scholarly journals 192 PROBLEMS USING JC-1 TO ASSESS MITOCHONDRIAL STATUS IN BROWN BEAR (URSUS ARCTOS) SEMEN

2005 ◽  
Vol 17 (2) ◽  
pp. 246 ◽  
Author(s):  
V. García-Macías ◽  
F. Martínez-Pastor ◽  
F. Martínez ◽  
N. González ◽  
M. Álvarez ◽  
...  
Keyword(s):  
Phase Contrast ◽  
Ursus Arctos ◽  
Brown Bear ◽  
Molecular Probes ◽  
Biological Resource ◽  
Germplasm Bank ◽  
Contrast Microscope ◽  
Before And After ◽  
Pearson Correlations ◽  
Epifluorescence Microscope

Brown bear is a highly endangered species in Spain and could benefit from biological resource banking. Currently, we are studying several reproductive aspects in order to aquire the knowledge for establishment of a germplasm bank for this species. One of our objectives is to develop adequate protocols for the evaluation of bear sperm before and after cryopreservation. We have used the fluorescent probe JC-1 protocol, which differentially stains mitochondria, according to its activity (Garner DL et al. 1997 Biol. Reprod. 57, 1401–1406). Here we describe one problem that arose using this staining for evaluation of extended bear semen. We electroejaculated 13 adult brown bears (Ursus arctos) (206–311 kg) housed in a half-freedom regime in the Cabarceno Park (Cantabria, Spain). Anesthesia was performed with tiletamine + zolazepan (Zoletil 100®, 7 mg/kg; Virbac, Spain), and ketamine (Imalgene 1000®, 2 mg/kg; Mericl, Sain). We used an electroejaculator (PT Electronics®; Boring, OR, USA) with a 3-electrode transrectal probe (26 mm in diameter, 320 mm long). Ejaculation occurred at 10 V/250 mA. Samples were extended (prepared in our laboratory, Anel L et al. 2003 Theriogenology 60, 1293–1308; M3 modified) and cooled to 5°C for 70 min (pre-freezing protocol). We analyzed individual (MI) and progressive (MP) motility by means of an automated motility analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA), using a phase contrast microscope (Nikon, ×10). Mitochondrial status was analyzed after diluting the sample 1:100 with buffered medium (20 mM HEPES, 153 mM NaCl, 2.5 mM KOH, 10 mM glucose; Sigma, Madrid, Spain) and adding JC-1 (6.8 μM final; Molecular Probes, The Netherlands). After 30 min at 37°C, 100 cells were counted with an epifluorescence microscope (Nikon, ×400), determining the percentage of sperm with orange-stained (active) mitochondria. We analyzed a total of 55 samples in three different models: fresh, pre-freezing, and thawed. We divided the samples into successful JC-1 staining (valids: V) or failed JC-1 staining (not valid: NV) (depending on the aspect of the stained cells). In not-valid samples we observed a greenish background, with almost no fluorescent spermatozoa. These observations were consistent in a given sample, giving the same V or NV result when we repeated the staining. In fresh and thawed groups there were no NV samples, but in the pre-freezing group there were 40 NV samples (73%). We calculated Pearson correlations (SAS; SAS Institute, Inc., Cary, NC, USA) between percent JC-1 orange population and MI and MP in fresh (r = 0.40 and 0.33; P < 0.001), thawed (r = 0.61 and 0.43; P < 0.001) and pre-freezing samples (r = −0.11 and −0.24; P > 0.05), all respectively. When pre-freezing samples were split between V and NV, the former had good correlations (r = 0.74 and 0.49; P < 0.05), and NV still did not (r = −0.17, −0.27; P > 0.05). We conclude that JC-1 staining is not reliable for the pre-freezing analysis of bear sperm, at least under the conditions described here. This could be due to the interaction of the extender or the refrigeration treatment with the sperm. However, this problem did not occur in the analysis of fresh and thawed samples. Nevertheless, it may be advisable to test other mitochondrial probes for analyzing this kind of samples.

