Simultaneous evaluation of superoxide content and mitochondrial membrane potential in stallion semen samples provides additional information about sperm quality

2018 ◽  
Vol 192 ◽  
pp. 290-297 ◽  
Author(s):  
A. Johannisson ◽  
M.I. Figueiredo ◽  
Z. Al-Kass ◽  
J.M. Morrell
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Sperm Quality ◽  
Additional Information ◽  
Simultaneous Evaluation

54 SINGLE LAYER CENTRIFUGATION BEFORE CRYOPRESERVATION IMPROVES BULL SPERM QUALITY

2017 ◽  
Vol 29 (1) ◽  
pp. 134
Author(s):  
T. Nongbua ◽  
A. Utta ◽  
N. Am-In ◽  
J. Suwimonteerabutr ◽  
A. Johannisson ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Chromatin Structure ◽  
Mitochondrial Membrane ◽  
Linear Mixed Model ◽  
Sperm Quality ◽  
Single Layer ◽  
Sperm Chromatin ◽  
Sperm Viability ◽  
Bull Sperm

Single layer centrifugation (SLC) with Bovicoll is a technique to enhance sperm quality. The purpose of this study was to investigate the effect of SLC before cryopreservation on bull sperm quality after thawing. Semen was collected from 8 bulls (American Brahman, n = 5 and Sahiwal, n = 3) at the North Eastern Bull Centre (KhonKaen, Thailand). The ejaculate was split: one part was prepared following the standard procedure at the bull centre (n = 88) as control. The other part was used for SLC with Bovicoll-B (Johannisson et al. 2016 Theriogenology 86, 140). The SLC-selected sperm samples were frozen using the same protocol as control (n = 88). After thawing at 37°C for 12 s, motility analysis was performed using the CEROS II® (Hamilton Thorne, Beverly, MA, USA); sperm chromatin structure, mitochondrial membrane potential, and sperm viability were assessed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Treatment means were compared using the linear mixed model (Proc MIXED, SAS®, 9.3, SAS Institute Inc., Cary, NC, USA). Results are reported as least-squares means ± standard error. The sperm kinematics for SLC samples were higher than controls for progressive motility (26.37 ± 1.59%, 19.56 ± 1.59%), Linearity (LIN) (52.80 ± 0.87%, 44.94 ± 0.87%), Straightness (STR) (83.06% ± 0.59, 76.20 ± 0.59%), beat cross frequency (BCF) (29.25 ± 0.50 Hz, 24.35 ± 0.50 Hz) and wobble (WOB) (61.78 ± 0.63%, 57.40 ± 0.63%) (all P < 0.0001) respectively, whereas SLC-selected samples were lower than controls for slow motility (13.61 ± 0.71%, 15.56 ± 0.71%; P < 0.05), Amplitude of lateral head displacement (ALH) (4.88 ± 0.18 μm, 6.67 ± 0.18 μm), velocity average path, (VAP) (61.17 ± 1.93μ/s, 67.88 ± 1.93μ/s), and curvilinear velocity (VCL) (99.78 ± 3.77 μ/s, 122.91 ± 3.77 μ/s) (all P < 0.0001), respectively. Other parameters of sperm quality were not different between treatments, although there was considerable variation among individual bulls in sperm chromatin structure assay, mitochondrial membrane potential, and sperm viability. These results suggest that SLC can be used before cryopreservation to improve the kinematics of thawed bull sperm samples without adversely affecting other parameters of sperm quality.

114 Omega-3-enriched diet improves fertilization competence of cryopreserved sperm

2020 ◽  
Vol 32 (2) ◽  
pp. 184
Author(s):  
D. Kalo ◽  
D. Reches ◽  
A. Komsky-Elbaz ◽  
U. Moallem ◽  
Y. Zeron ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Dna Fragmentation ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Semen Quality ◽  
Sperm Quality ◽  
Fatty Acid Content ◽  
Moderate Increase ◽  
Omega 3 ◽  
Feeding Trial

