Matthias Krienke

Matthias KrienkeFACS cell sorting lab

12.02
· Dr. med. vet.
  • About
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    Education
    Feb 1983 - Oct 1989
    Freie Universität Berlin and RUCA Antwerpen (Belgien)
    Tiermedizin
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    Research Items (6)
    Question - How can I remove unwanted cell "trash" from hepatocyte Isolation?
    Answer
    Can you show us your SSC/FSC dot plot with gate
    Question - Does anyone have experience in preparing strongly adherent cells (U2OS) for FACS without using acute dissociation treatments such as trypsin?
    Answer
    you can use EDTA and a potter
    Question - I would like to analyse apoptosis by flow cytometry technique. Can anybody suggest the dye which can be utilized for this purpose?
    Answer
    I tink Using PI + annexin V-FITC in cominatiom with tunel assy. Also MMP with JC-1 gives results for apoptosis.
    Question - Do you have experience with LumiSort?
    Answer
    see attachment and internet!
    It looks verry interesting: no elektric charge and the orientation from sperms are no longer importent so better fertility (not only hifers) and better yield
    Question - How can I set PMT voltage of FL1 and FL2 channels?
    Answer
    what fluorochrome do u use in FL1 and FL2 ?
    Question - How can I set PMT voltage of FL1 and FL2 channels?
    Answer
    what cytomyter do you use?
    Can you show us a dot plot FS vs. SSC please.
    Question - Why do my NIH 3T3 cells die after single cell sorting?
    Answer
    And dont sort verry high GFP pos cells. Try it with lower green cells
    Question - Why do my NIH 3T3 cells die after single cell sorting?
    Answer
    Medium to put the cells in the sorter: 1X PBS by adding HEPES to 25 mM or using HBSS (Hanks's), or cultur medium with out phenolrot,
    Medium in the wells: cultur medium or HBSS 20 % FCS or more. So here you can use pure serum. Some medium where are the cells be happy
    Question - Why do my NIH 3T3 cells die after single cell sorting?
    Answer
    You can try to sort in pure serum
    Question - Why do my NIH 3T3 cells die after single cell sorting?
    Answer
    So in what medium your cells went into the sorter?
    Question - Can we compare the mean fluorescence intensity from different samples in FACS?
    Answer
    Simply yes
    Question - Is there an important difference between PFH fixation and BD's Cytofix?
    Answer
    What is a Filipin staining?
    Question - Can I use two different antibodies from the same host but conjugated with different fluorescent dyes?
    Answer
    Yes
    Question - Is it possible to stain glycogen in hepatocytes with fluorescence dye?
    Answer
    I cant use glycogen-AB because I need the cells vital
    Question - What is the best way to check for mycoplasma in cell culture?
    Answer
    I use MycoAlert™ PLUS Mycoplasma Detection Kit from Lonza. But you need a luminometer. It works verry well and verry sensitiv.
    Question - Can anyone suggest a fixation/permeabilization protocol effective for FACS on intracellular markers but results in minimal RNA leakage from the cells?
    Answer
    Saponin is the softes way to permeabilization and the permeabilition is reversible if you removed saponin from your cells.
    Question - Is it normal to find two big populations when you exclude doublets in flow cytometry (FSC-H vs FSC-W)?
    Answer
    Now i have 3 Population only with FS-W. But SS-W vs SS-H looks normal (1 population and 5% dublettes).
    I phoned with Mr. Egner (application specialist from Beckman/Germany) and he told me that I dont have to use the Parameter FS-Width by MoFlo machines to discriminate doublets, because FS-W is realy not the time-of-flight but only the half of FS-H. It is not the same like BD machines. So I had to use FS-A vs FS-H and discriminate all events which are not located on the diagonal. So I am sorting my schwann cells with both plots SS-W vs SS-H and FS-A vs FS-H and I am discriminate 10% doubletes and the purity of the sorting was 99%. So I think i will prefer this way of discriminating doubletes when sorting with my MoFlo.