245 USE OF A TRIPLE STAIN (SYBR-14/PI/MC540) FOR VIABILITY AND CAPACITATION ASSESSMENT IN THAWED SEMEN FROM BROWN BEAR (URSUS ARCTOS)

2007 ◽  
Vol 19 (1) ◽  
pp. 239 ◽  
Author(s):  
V. Garcia-Macias ◽  
F. Martinez-Pastor ◽  
M. Alvarez ◽  
P. Paz ◽  
S. Borragan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ursus Arctos ◽  
Brown Bear ◽  
Emission Spectra ◽  
Egg Yolk ◽  
Membrane Lipid ◽  
Molecular Probes ◽  
Lower Percentage ◽  
Sperm Viability ◽  
Lipid Disorder

Application of new sperm assessment techniques would improve our capability to determine the effects of cryopreservation on sperm function. This becomes relevant when germplasm banks are established for endangered species, as in the case of the brown bear in Spain. Different triple stain techniques have been used in conjunction with flow cytometry to assess various sperm attributes including viability and acrosome status. However, fluorochromes with similar emission spectra may interfere with data resolution, making acquisition and interpretation of data difficult. The double stain combination of SYBR-14 and PI (propidium iodide; max λ 617) has been widely used to differentiate live from dead spermatozoa (spz), and, more recently, merocyanine 540 (MC; max λ 555) has been used to detect a sperm membrane lipid disorder associated with sperm capacitation. In the present study, we analyzed the suitability of combining SYBR-14/PI with MC for simultaneous determination of the viability and capacitation status of frozen–thawed spermatozoa of brown bears (n = 10; semi-free ranging; Cabarceno Park, Cantabria, Spain) obtained by electroejaculation under general anesthesia (7 mg kg-1 tiletamine + zolazepan and 2 mg kg-1 ketamine). Semen was diluted (Tes-Tris-fructose, 8% glycerol, 20% egg yolk, EDTA, and Equex paste), loaded in 0.25-mL straws, and frozen in a biofreezer at 20�C min-1 to -100�C. After storage in liquid nitrogen, samples were thawed at 65�C for 6 s, divided into 2 aliquots (1–2 million spz mL-1), extended with 300 �L PBS, and stained with SYBR14 (1.2 �L) and PI (3 �L; LIVE/DEAD� Sperm Viability Kit; Molecular Probes, Inc., Eugene, OR, USA). To evaluate the possible interaction of MC on sperm viability, half of the aliquots were counterstained with 1.5 �L of MC (diluted with 2.7 �M of DMSO); the other half were not counterstained (control). All tubes were incubated at 37�C for 30 min, and assessed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA). Data were analyzed with Bland-Altman. Results indicated that MC staining was mainly confined to dead spermatozoa (22.2 � 7.7%), whereas a lower percentage of live spermatozoa (5.7 � 1.6%; P &lt; 0.05) were also stained with MC. Possibly, the staining of dead spermatozoa with MC was due to capacitation changes induced by cryopreservation. The percentage of live spermatozoa was not different between samples counterstained with MC (68.9 � 9.2) and non-MC-stained control samples (68.6 � 8.8). Thus, we consider that MC does not influence SYBR14/PI discrimination of viable spermatozoa, and that the 3 stains can be used simultaneously. However, more studies are necessary to determine whether MC can be used to distinguish the capacitation status of brown bear thawed spermatozoa. This work was supported by CANTUR S.A. and CICYT (CGL 2004-0278/BOS).

Brown Bear (Ursus arctos) Habitat Use and Food Resources on Elmendorf Air Force Base, Alaska

2007 ◽  
Author(s):  
Sean D. Farley ◽  
Herman Griese ◽  
Rick Sinnott ◽  
Jessica Coltrane ◽  
Chris Garner ◽  
...  
Keyword(s):  
Habitat Use ◽  
Air Force ◽  
Ursus Arctos ◽  
Brown Bear ◽  
Food Resources ◽  
Air Force Base

scholarly journals Using Landscape Change Analysis and Stakeholder Perspective to Identify Driving Forces of Human–Wildlife Interactions

Land ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 146
Author(s):  
Mihai Mustățea ◽  
Ileana Pătru-Stupariu
Keyword(s):  
Landscape Change ◽  
Ursus Arctos ◽  
Driving Forces ◽  
Brown Bear ◽  
Mountain Range ◽  
Change Analysis ◽  
Local Stakeholders ◽  
Stakeholder Perspective ◽  
Forested Ecosystems ◽  
Forest Habitats