Intensive reproductive management in dairy herds is mostly based on AI using high-merit bulls. Therefore, semen quality of bulls is of high importance. An association between semen quality and fatty acid content in feed has been suggested. Accordingly, the aim of this study was to examine the effect of omega-3 supplementation on sperm traits and fertilization competence. Fifteen Israeli Holstein bulls were assigned to three experimental groups. Bulls were fed over 13 weeks with a standard ration top-dressed with encapsulated-fat supplementation-fish oil (FO) or flaxseed oil (FLX; i.e. omega-3 sources), or saturated fatty acids (SFA, control). Ejaculates were collected before initiation of the study, during the feeding trial, and 1 month after feeding trial. Ejaculates were treated according to the routine procedure of the Israeli AI centre (Sion Ltd.), frozen, and stored in straws. Frozen-thawed samples were subjected to “swim-up” procedure, and spermatozoon viability, mitochondrial membrane potential, reactive oxygen species (ROS) level, acrosome membrane integrity, and DNA fragmentation were evaluated via flow cytometry, using sperm-specific kits (EasyCyte, IMV Technologies). Feeding with FO, FLX, or SFA did not affect the viability or mitochondrial membrane potential of sperm collected before, during, or after the feeding trial. On the other hand, a reduced proportion of sperm with ROS expression was recorded in the FLX samples compared to the SFA sample at the end of the feeding trial (42.2±1.2 vs. 47.3±4.3%, respectively; P&lt;0.05) and one month later (36.3±2.2 vs. 41.6±4.6%, respectively; P&lt;0.05). A low proportion of sperm with damaged acrosomal membrane was observed in both FLX and FO samples compared with SFA at the end of the feeding trial (48.8±3.4 and 41.7±2.7 vs. 59.8±3.4%, respectively; P&lt;0.05). The proportion of sperm with fragmented DNA was lower in the FLX group than in the SFA group, collected one month after the end of the feeding trial (2.3±0.6 vs. 5.4±1.2%, respectively; P&lt;0.05). To examine fertilization competence, oocytes were aspirated from ovaries collected from a local abattoir, then matured (n=216; 3 replicates) and fertilized invitro with a pool of samples from each group, collected one month after the end of the feeding trial (n=5 samples per group). The proportions of 2- to 4-cell-stage embryos and of blastocysts were determined 42h and 8 days after fertilization, respectively. Although the proportion of cleaved embryos did not differ between groups, a higher blastocyst formation rate was recorded in the FLX group (P&lt;0.05), and a moderate increase was noted in the FO group, relative to the SFA group (28.1±4.4, 19.1±2.6, and 11.9±3.4%, respectively). Results imply that feeding bulls with omega-3 originating from FLX improves sperm quality, most likely due to improved redox status and decreased DNA fragmentation. This nutritional approach seems to be an effective tool for improving bull fertility competence. Nevertheless, invivo examination is required.

scholarly journals Flurochloridone Induced Abnormal Spermatogenesis by Damaging Testicular Sertoli Cells in Mice

2021 ◽  
Author(s):  
Weiqi Sun ◽  
Fang Tian ◽  
Hongjie Pan ◽  
Xiuli Chang ◽  
Minjie Xia ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Mitochondrial Membrane ◽  
Cell Damage ◽  
Sperm Quality ◽  
Reproductive Toxicity ◽  
Sperm Concentration ◽  
Mitochondrial Damage

Abstract BackgroundFlurochloridone (FLC), a selective herbicide used on a global scale, has been reported to have male reproductive toxicity which evidence is limited and the mechanism is still unclear. The present study was conducted to systematically explore the male reproductive toxicity of FLC, including sperm quality, spermatogenesis process, toxicity targets and possible mechanisms. MethodsMale C57BL/6 mice aged 6-7 weeks received gavage administration of FLC (365/730 mg/kg body weight) for 28 consecutive days. Then the tissue and sperm of mice were collected for analysis. We measured the coefficient of male reproductive organs, and analyzed sperm concentration, motility, malformation rate and mitochondrial membrane potential. Spermatocyte immunofluorescence staining was performed to analyze meiosis processes. At the same time, we performed pathological staining on the testis and epididymis tissue, and performed TUNEL staining, immunohistochemical analysis and ultrastructural observation on the testicular tissue.ResultsThe results showed that FLC caused mice testicular weight reduction, dysfunction and architectural damage, but no significant adverse effect was found in epididymis. The exposure interfered with the proliferation of spermatogonia and the process of meiosis, affecting sperm concentration, motility, kinematic parameters, morphology and mitochondrial membrane potential, leading to sperm quality decline. Furthermore, mitochondrial damage and apoptosis of testicular Sertoli cells were observed in mice treated with FLC. ConclusionWe found that FLC has significant adverse effects on spermatogonia proliferation and meiosis. Meanwhile, apoptosis and mitochondrial damage may be the potential mechanism of Sertoli cell damage. Our study demonstrated that FLC could induce testicular Sertoli cell damage, leading to abnormal spermatogenesis which resulted in sperm quality decline and provided a methodological reference for related studies.

