PSX-A-7 Late-Breaking: Correlation of mitochondrial membrane potential and rough motility scores in cryopreserved bovine spermatozoa

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 362-363
Author(s):  
Lauren Clark ◽  
Jolena N Waddell ◽  
David Roper ◽  
William B Smith ◽  
Cheyenne L Runyan
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Reproductive Tract ◽  
Pearson Correlation ◽  
Reproductive Technologies ◽  
Female Reproductive Tract ◽  
Strong Positive Correlation ◽  
University Of Texas ◽  
Progressive Motility

Abstract Spermatozoon motility is an important factor in successful artificial reproductive technologies. Successful reproduction requires properly developed spermatozoa with adequate forward, progressive motility that allows for transport through the female reproductive tract. Motility is driven by production of ATP; however, cryopreservation is known to have damaging effects on spermatozoa. Mitochondria utilize oxidative phosphorylation to synthesize ATP through an electrochemical proton motive force that is composed primarily of mitochondrial membrane potential (Δψm). The mitochondrial membrane potential can be measured using a fluorescent, carbocyanine JC-1 dye (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide). The objective of this study was to examine correlations between the mitochondrial membrane potential and motility score. Cryopreserved semen samples from three bulls were donated to Tarleton State University for research purposes. Samples were thawed, placed under a phase contrast microscope, and analyzed by three individuals with varying levels of training for rough motility. These samples were then prepared with JC-1 dye according to manufacturer’s instructions. Two microliters of dye were added to a stock preparation of each sample. Samples were examined on a FACS Calibur flow cytometer at University of Texas Southwestern Core in Dallas, TX. Flow cytometry analysis was performed using FlowJo V10.7. Statistical analysis was performed using SAS v9.4. The Pearson correlation coefficient showed a strong, positive correlation (r = 0.90) between the mitochondrial membrane potential and motility (P = 0.28), thus indicating as the mitochondrial membrane potential increases, so does the rough motility score. These data represent a subset of a population that demonstrates the need of further research on the ability for spermatozoa to produce ATP and the correlation in forward, progressive motility. This research can provide a foundation in which future researchers may develop an assay that allows for testing of mitochondrial membrane potential by producers to select bulls with greater breeding potential.

63 STEPWISE THAWING PRESERVES MITOCHONDRIA MEMBRANE POTENTIAL OF BOAR SPERMATOZOA EXTENDED IN GLYCEROL-FREE MEDIUM CONTAINING TREHALOSE

2015 ◽  
Vol 27 (1) ◽  
pp. 124
Author(s):  
R. Athurupana ◽  
H. Funahashi
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Semen Analysis ◽  
Pearson Correlation ◽  
Computer Assisted ◽  
High Fertility ◽  
Positive Trend ◽  
Mitochondria Membrane Potential ◽  
Boar Spermatozoa

Motility and penetrability of spermatozoa are significantly affected by the energy produced in mitochondria. The survival of cryopreserved spermatozoa is influenced by the warming rate as well. The objective in the present study was to evaluate the effect of stepwise thawing on post-thaw motility and mitochondria activity of boar spermatozoa. The sperm samples collected from a Berkshire boar with high fertility at an AI centre were diluted in egg yolk-based and glycerol-free freezing extender containing 100 mM trehalose and 0.25% Equex STM™. The samples were cryopreserved using the straw freezing procedure. Best thawing durations in high temperatures were examined in a previous study. Straws were immersed in 80, 70, and 60°C water for 6, 8, and 10 s and continued to thaw in 39°C for 54, 52, and 50 s, respectively. Sperms thawed at 39°C for 60 s were considered as the control. Frozen-thawed spermatozoa were analysed for motility with computer-assisted semen analysis and mitochondrial membrane potential (MMP) with JC-1/PI staining (Table 1). Data were analysed with ANOVA and Pearson correlation. Motility and MMP of frozen-thawed sperm were significantly lower than fresh samples. Motility was fairly high compared with frozen-thawed controls, when thawed rapidly in 2-steps, though the difference was not significant. The MMP was also significantly higher when thawed in 2-steps from 80 or 70°C, then to 39°C, as compared with frozen-thawed controls, and it was positively correlated with the thawing temperature (r = 0.64; P < 0.01). There was a positive trend between thawing temperature and motility, but it was not significant (r = 0.37; P = 0.11). In conclusion, our results suggest that stepwise thawing with a rapid thawing at the beginning preserves post-thaw motility and MMP of boar spermatozoa. Table 1.Effect of thawing temperature on post-thaw motility and mitochondrial membrane potential (MMP)1

scholarly journals Cryopreservation of boar semen in 0.5mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability

2018 ◽  
Vol 38 (9) ◽  
pp. 1726-1730 ◽  
Author(s):  
Gisele M. Ravagnani ◽  
Mariana A. Torres ◽  
Diego F. Leal ◽  
Simone M.M.K. Martins ◽  
Frederico O. Papa ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Membrane Lipid ◽  
Membrane Lipid Peroxidation ◽  
Progressive Motility ◽  
Acrosomal Membrane ◽  
Boar Spermatozoa

ABSTRACT: To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.

scholarly journals Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes

Reproduction ◽  
2017 ◽  
Vol 153 (3) ◽  
pp. 293-304 ◽  
Author(s):  
C Ortega-Ferrusola ◽  
L Anel-López ◽  
P Martín-Muñoz ◽  
J M Ortíz-Rodríguez ◽  
M C Gil ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Reproductive Tract ◽  
Relevant Information ◽  
Computational Techniques ◽  
Reduction Techniques ◽  
Cellular Expression

The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes. However, there is a lack of studies investigating both phenomena simultaneously. In order to improve our knowledge in this particular point, we studied in raw and frozen-thawed samples apoptotic and capacitative markers using a wide battery of test based in flow cytometry. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. Conventional and computational flow cytometry using nonlinear dimensionally reduction techniques (t-distributed stochastic neighbor embedding (t-SNE)) and automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) were used. Most of the changes induced by cryopreservation were apoptotic, with increase in caspase 3 activation (P < 0.01), PS translocation to the outer membrane (P < 0.001), loss of mitochondrial membrane potential (P < 0.05), and increase in intracellular Na+ (P < 0.01). Average values of markers of capacitative changes were not affected by cryopreservation; however, the analysis of the phenotype of individual spermatozoa using computational flow cytometry revealed the presence of subpopulations of spermatozoa experiencing capacitative changes. For the first time advanced computational techniques were applied to the analysis of spermatozoa, and these techniques were able to disclose relevant information of the ejaculate that remained hidden using conventional flow cytometry.

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

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