    If you change the Forward-scatte hight your 2 populations change to one population and every looks normal. But I will use in the future FS-A vs FS-H and SS-W vs SS-H to discriminate dublettes.
    Question - Is there an easy way to count cells?
    Answer
    With FACS ARIA from BD or MoFlo from Beckman you can deposit single cell or another count of cells in any well plate you want.
    Question - How can we check the proliferation rate in the normal cell lines?
    Question - Is there an easy way to count cells?
    Answer
    If you can use a flow cytometer cell sorter you can deposit exactly 3000 (vital that means by example PI neg.) per well.
    Question - Does anyone use propidium iodide for canine spermatozoa plasma membrane integrity assessment?
    Question - Does anyone use propidium iodide for canine spermatozoa plasma membrane integrity assessment?
    Answer
    Yes you can use the exactly the same protocol for PI staining for canine sp. as discribed for bulls.
    Greatings from Germany
    Question - Can anyone help with CD31 (PECAM-1) immunofluorescence protocol in PFA fixed tissues?
    Answer
    sorry i have no new idear but its known thats a big problem with samples fixed so long in PFA
    Question - Is it normal to find two big populations when you exclude doublets in flow cytometry (FSC-H vs FSC-W)?
    Answer
    I have the same problem with my MoFlo-XDP. When I chanche the hight of the FS than the 2 populations goes together to one nice population.
    No one can give me an explanation for this phenomenon.
    Question - Can anyone help with CD31 (PECAM-1) immunofluorescence protocol in PFA fixed tissues?
    Answer
    Hi
    I only know DyLight 549 or Alexa 546. But anyway for both you need a green laser (%60 nm). Do you use such a green laser for exitation?
    Question - Can anyone help with CD31 (PECAM-1) immunofluorescence protocol in PFA fixed tissues?
    Answer
    page 9
    What Fluorochrom do yue use with you antibody
    Question - Can anyone help with CD31 (PECAM-1) immunofluorescence protocol in PFA fixed tissues?
    Answer
    Why do you fix your bone marrow samples so long (24 h) in PFA?
    Question - Why do H357 cells die after FACS?
    Answer
    Hi Holger
    can you tell me something about the concentration of Y-27632 . dihydrochloride as a culture media supplement
    Question - Can we store Annexin V FITC stained cells and use after a week?
    Answer
    FITC bleach very quickly and strong
    Question - Why do H357 cells die after FACS?
    Answer
    how high was the pressure during the sort? (PSI)
    Question - Can anyone help me find my population?
    Answer
    can you please schow us your Dot plots
    Question - How can I prepare a Stock and Work Solution of Acridine Orange Dye (for Integrity of DNA) and PNA-Alexa 488 (for acrosomal integrity)?
    Answer
    it is the same for bulls and stallions sperm but the use of syto 17 is a fault sorry. You have to leave the living sperm unstained or you had use a red laser for syto 17.
    Good luck
    (but please think at the few DFI-sperms by bull sperma (0-3%) stallion sperms like human sperms the cut off is arround 10%)
    Question - How can I prepare a Stock and Work Solution of Acridine Orange Dye (for Integrity of DNA) and PNA-Alexa 488 (for acrosomal integrity)?
    Question - Can I store PBMC suspended in PBS at 4 degrees or -80*C?
    Answer
    I did bovine PBMC after Ficoll centrifugation suspended in PBS at 4 degree for 5 day and after this 70% are vital (PI neg). For store at - 80 degree you had to use DMSO 10% I think
    Question - Can I make pmCherry and GFP express in the same cell (stable cell line) and use the same to FACS cherry+GFP+ cells?
    Answer
    You will need 2 lasers (blue 488 nm and yellow 560 nm) for excitation.
    Question - Does anyone know a good blocking reagent to reduce the background signal from mouse hepatocyte?
    Answer
    hepatocyte have high level of autofluo. in the green spectrum. Better use an antibody with Fluoch. red or far red
    Question - Is acridine orange a proper marker for lysosomes?