Human–wildlife interactions (HWI) were frequent in the post-socialist period in the mountain range of Central European countries where forest habitats suffered transitions into built-up areas. Such is the case of the Upper Prahova Valley from Romania. In our study, we hypothesized that the increasing number of HWI after 1990 could be a potential consequence of woodland loss. The goal of our study was to analyse the effects of landscape changes on HWI. The study consists of the next steps: (i) applying 450 questionnaires to local stakeholders (both citizens and tourists) in order to collect data regarding HWI temporal occurrences and potential triggering factors; (ii) investigating the relation between the two variables through the Canonical Correspondence Analysis (CCA); (iii) modelling the landscape spatial changes between 1990 and 2018 for identifying areas with forest loss; (iv) overlapping the distribution of both the households affected by HWI and areas with loss of forested ecosystems. The local stakeholders indicate that the problematic species are the brown bear (Ursus arctos), the wild boar (Sus scrofa), the red fox (Vulpes vulpes) and the grey wolf (Canis lupus). The number of animal–human interactions recorded an upward trend between 1990 and 2018, and the most significant driving factors were the regulation of hunting practices, the loss of habitats, and artificial feeding. The landscape change analysis reveals that between 1990 and 2018, the forest habitats were replaced by built-up areas primarily on the outskirts of settlements, these areas coinciding with frequent HWI. The results are valid for both forest ecosystems conservation in the region, wildlife management, and human infrastructures durable spatial planning.

Plasma ochratoxin A in the European brown bear ( Ursus arctos L.) from Croatia

2017 ◽  
Vol 280 ◽  
pp. S198
Author(s):  
Dubravka Rašić ◽  
Maja Lazarus ◽  
Đuro Huber ◽  
Slaven Reljić ◽  
Maja Peraica
Keyword(s):  
Ochratoxin A ◽  
Ursus Arctos ◽  
Brown Bear

scholarly journals 209FUNCTIONAL ASSESSMENT OF WHITE RHINOCEROS CERATHOTERIUM SIMUM EPIDIDYMAL SPERMATOZOA BEFORE AND AFTER CRYOPRESERVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 226 ◽  
Author(s):  
F. Martinez-Pastor ◽  
F. Olivier ◽  
T. Spies ◽  
L. Anel ◽  
P. Bartels
Keyword(s):  
Plasma Membrane ◽  
Assisted Reproduction ◽  
Phase Contrast ◽  
Egg Yolk ◽  
Phase Contrast Microscopy ◽  
White Rhinoceros ◽  
Contrast Microscopy ◽  
Before And After ◽  
Epididymal Spermatozoa

Biological Resource Banks represent a potentially valuable tool for species conservation. It is, however, necessary to understand the species-specific cryopreservation process and its consequences for spermatozoa to aid in the development of assisted reproduction as a future conservation tool. The aim of this study was to assess the in vitro functionality of white rhinoceros Cerathoterium simum epididymal spermatozoa both before and after cryopreservation. Testes from a harvested white rhino bull were removed and transported at 5°C to the laboratory within 4h. The cauda epididymis was dissected out and flushed with 2mL of Tris-citrate egg yolk extender (fraction A, Biladyl, Minitüb, Germany). A 0.1mL aliquot was removed for analysis and the balance (9mL; 2mL fraction A+7mL sperm sample) mixed with an additional 27.2mL of Tris-citrate egg yolk with glycerol (fraction B, Bidadyl). The extended sample was allowed to cool to 4°C over a 6-h period before an additional 29.2mL of cooled fraction B were added (final sperm concentration=150×106mL−1). Sperm samples were loaded into 0.25-mL straws and frozen over LN2 vapor (4cm for 20min) for later assessment. Sperm straws were thawed by placing the straws in water at 37°C for 30s. Pre-freeze and post-thaw evaluations were carried out in the same manner. Media used included: HEPES for washing (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH) and HEPES saline (197mM NaCl, instead of sucrose). An aliquot was diluted with HEPES (washing) and centrifuged for 5min at 600×g; the pellet was resuspended in HEPES saline. Sperm motility (total motility %, TM;; and progressive motility %, PM) was assessed using phase contrast microscopy (×200; 37°C). Sperm plasma membrane status was assessed using the fluorescent dye, propidium iodide (50ngmL−1 in HEPES saline;; 10min, RT). Percentage of cells with plasma membranes intact (unstained;; PMI) was recorded. Mitochondrial status was assessed with the fluorescent dye, JC-1 (7.5μM in HEPES saline;; 30min, 37°C). The % of cells with an orange-stained midpiece was recorded (active mitochondria;; MIT). Resilience to hypoosmotic shock (HOS test) was assessed by diluting a sample in 100mOsm/kg HEPES saline (1:20; 15min, RT). An aliquot was stained with PI to assess plasma membrane status (HOSPMI), and the rest was fixed with formaldehyde, and % coiled tails (positive endosmosis;; HOST) was estimated using phase contrast microscopy (×400). Evaluations of PMI, MIT and HOSPMI were performed using fluorescence microscopy (×400, 450–490nm excitation filter). The results indicated that quality was good pre-freezing (TM: 60%; PMI: 86%; MIT: 100%), except for a PM value of 15%. After thawing, although there was a drop in TM (30%), there was no decrease in PM (20%). Our in vitro functional assessment indicated a loss of quality between the pre-freeze and post-thaw evaluations, but PMI and MIT maintained their pre-thaw levels (60% and 72%, respectively). The HOS test, which indicates plasma membrane integrity, decreased from the pre-freeze level (91%) to a post-thaw value of 70%. HOSTPMI was 72% pre-freeze, and decreased to 54% post-thaw. In conclusion, epididymal spermatozoa from the white rhino may retain its functionality after cryopreservation in a commerically available cryo-extender (Bidadyl). The use of assisted reproduction techniques could someday play a role in the management and conservation of the white rhinoceros and related species.