156 Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

2019 ◽  
Vol 31 (1) ◽  
pp. 203
Author(s):  
R. Felmer ◽  
C. Arroyo-Salvo ◽  
F. Fuentes ◽  
P. Cabrera ◽  
F. Treulen ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Tyrosine Phosphorylation ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Sperm Quality ◽  
Membrane Damage ◽  
Sperm Capacitation ◽  
Fluid Medium ◽  
Motility Parameters ◽  
Human Tubal Fluid

Conventional IVF has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. Thus, the first part of this study aimed to compare HTF (Summers and Biggers, 2003 Hum. Reprod. Update9, 557-582, DOI: 10.1093/humupd/dmg039) and Whitten’s (McPartlin et al. 2009 Biol. Reprod.81, 199-206, DOI: 10.1095/biolreprod.108.074880) media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine on sperm motility parameters was evaluated in both media at different incubation times. Fresh semen from 3 Chilote stallions was collected, diluted to 10×106 sperm mL−1 in capacitating (7mg mL−1 BSA and 25mM NaHCO3) and non-capacitating (without BSA and NaHCO3) HTF and Whitten’s media and incubated for 30 and 120min at 38°C in air atmosphere. Integrity and destabilisation of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (ΔΨm) using tetramethylrhodamine methyl ester perchlorate, acrosome membrane integrity by peanut agglutinin/fluorescein isothiocyanate and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® in a FACSCanto II flow cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ, USA). A total of 10,000 sperm events were acquired from each measurement (n=3 replicates for each stallion). Motility parameters were evaluated using the integrated semen analysis system (ISAS®, Selinion Medical, Brussels, Belgium). Percentage data were arcsine transformed and subjected to a 2-way ANOVA with Bonferroni’s post hoc test using Prism 7 software (GraphPad Software, La Jolla, CA, USA). We found no differences between Whitten’s and HTF media in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30 and 120min of incubation. Membrane fluidity (MC540) increased in both media at 30 and 120min of incubation compared with non-capacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2 and 4h of incubation compared with non-capacitating conditions, without differences between media. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm. Funding support was received from FONDECYT 1160467 CONICYT, Chile.

Ram spermatozoa migrating through artificial mucus in vitro have reduced mitochondrial membrane potential but retain their viability

2015 ◽  
Vol 27 (5) ◽  
pp. 852 ◽  
Author(s):  
Carmen Martínez-Rodríguez ◽  
Mercedes Alvarez ◽  
Elena López-Urueña ◽  
Susana Gomes-Alves ◽  
Luis Anel-López ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Mucus Penetration ◽  
Sperm Counts ◽  
Sample Test

Sperm motility in vitro is one of the most common predictors of fertility in male screening. We propose that a mucus-penetration assay can isolate a cellular subpopulation critical to reproductive success. To this end, a device was designed with three modules (sample, test and collection) and its conditions of use evaluated (length of mucus, incubation time, mucus medium, sperm concentration and position in relation to the horizontal). The number of spermatozoa migrating and the viability and acrosomal status of the spermatozoa not migrating were calculated. The second objective was to evaluate the qualitative parameters of the spermatozoa migrating in 1.6% polyacrylamide for 30 min. The number of spermatozoa migrating and the sperm motility, viability and the acrosomal and mitochondrial status of three sperm populations (fresh, not migrating and migrating) were determined. A higher number of migrating spermatozoa were observed after 60 min of incubation, but this situation adversely affected sperm quality. The methylcellulose-based test showed a significantly lower number of migrating spermatozoa than the polyacrylamide test. The position at an angle of 45° resulted in a higher number of migrating spermatozoa in the polyacrylamide-based test. The sperm counts for three consecutive assays indicated an acceptable repeatability of the method. The viability and acrosomal status of the migrating spermatozoa showed no significant changes with regard to the control when the device was placed at 45°, whereas these parameters showed lower values at 0°. The percentage of high mitochondrial membrane potential spermatozoa was significantly reduced in the population of migrating spermatozoa.