    Answer
    AO shows green fluorescence when bound to d-DNA and red when bound to s-DNA
    Question - What is the optimal cell concentration for a HepG2 cell culture?
    Answer
    Hi
    What is the respons stimulating with hGH ??????
    Question - Cell cycle measurement facs?
    Answer
    Thanks a lot
    Question - Cell cycle measurement facs?
    Answer
    how long do you incubate your cells with PI
    Question
    Question - How to do FACS on primary mouse hepatocytes?
    Answer
    we did diskriminate gfp+ cells with high autofluorescene. so we made a plot fl1 vs. fl2
    and so you can see verry fine 3 populations (importent make no compensation). see atachment
    So you can discrimeanate autoflourecente
    Question - What is the possible explanation for plasticity of CD25loFoxp3+ Treg cells?
    Answer
    Th17 cells also have regulatory function too ? In the future I had to sort Tregs so they schoud converts into Th17 cells. Is it right that I had to sort T-cells CD4+ CD 25+ low not high
    Question - For measurement of cell cycle with FACS and PI, is it better to do the measurement on Fl2 (580/30) or Fl3 (613/30) or is the same?
    Answer
    Thanks
    Question - SCSA Software
    Answer
    We too
    Question - Acridine orange DNA fragmentation test?
    Answer
    And do the SCSA evrry time 0h directly after thawing, 3h after incubation by 37 C like Evenson. We did it also 6h after incubation by 37 C
    Question - Acridine orange DNA fragmentation test?
    Answer
    But if you want to do your samples with FACS look for a cheep lab and sent your probs schochfrozen in epis in liquet N2 to do the scsa in the lab. How many samples do you want to investigate?
    Question - Acridine orange DNA fragmentation test?
    Answer
    I think with microscop is not a good method, becaus the red or orange cuolore is exspacialy by bulls verry verry weak because bulls have so strong chromatinstructur. We did here in hannover als SCSA with bulls and a longer expose of the acid and the result are lower than by men and horses.
    Question - Is it okay to stain cells for both intercellular and surface marker for FACS analysis? How can you standardize your FACs measurement ?
    Answer
    PFA alone is only fix
    methanol and Ethanol is fix and permea
    Question - Is it okay to stain cells for both intercellular and surface marker for FACS analysis? How can you standardize your FACs measurement ?
    Answer
    did you use 70 % methalon (with PBS not with aqua bidest) cold, dropwise
    Question - Is it okay to stain cells for both intercellular and surface marker for FACS analysis? How can you standardize your FACs measurement ?
    Answer
    It is possible that your fixation decrease the florescent detection for surface markers.
    I would compare the samples with fixation and without fixation. So you see if it is the same fluoreszenzintensity. You can standardize your measurement with measure at the beginning of your work beads. So with changing the volts of your PMT's your beads have allways the same fluoreszenzintensity.
    Question - Does anyone know a protocol for human NK cells isolation that does not need MACS or FACS systems?
    Question - How to separate live and dead rat cardiomyocytes?
    Answer
    color cells with PI or DAPI or sytox green and sort teh live cells with FACS
    Question - Acridine orange DNA fragmentation test?
    Answer
    I did the scsa with stallion sperm and it has worked well. We made from one ejaculat from a healthy doner ca 100 probes and tested this after every 10. sample so if you have alway the same MFI your scsa-test works well.
    Question - Acridine orange DNA fragmentation test?
    Answer
    but % DFI sperms are verry low in bovine sperm (Around 2-5%) because the chromatin structure .
    Question - Why is DAPI unable to penetrate the cell membrane but Hoechst stain does?
    Answer
    Molecular Probes call DAPI semi-permeable it depense on concentration
    Question - Why is DAPI unable to penetrate the cell membrane but Hoechst stain does?
    Answer
    But if the DAPI concentration is too high live cells are stained too
    Question - Can someone advise on problems with gating on FACSCalibur?
    Answer
    What program are you using to analyze the data?
    Question - How to stop of shifting of cell clusters on FACS profile?
    Answer
    What kind of sorter do you use
    Question - Easy, fast, routine mycoplasma detection assay for cell culture contamination?