scholarly journals Fallot's Tetralogy in a European Brown Bear (Ursus arctos)

2005 ◽  
Vol 41 (4) ◽  
pp. 825-828 ◽  
Author(s):  
Erik Ågren ◽  
Arne Söderberg ◽  
Torsten Mörner
Keyword(s):  
Ursus Arctos ◽  
Brown Bear ◽  
Fallot's Tetralogy ◽  
Fallot’S Tetralogy

Histochemical and Morphological Studies of Lipids in the Oogenesis of Pheretima posthuma

1958 ◽  
Vol s3-99 (48) ◽  
pp. 475-484
Author(s):  
VISHWA NATH ◽  
BRIJ L. GUPTA ◽  
S. L. MANOCHA
Keyword(s):  
Phase Contrast ◽  
Chemical Change ◽  
Follicular Epithelium ◽  
Cholesteryl Esters ◽  
Lipid Bodies ◽  
Interference Microscope ◽  
Phase Contrast Microscope ◽  
Morphological Studies ◽  
Contrast Microscope ◽  
Second Category

A study of the oocytes of the earthworm, Pheretima posthuma, examined fresh under the phase-contrast and interference microscopes as well as by histochemical techniques, has revealed that there are two types of lipid bodies in the cytoplasm. The lipid bodies of the first type (L1) are smaller, appear as homogeneous, dark granules under the phase-contrast microscope, and have a protein-phospholipid core surrounded by a thick sheath of phospholipids only. The lipid bodies of the second category (L2), which arise as a result of growth and chemical change in L1 bodies, have a pure phospholipid core surrounded by a thick triglyceride sheath. They give a ringed appearance under the phase-contrast microscope. The study under the interference microscope shows that this ringed appearance is an optical artifact. The lipid spheres present in the follicular epithelium contain phospholipids only. The mitochondria are in the form of minute granules. They remain unchanged throughout oogenesis. Some vacuoles devoid of any lipids, proteins, or carbohydrates have been observed. They also remain unchanged. Pure triglyceride spheres, yolk globules, nucleolar extrusions, as well as cholesterols and cholesteryl esters are absent.

Automated Confluence Measurement Method for Mesenchymal Stem Cell from Brightfield Microscopic Images

2021 ◽  
pp. 1-9
Author(s):  
Zenan Wang ◽  
Rucai Zhan ◽  
Ying Hu
Keyword(s):  
Cell Culture ◽  
Phase Contrast ◽  
Low Cost ◽  
Cost Effective ◽  
Cell Phenotype ◽  
Cell Culture Process ◽  
Reconstruction Method ◽  
Image Reconstruction Method ◽  
Contrast Microscope ◽  
Culture Process

Cell confluence is an important metric in cell culture, as proper timing is essential to maintain cell phenotype and culture quality. To estimate cell confluence, transparent cells are observed under a phase-contrast or differential interference contrast microscope by a biologist, whose estimations are error-prone and subjective. To overcome the necessity of using the phase-contrast microscope and reducing intra- and inter-observer errors, we have proposed an algorithm that automatically measures cell confluence by using a commonly used brightfield microscope. The proposed method consists of a transport-of-intensity equation-based brightfield microscopic image processing, an image reconstruction method, and an adaptive image segmentation method based on edge detection, entropy filtering, and range filtering. Experimental results have shown that our method has outperformed several popular algorithms, with an F-score of 0.84 ± 0.07, in images with various cell confluence values. The proposed algorithm is robust and accurate enough to perform confluence measurement with various lighting conditions under a low-cost brightfield microscope, making it simple and cost-effective to use for a fully automated cell culture process.

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