scholarly journals Integrity of mitochondrial membrane potential reflects human sperm quality

Andrologia ◽  
2009 ◽  
Vol 41 (1) ◽  
pp. 51-54 ◽  
Author(s):  
J. A. Espinoza ◽  
M. A. Schulz ◽  
R. Sánchez ◽  
J. V. Villegas
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Sperm Quality ◽  
Human Sperm

scholarly journals Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

2016 ◽  
Vol 36 (1) ◽  
Author(s):  
Subir Roy Chowdhury ◽  
Jelena Djordjevic ◽  
Benedict C. Albensi ◽  
Paul Fernyhough
Keyword(s):  
Oxygen Consumption ◽  
Membrane Potential ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Mitochondrial Bioenergetics ◽  
Respiration Rates ◽  
Pathological Conditions ◽  
Simultaneous Evaluation ◽  
Consumption Rates

Simultaneous evaluation of two mitochondrial bioenergetics parameters, respiration rates and mitochondrial membrane potential (mtMP) can be useful to determine the mitochondrial dysfunction under various pathological conditions including neurodegenerative diseases and diabetes.

scholarly journals Influence of Non-conventional Sperm Quality Parameters on Field Fertility in Ovine

2021 ◽  
Vol 8 ◽  
Author(s):  
Noelia Mendoza ◽  
Adriana Casao ◽  
Juan Domingo ◽  
Francisco Quintín ◽  
Adolfo Laviña ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Sperm Quality ◽  
Multiple Logistic Regression Analysis ◽  
Membrane Integrity ◽  
Quality Analysis ◽  
Reproductive Parameters ◽  
Economic Point ◽  
Intact Membrane

The prediction of the fertilizing ability of a seminal dose continues to be a primary aim in the field of artificial insemination (AI). To achieve this goal, in this study we have included the evaluation of some non-conventional sperm quality markers. A total of 3,906 ewes from 52 different farms were inseminated with 357 refrigerated seminal doses obtained from 45 mature Rasa Aragonesa rams. The same samples were used for sperm quality analysis including membrane integrity, capacitation status, oxygen consumption and apoptotic-like markers such as phosphatidylserine translocation (PS), plasmalemma disorganization/mitochondrial membrane potential, caspase activation and DNA damage. Seminal doses from the breeding (B) season presented higher percentages of intact membrane (IM), non permeant (NP) membrane with high mitochondrial membrane potential (ΔΨm) and IM without PS translocation spermatozoa than those from the non-breeding (NB) season. Therefore, we can conclude that there were less spermatozoa showing apoptotic-like features in the seminal doses from the B than the NB season, although these differences did not affect field fertility. Only the percentage of intact membrane, non-capacitated (IM-NC) spermatozoa showed a significant correlation with in vivo fertility (P = 0.005) and fecundity (P = 0.007) values obtained after cervical AI when all data were evaluated. When the data were sorted by season and distance to the farms where AI was performed, the correlation between the percentage of IM-NC spermatozoa and reproductive parameters increased in the NB season and progressively with remoteness from the farms. Some other sperm parameters, like NP with high ΔΨm, IM sperm without active caspases and DNA-intact spermatozoa, also showed significant correlations with the reproductive parameters in the sorted data. Moreover, the increment in both the percentage of IM-NC and DNA-intact spermatozoa would increase the probability of obtaining a fertility higher than the mean (&gt;52%), as revealed by a multiple logistic regression analysis. In conclusion, we have identified two seminal markers—the percentage of intact membrane, non-capacitated spermatozoa, and DNA intact spermatozoa—which could be used as a test to discard males in AI programs, which is highly important from an economic point of view and can contribute to achieving satisfactory fertility rates.

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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