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.

scholarly journals Effects of season and single layer centrifugation on bull sperm quality in Thailand

2020 ◽  
Vol 33 (9) ◽  
pp. 1411-1420
Author(s):  
Thanapol Nongbua ◽  
Apirak Utta ◽  
Nutthee Am-in ◽  
Junpen Suwimonteerabutr ◽  
Anders Johannisson ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane ◽  
Sperm Morphology ◽  
Sperm Quality ◽  
Single Layer ◽  
Normal Morphology ◽  
Fragmentation Index ◽  
Semen Characteristics ◽  
Bull Sperm ◽  
Dna Fragmentation Index

Objective: The aim of study was to investigate the effects of season and single layer centrifugation (SLC) before cryopreservation on post-thaw bull sperm quality in Thailand.Methods: Semen was collected from 6 bulls (Bos indicus) in summer, rainy season and winter 2014 through 2016. Semen characteristics, sperm morphology, sperm kinematics, viability, chromatin structure and mitochondrial membrane were evaluated. Meteorological data were available from the local meteorological station;Results: Season had an effect on semen characteristics in the raw ejaculate, with higher proportions of normal spermatozoa and lower abnormalities in winter than in the other two seasons. Sperm kinematics, viability, DNA fragmentation index, and mitochondrial membrane potential were not different between seasons. Sperm samples selected by SLC had greater normal morphology and a lower proportion with bent tails than controls and higher values of progressive motility (PRO), beat cross frequency, linearity, straightness, wobble (WOB), and lower values of slow motility, velocity average path (VAP), velocity curved line, and amplitude of lateral head displacement than controls. In addition, SLCselection had a favorable effect on PRO, VAP, and WOB that differed among seasons.Conclusion: Our results suggested that these bulls were well adapted to their location, with season having an effect on sperm morphology. Moreover, SLC could be used prior to cryopreservation, regardless of season, to enhance normal morphology and kinematics of bull sperm samples without adversely affecting other parameters of sperm quality. However, there was considerable variation among bulls in DNA fragmentation index, mitochondrial membrane potential and sperm viability. In addition, SLC had a positive effect on sperm morphology and sperm kinematics, which could be expected to influence fertility.

scholarly journals Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2128
Author(s):  
Marion Papas ◽  
Jaime Catalán ◽  
Sandra Recuero ◽  
Jane M. Morrell ◽  
Marc Yeste ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Seminal Plasma ◽  
Plasma Proteins ◽  
Mitochondrial Membrane ◽  
Single Layer ◽  
Polymorphonuclear Neutrophils ◽  
Lipid Disorder ◽  
Seminal Plasma Proteins

This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding.

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.

Effect of Single Layer Centrifugation on reactive oxygen species and sperm mitochondrial membrane potential in cooled stallion semen

2017 ◽  
Vol 29 (5) ◽  
pp. 1039 ◽  
Author(s):  
J. M. Morrell ◽  
A. Lagerqvist ◽  
P. Humblot ◽  
A. Johannisson
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Reactive Oxygen ◽  
Single Layer ◽  
Ros Production ◽  
Oxygen Species ◽  
Species Production

Additional means are needed for evaluating the quality of stallion spermatozoa in semen doses for AI. Mitochondrial membrane potential (ΔΨm) has been linked to fertility in some species, but is rarely used in the evaluation of cooled stallion semen; metabolic activity may be associated with reactive oxygen species production (ROS). In the present study, ΔΨm and ROS production were measured in doses of cooled stallion semen. The effect of colloid centrifugation on these parameters was also investigated. In this case, colloid centrifugation involves centrifuging a sperm sample through a silane-coated silica colloid formulation to retrieve the most robust spermatozoa. High and low ΔΨm in cooled stallion semen varied between stallions and between ejaculates, but was not affected by single-layer centrifugation (SLC). The SLC-selected spermatozoa produced significantly less hydrogen peroxide than controls (P < 0.001), which could explain the increased longevity and retention of fertilising capacity seen in previous studies. For SLC samples, ΔΨm was positively associated with viable spermatozoa that were not producing reactive oxygen species (r = 0.49; P < 0.001) and negatively associated with ROS production (for superoxide: r = –0.4, P < 0.01; for hydrogen peroxide: r = –0.39, P < 0.05). There was no clear association between ΔΨm and ROS production in control samples.

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.

Bovine sperm cells viability during incubation with or without exogenous DNA

Zygote ◽  
2009 ◽  
Vol 17 (4) ◽  
pp. 315-320 ◽  
Author(s):  
Weber Beringui Feitosa ◽  
Marcella Pecora Milazzotto ◽  
Renata Simões ◽  
Mariana Rovegno ◽  
Alessandra Coralo Nicacio ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Incubation Time ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Sperm Viability ◽  
Hoechst 33342 ◽  
Exogenous Dna ◽  
Bovine Sperm ◽  
Simultaneous Assessment

SummaryThe aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 × 106/ml with or without plasmid pEYFP–NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC–PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.9% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

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