    Answer
    I use Lonza kit, MycoAlert too. IT works well, but you need a luminometer
    Question - What could be the reason for transfected K562s to die during cell sorting?
    Answer
    what nozzle size and pressure (PSI) do you work
    Question - What is the best or suitable protocol to detect the accumulation of reactive oxygen species in cell culture?
    Answer
    You can also use a FACS to meeasure fluorescent intensity.
    You can use also other dyes like DCFH, DCFH-DA/PI Assay; DHR, DHR/PI Assay; NO, DAF-2-DA/PI Assay; MITOSOX, MITOSOX/Sytox Green Assay.
    Question - How can you sterilize a liposuction cannula?
    Answer
    no povidone Iodine
    Question - How can you sterilize a liposuction cannula?
    Answer
    commercial instruments disinfection n povidone Iodine
    Question - How can I change the scale from log to 1024 in FACScanto II?
    Answer
    You must use FL-xx lin not FL-xxx log. Right mouse click on the parameter FL-xxx or SSC or FSC. What Program do you use DIVA ????
    Question - Does anyone has an idea how small particles (subcell dimension) can be separate on cell sorter? Is it for example range of 100 nm or 10 nm?
    Answer
    but dont use for forward scatter a diode but a photomultiplier (PMT)
    Question - How can I sort regulatory T cells out of a population of PBMCs? How good is the perfomance of a MACS system?
    Answer
    you can isolate regulatory T cells by cd4+ cd25+ (high) withe a sorter like moflo-xdp or aria bd from lymphocytes population with a purity around 90%,
    Question - How can I sort regulatory T cells out of a population of PBMCs? How good is the perfomance of a MACS system?
    Answer
    you can isolate regulatory T cells by cd4+ cd25+ (high) withe a sorter like moflo-xdp or aria bd from lymphocytes population with a yeald by 90%,
    Question - Does anyone has an idea how small particles (subcell dimension) can be separate on cell sorter? Is it for example range of 100 nm or 10 nm?
    Answer
    I only know it is possible with moflo-xdp sorting chromosomes
    Question - Is there any easy and low cost method to check smooth muscle cells viability?
    Answer
    Sytox green works verry well. Analysis with flow cytometry or fluorescent microscope.
    What is your assay for intracellular calcium level. Do you use flurescent dye too?
    Question - Can one freeze CFSE labelled cells?
    Answer
    CFSE fluorescence resists freezing
    Question - Live/Dead cell vitality assay that doesn't kill cells within scaffold
    Answer
    Hi Moritz,
    I use Hoechet 33342 and Allura Red AC (Sigma-Aldrich 458848) for Live/Dead cell vitality assay for sperm sex-sorting. It's perfect. So live sperms are Hoechst 33342 pos. and Allura Red AC (Food Dye o,01 g/ml FD&C #40) wich quenches the fluorescence of hoechst 33342 in cells with compromised cell membranes.
    Question - How can I read the results of a FITC-PNA/PI, CTC, SCSA and SYBR-14/PI assays in mammalian sperm?
    Answer
    So if you don't understand anything because its gernan please ask me
    Question - SCSA Software
    Answer
    Ask Prof. Heinrich Bollwein hbollwein@vetclinics.uzh.ch
    Kontakt: Direktor
    Klinik für Reproduktionsmedizin
    Departement für Nutztiere
    Vetsuisse-Fakultät Universität Zürich
    Winterthurerstrasse 260
    CH-8057 Zürich
    Question - How can I read the results of a FITC-PNA/PI, CTC, SCSA and SYBR-14/PI assays in mammalian sperm?
    Answer
    Hi Juan,
    look at my thesis http://edoc.ub.uni-muenchen.de/1338/. But note the verry few SCSA pos. (red) sperms by bulls (only 1-3 %) because the verry compacte chromatin structure.
    Question - Ramos cells dying after FACS sorting
    Answer
    Try 100 um nozzle and 20-25 PSI
    Question
    Question - If I set up a FACS cell sorter to deposit one cell per well of a microtiter plate, what rate of errors (e.g. zero or two cells per well) can I expect?
