Mitochondrial activity of frozen-thawed spermatozoa assessed by MitoTracker Deep Red 633

Hallap, Triin; Nagy, Szabolcs; Jaakma, Ülle; Johannisson, Anders; Rodriguez‐Martinez, Heriberto

doi:10.1016/j.theriogenology.2004.10.010

Cited by 80 publications

(65 citation statements)

References 13 publications

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“…Mitochondrial transmembrane potential was evaluated with Mitotracker Deep Red FM (Life Technologies, M22426) fluorochrome according to Hallap et al [12]. The fluorescent probe indicates high mitochondrial membrane potential with a high far red signal (Fig.…”

Section: Mitochondrial Activitymentioning

confidence: 99%

Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity — A flow cytometric study

Nagy

Kakasi

Pál

et al. 2016

Acta Biologica Hungarica

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Local extreme climatic conditions occurring as a result of global climate change may interfere with the reproduction of animals. In the present study fish spermatozoa were incubated at different temperatures (20, 25, 30 and 40 °C) for 10 and 30 minutes, respectively and plasma membrane integrity and mitochondrial membrane potential changes were evaluated with flow cytometry using SYBR-14/PI and Mitotracker Deep Red FM fluorescent dyes. No significant differences were found in plasma membrane integrity at either incubation temperatures or time points. Mitotracker Deep Red FM histogram profiles indicating mitochondrial activity showed significant (p < 0.001) alterations in all cases of higher (25, 30 and 40 °C) temperature treatments as compared to the samples incubated at 20 °C. Our results indicate that fish spermatozoa exposed to high temperatures suffer sublethal damage that cannot be detected with conventional, vital staining techniques.

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Section: Mitochondrial Activitymentioning

confidence: 99%

Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity — A flow cytometric study

Nagy

Kakasi

Pál

et al. 2016

Acta Biologica Hungarica

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“…77 There are approximately 100 mitochondria in the mid-piece of the spermatozoon, depending on the species. 78 Most of these dyes applied in the evaluation of mitochondrial status work by diffusing into living cells and accumulating in mitochondria. The most widely used mitochondrial-specific probe, rhodamine 123 (R123), is a cationic compound that accumulates in the mitochondria as a function of transmembrane potential.…”

Section: Sperm Intactnessmentioning

confidence: 99%

“…They hypothesized that cryopreserved sperm cells have damaged metabolic capacity, probably due to glycerol. We developed an alternative labeling technique, using MitoTracker Deep Red, 78 which needs only one detector to identify spermatozoa with high and low mitochondrial membrane potential ( Figure 6). Unfortunately, the spectral properties of this particular dye do not fit to the smaller bench top FCs equipped with a 488-nm laser source only.…”

Section: Sperm Intactnessmentioning

confidence: 99%

“…In our laboratory, where we use a BD LSR FC equipped with three laser lines, we usually apply a DNAspecific, membrane permeable dye to label every sperm cell (as with the exception of pathological cases we do not expect to have other particles with DNA in the sperm samples) and add a fluorescent gate to the scatter gate to narrow our analysis to spermatozoa only, for example, Merocyanine 540/YO-PRO-1/Hoechst 33342 50 where the Hoechst signal was used as fluorescent gate, or MitoTracker Deep Red 633 in combination with SYBR14, where the later was used to label every DNA-containing event. 78 Obviously, this approach needs a larger, more expensive instrument, and cannot always be followed in laboratories equipped with smaller, single-laser FCs. As an alternative, the use of pretreatments and a mathematical model of alien particles can reduce particle misidentification, 173 and can be performed in parallel with the functional assessment in the same FC.…”

Section: Detection Of Oxidative Stress and Lipid Peroxidationmentioning

confidence: 99%

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Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

Hossain¹,

Johannisson²,

Wallgren³

et al. 2011

Asian J Androl

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135

108

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Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of 'bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.

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“…The recently developed MitoTracker V R dyes can be used to stain active mitochondria in a highly specific manner. These fluorochromes are available in a wide range of emission wavelengths and could be applied in multicolor studies of spermatozoa (13)(14)(15)(16). Egg yolk particles do not have mitochondria, a fact that is exploited in the present study to enable differentiation of spermatozoa from the egg yolk particles or subpopulations of non-sperm events based on a fluorescent marker.…”

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confidence: 99%

Comparison of electronic volume and forward scatter principles of cell selection using flow cytometry for the evaluation of acrosome and plasma membrane integrity of bull spermatozoa

Standerholen

Myromslien²,

Kommisrud³

et al. 2014

Cytometry Pt A

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The objective of the study was to compare two different flow cytometers to reveal if there are differences between them and to find the most suitable protocol for analysis of spermatozoa. These two flow cytometers; Cell Lab Quanta TM and Coulter Epics XL, have different principles to calculate cell size, electric volume, and forward scatter (FS), respectively. Flow cytometry is a valuable tool to assess various spermatozoa quality traits simultaneously, such as plasma membrane and acrosome integrity. A double-and triple-stain combination was performed to compare evaluation of these two parameters by both flow cytometers and to assess the need of a fluorescent probe to identify the spermatozoa. Propidium iodide was used to assess the proportion of dead spermatozoa, whereas Alexa Fluor V R 488 conjugated peanut agglutinin (PNA-Alexa 488) was used to evaluate the percentage of acrosome intact and acrosome-reacted cells or degenerated cells. In the triple-stain protocol, MitoTracker V R Orange (MO) was included to test the capacity of this probe to discriminate spermatozoa from egg yolk and debris particles present in the semen sample. Cryopreserved semen from 13 Norwegian Red bulls was included in the study and the semen was evaluated immediately after thawing and after 3 hr incubation at 37 C. The results show that there is good agreement between the instruments. Nevertheless, a significant difference was found in percentages of acrosome intact live spermatozoa (% AIL) when including MO as a spermatozoa identification probe, compared to assessment without MO, with the Coulter Epics XL, while no significant difference was found when including the probe with the Cell Lab Quanta. In conclusion, the results show that cell size measurement based on electronic volume used by the Cell Lab Quanta flow cytometer is more accurate than FS used by the Coulter Epics XL flow cytometer in identification of spermatozoa. V C 2014 International Society for Advancement of Cytometry Key terms flow cytometry; sperm analysis; electronic volume; forward scatter; plasma membrane integrity; acrosome integrity FLOW cytometry has become a powerful tool for characterization of cell populations. The instrument has the capability to measure multiple parameters of individual cells, thus it is possible to detect abnormal cells in a short time (1-3). The flow cytometric analysis is based on light-spreading and fluorescence signals. Light that is scattered at high angles is termed side scatter (SS) and will provide information about such intracellular properties as cell granularity (4). Scattered light with a small angle is termed forward scatter (FS) and depends on particle size and the refractive

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Mitochondrial activity of frozen-thawed spermatozoa assessed by MitoTracker Deep Red 633

Cited by 80 publications

References 13 publications

Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity — A flow cytometric study

Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity — A flow cytometric study

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

Comparison of electronic volume and forward scatter principles of cell selection using flow cytometry for the evaluation of acrosome and plasma membrane integrity of bull spermatozoa

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