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 < 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).

scholarly journals The antioxidant effects of soybean lecithin- or low-density lipoprotein-based extenders for the cryopreservation of brown-bear (Ursus arctos) spermatozoa

2013 ◽  
Vol 25 (8) ◽  
pp. 1185 ◽  
Author(s):  
M. Alvarez-Rodríguez ◽  
M. Alvarez ◽  
L. Anel-López ◽  
C. Martínez-Rodríguez ◽  
F. Martínez-Pastor ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Ursus Arctos ◽  
Soybean Lecithin ◽  
Density Lipoprotein ◽  
Brown Bear ◽  
Egg Yolk ◽  
Type A ◽  
Low Density ◽  
Sperm Viability ◽  
Anti Oxidant

Egg yolk low-density lipoproteins (LDL) and soybean lecithin were evaluated as replacements for egg yolk in extenders used for the cryopreservation of brown-bear spermatozoa. The motility, viability and acrosomal status of post-thawed spermatozoa were analysed, and an egg-yolk extender was used as a control. The total antioxidant capacity of these extenders was tested. Soybean lecithin showed an effect that was dependent on the soybean concentration (2%, 3.5% and 5%) and source (Type A: 24% l-α-phosphatidylcholine, and Type B: 14–23% l-α-phosphatidylcholine). Only semen cryopreserved with 5% Type A soybean exhibited a sperm motility similar to that of semen cryopreserved in egg-yolk-based extender after thawing, although the sperm viability and acrosome status were not as high. Semen frozen in an extender containing LDL (10–15%) exhibited improved sperm viability in comparison with the control, but sperm motility was lower. The LDL-based extender exhibited a higher anti-oxidant activity than the egg-yolk extender and soy lecithin-based extenders. The extenders with higher anti-oxidant activity showed improvements in frozen sperm viability but lower semen motility. These results indicate that soybean lecithin did not have the same protective effect as egg yolk during the freezing of brown-bear spermatozoa but suggest that LDL (10–15%) could be a useful substitute for egg yolk in these extenders.

scholarly journals HVCN1 Channels Are Relevant for the Maintenance of Sperm Motility During In Vitro Capacitation of Pig Spermatozoa

2020 ◽  
Vol 21 (9) ◽  
pp. 3255
Author(s):  
Marc Yeste ◽  
Marc Llavanera ◽  
Yentel Mateo-Otero ◽  
Jaime Catalán ◽  
Sergi Bonet ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Membrane Lipid ◽  
Sperm Head ◽  
Sperm Viability ◽  
Lipid Disorder ◽  
Acrosomal Exocytosis ◽  
Control Samples

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca2+ entrance to the sperm head.

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.

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 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

scholarly journals Cooling of equine semen at 16°C for 36 hours with addition of different glutathione concentrations

2015 ◽  
Vol 36 (6) ◽  
pp. 3699
Author(s):  
Rodrigo Arruda de Oliveira ◽  
Marco Antônio De Oliveira Viu ◽  
Maria Lúcia Gambarini
Keyword(s):  
Lipid Peroxidation ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Membrane Lipid ◽  
Sperm Cells ◽  
Sperm Viability ◽  
Membrane Lipid Peroxidation ◽  
Acrosomal Membrane ◽  
In Vitro Effects

Handling equine semen during the refrigeration process reduces sperm viability, and consequently causes membrane lipid peroxidation, among other challenges. The present study aimed to evaluate the in vitro effects of glutathione (control, 1. 0, 1. 5, and 2. 5 mM) on equine semen in a refrigeration protocol of 16ºC for 36 hours. The following variables were evaluated after 0, 12, 24, and 36 hours refrigeration: total sperm motility, vigor, viability, and plasma and acrosomal membrane integrity. Motility was higher with 2. 5mM of glutathione (57. 8 ± 7. 3) after 12 hours of refrigeration compared to the control (53. 2 ± 8. 3) (P < 0. 05). After 36 hours of refrigeration, motility was higher with 1. 5 mM (43. 4 ± 12. 7) and 2. 5mM glutathione (45. 5 ± 6. 2), than it was with 1mM glutathione (38. 2 ± 9) and the control (35. 5 ± 18. 4) (P < 0. 05), respectively. Vigor was highest with 1. 5mM glutathione (3. 7 ± 0. 3) after 36 hours compared to the control (3. 2 ± 1. 1), (P < 0. 05). Viability differed between control and 1mM treatments (79. 5 ± 1. 8) only after 24 hours (75. 5 ± 9. 7) (P < 0. 05). Throughout the investigation, no significant differences were noted in plasma and acrosomal membrane integrity (P > 0. 05). The 1. 5 and 2. 5mM glutathione levels were more efficient in protecting sperm cells and yielded higher total motility values after 36 hours of refrigeration.

scholarly journals Phenological synchronization disrupts trophic interactions between Kodiak brown bears and salmon

2017 ◽  
Vol 114 (39) ◽  
pp. 10432-10437 ◽  
Author(s):  
William W. Deacy ◽  
Jonathan B. Armstrong ◽  
William B. Leacock ◽  
Charles T. Robbins ◽  
David D. Gustine ◽  
...  
Keyword(s):  
Sockeye Salmon ◽  
Ursus Arctos ◽  
Large Body ◽  
Brown Bear ◽  
Switching Behavior ◽  
Air Temperatures ◽  
Trophic Resources ◽  
Energy Flows ◽  
Strength Of Interaction ◽  
Prey Interaction

Climate change is altering the seasonal timing of life cycle events in organisms across the planet, but the magnitude of change often varies among taxa [Thackeray SJ, et al. (2016) Nature 535:241–245]. This can cause the temporal relationships among species to change, altering the strength of interaction. A large body of work has explored what happens when coevolved species shift out of sync, but virtually no studies have documented the effects of climate-induced synchronization, which could remove temporal barriers between species and create novel interactions. We explored how a predator, the Kodiak brown bear (Ursus arctos middendorffi), responded to asymmetric phenological shifts between its primary trophic resources, sockeye salmon (Oncorhynchus nerka) and red elderberry (Sambucus racemosa). In years with anomalously high spring air temperatures, elderberry fruited several weeks earlier and became available during the period when salmon spawned in tributary streams. Bears departed salmon spawning streams, where they typically kill 25–75% of the salmon [Quinn TP, Cunningham CJ, Wirsing AJ (2016) Oecologia 183:415–429], to forage on berries on adjacent hillsides. This prey switching behavior attenuated an iconic predator–prey interaction and likely altered the many ecological functions that result from bears foraging on salmon [Helfield JM, Naiman RJ (2006) Ecosystems 9:167–180]. We document how climate-induced shifts in resource phenology can alter food webs through a mechanism other than trophic mismatch. The current emphasis on singular consumer-resource interactions fails to capture how climate-altered phenologies reschedule resource availability and alter how energy flows through ecosystems.

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