    Answer
    So I woked with facs Aria III and Moflo-XDP. If you sort on Moflo-XDP please use the single mode (all negative events are aborted and all sorted drops must contain only one cell) not as normal purify mode. And you select droplet envelop 0,5 Drop. Verry importent is to discriminate oublets with gating FCS width and SSC width. All events with a higher FCS width or SSC width are doublets. Dont sort the doublets!!! So you can expect only one cell per well and no empty well. And calibrate the Home position (first well) and the End position (last well) accurately with the CyCLONE Configuration.so your cells fall in the center of the wells.
    Good luck
    Question - How do I perform a 1:2 antibody dilution in 200ul of FACS buffer?
    Answer
    Hi Aroon if u put 20 ul in 200 (exactly 180 ul) u get a dilution 20 ul Ab) in 200 ul so it is a dilution 1:10
    Question - Can normal primary antibodies and FITC conjugated secondary antibodies be used to isolate Cancer Stem Cells by the FACS method?
    Answer
    But work in trhe darkness sorting too because fitc is very photosensitive
    Question - Can normal primary antibodies and FITC conjugated secondary antibodies be used to isolate Cancer Stem Cells by the FACS method?
    Answer
    Yes why not?
    Question - Is MoFlo cytometer and sorter reliable, durable, friendly, or simply better than other sorters?
    Answer
    i think for S2 ( biosafety cabinet)lab the Facs Aria III is the best
    Question
    Question - SCSA Acridine orange prtocol
    Answer
    Please remember DFI sperms % by men and horses are arount 10-20% and some sperms are verry red but by bulls it is normal 2-4 % DFI sperms and weak red. The reason is the chromatin structur (Protamin) Men and stallions have less Disulfid bindings.
    Although sex-sorted sperm have been used for AI and IVF for over a decade there is still need to improve the technology as the results are highly variable. The goal of the present study was to assess the effect of seminal plasma and seminal plasma proteins as a supplement to sorted sperm on subsequent embryonic development, as a beneficial effect of these substances has been reported. In vitro matured oocytes were fertilized in vitro with either unsorted sperm (n = 215; Group 1), bulk sorted sperm (n = 226; Group 2), bulk sorted sperm extended in the presence of 1% seminal plasma (n = 185; Group 3) or bulk sorted sperm supplemented with seminal plasma proteins (4 mg mL(-1); n = 254; Group 4). An additional group of oocytes (n = 307; Group 5) was fertilized with the semen of another bull routinely used for IVF and served as a laboratory standard control. Subsequently, the presumptive zygotes were cultured for 8 days under standard conditions (SOFaa, 39 °C, 5% CO(2), 5% N(2)). Cleavage rates were assessed on day 3 p.i. (post insemination; group 1: 30.5 ± 14.7%; group 2: 28.8 ± 9.8%; group 3: 20.8 ± 14.9%; group 4: 25.7 ± 8.2%; group 5: 54.8 ± 11.5%). Development rates were documented on days 7 p.i. (group 1: 7.3 ± 6.6%; group 2: 5.6 ± 3.1%, group 3: 6.2 ± 7.7%, group 4: 6.7 ± 5.9%, group 5: 20.2 ± 6.9%) and 8 p.i. (group 1: 8.9 ± 7.0%; group 2: 6.0 ± 2.9%; group 3: 8.6 ± 11.3%; group 4: 7.8 ± 6.2%; group 5: 23.3 ± 7.8%), respectively. Significant differences among cleavage and development rates could only be seen for Group 5 compared to all other groups. However, this difference between Groups 1-4 vs. Group 5 regarding the development rates on Day 8 could not be detected when assessing the development rates on base of the number of cleaved embryos instead of the number of oocytes fertilized (group 1: 31.4 ± 17.2%; group 2: 26.0 ± 21.0%; group 3: 33.3 ± 19.05%; group 4: 26.6 ± 17.8%; group 5: 42.6 ± 11.3%). The relative abundance of six different developmentally important gene transcripts (G6PD, HSP1A1, SLC2A3, BAX, BCL2L1, DNMT3A) was determined using single Day 8 expanded blastocysts of all five groups. No significant differences were seen among the embryos of the five groups. Our results show that neither the bulk sorting procedure nor the addition of seminal plasma or seminal plasma proteins, respectively, affected cleavage and development rates when sperm from a specific bull was used. Additionally, sorting and subsequent exposure of sperm to either seminal plasma or seminal plasma proteins did not influence mRNA expression in bovine IVP embryos.
