ultrastructural and histological changes of the endometrium at day 5 after ovulation in cyclic and inseminated mares. Mares were daily examined by transrectal palpation and ultrasonographic examination of the reproductive tract until ovulation was detected. In the first cycle, endometrial biopsies from 10 cyclic mares (Cyclic group) were collected on day 5 post-ovulation. In the second cycl...
Vitrifying immature equine oocytes impairs their ability to correctly align the chromosomes on the MII spindle.
Reproduction, fertility, and development    April 11, 2019   Volume 31, Issue 8 1330-1338 doi: 10.1071/RD18276
Ducheyne KD, Rizzo M, Daels PF, Stout TAE, de Ruijter-Villani M.Vitrified-warmed immature equine oocytes are able to complete the first meiotic division, but their subsequent developmental competence is compromised. Therefore, the present study investigated the effects of vitrifying immature horse oocytes on the chromosome and spindle configuration after IVM. Cumulus-oocytes complexes (COCs) were collected and divided into two groups based on mare age (young ≤14 years; old ≥16 years). COCs were then either directly matured invitro or vitrified and warmed before IVM. Spindle morphology and chromosome alignment within MII stage oocytes were assessed usin...
Equine Adipose-Derived Mesenchymal Stromal Cells Release Extracellular Vesicles Enclosing Different Subsets of Small RNAs.
Stem cells international    March 18, 2019   Volume 2019 4957806 doi: 10.1155/2019/4957806
Capomaccio S, Cappelli K, Bazzucchi C, Coletti M, Gialletti R, Moriconi F, Passamonti F, Pepe M, Petrini S, Mecocci S, Silvestrelli M, Pascucci L.Equine adipose-derived mesenchymal stromal cells (e-AdMSC) exhibit attractive proregenerative properties strongly related to the delivery of extracellular vesicles (EVs) that enclose different kinds of molecules including RNAs. In this study, we investigated small RNA content of EVs produced by e-AdMSC with the aim of speculating on their possible biological role. Methods: EVs were obtained by ultracentrifugation of the conditioned medium of e-AdMSC of 4 subjects. Transmission electron microscopy and scanning electron microscopy were performed to assess their size and nanostructure. RNA was is...
Cryopreservation (-20°C) of equine corneoscleral tissue: Microbiological, histological, and ultrastructural study.
Veterinary ophthalmology    February 8, 2019   Volume 22, Issue 4 493-501 doi: 10.1111/vop.12618
Costa D, Leiva M, Naranjo C, Ru00edos J, Peu00f1a MT.To evaluate microbiological, histological, and ultrastructural characteristics of short-term cryopreserved (STC) equine corneoscleral tissue (7 years). Methods: Thirty-four healthy equine globes. Methods: After a decontamination protocol, globes were enucleated and stored at -20°C in broad-spectrum antibiotics. Corneoscleral tissue was evaluated at different storage periods: 1 month-1 year (20 eyes) and 7-9 years (12 eyes). Two eyes were used as controls. Microbiologic study included direct (blood, McConkey, and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopr...
Ultrastructural and histological characteristics of the endometrium during early embryo development in mares.
Theriogenology    September 18, 2018   Volume 123 1-10 doi: 10.1016/j.theriogenology.2018.09.018
Camozzato GC, Martinez MN, Bastos HBA, Fiala-Rechsteiner S, Meikle A, Jobim MIM, Gregory RM, Mattos RC.The aim of this study was to evaluate ultrastructural and histological changes in the endometrium on days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. Mares were routinely examined by transrectal palpation and ultrasonographic examination of the reproductive tract until estrus was detected. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sampl...
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