    The aim of these examinations was to prove the suitability of various flow cytometric methods for the evaluation of sperm quality in stallions. The SYBR 14/PI staining was carried out for the determination of plasma membrane integrity, the JC-1 assay for the measurement of mitochondrial membrane potential, the SYTO 17/FITC-PNA/PI staining for the evaluation of plasma membrane integrity as well as acrosomal status and the sperm chromatin structure assay (SCSA) for the assessment of integrity of chromatin structure. All flow cytometric sperm parameters showed a good reproducibility. The results of measurements of three different straws of an ejaculate carried out independently of each other were similar (0.88 ≤ Intra-Class-Correlation- Coefficient ≤ 0.98). Sperm viability measured by SYBR 14/PI-, JC-1- and SYTO 17/FITC-PNA/PI-assay, did not differ (p > 0.05) and was highly related with each other (r ≥ 0.97; p < 0.0001). Only moderate relationships (-0.65 ≤ r ≤ -0.57; p < 0.0001) occurred between acrosomal status and viability of sperm. No correlations (p > 0.05) could be detected between changes in chromatin structure and disturbances in plasma membrane integrity and acrosomal status, respectively. As all viability assays (SYBR14/PI, JC-1 and SYTO 17/FITC-PNA/PI) provided similar results and the SYTO 17/FITC-PNA/PI staining is the only one of these assays, yielding additional information about the acrosomal status, the last mentioned triple staining is recommended in combination with the sperm chromatin structure assay for the flow cytometric analysis of sperm quality in stallions.
    The aim of these examinations was to check various flow cytometric methods to establish whether they are suitable objective methods for assessing the quality of cryopreserved sperm from stallions. The plasma membrane integrity, the mitochondrial membrane potential, the acrosomal status and the integrity of the chromatin structure in cryopreserved sperm from stallions were assessed. For this purpose various fluorescent stainings were carried out both immediately after the semen samples were thawed and after a three-hour period of incubation at 37 C with SYBR® 14, JC-1 and SYTO® 17 / propidium iodide / FITC-PNA and a sperm chromatin structure analysis (SCSA™) and were then evaluated. The sperm quality parameters obtained by flow cytometry showed good reproducibility. The results of measurements of three different straws of an ejaculate which were carried out independently of each other did not differ significantly. The intra-class correlation coefficients were between 0.88 and 0.98. The flow cytometric tests evaluated in this examination were therefore considered to be reliable. In order to demonstrate the correlations between the routine examination methods for assessing the sperm quality and the analysis processes evaluated in this study, the viability and the morphology of the sperms after thawing were assessed by light microscopy using bromide phenol blue nigrosine smears. A computer controlled motility analysis was also carried out. Moderate correlations (0.60 > r > 0.58; p < 0.0001) were obtained between the viability assessed by light microscopy and the percentage of vital sperms obtained by flow cytometry. The proportion of progressively forward moving sperms correlated positively (r = 0.50; p < 0.01) to the proportion of sperms with a high mitochondrial membrane potential. The relationships between the results of the SCSA™ test and the proportions of both primary and secondary sperm anomalies were weakly pronounced (r = 0.24; p < 0.01). In the flow cytometric assessment of the viability, the mitochondrial membrane potential and the integrity of the acrosome it was shown that the ejaculate-related variations were approximately the same as the variations between the stallions. Regarding the results of the sperm chromatin structure the individual variations (88 - 91%) were considerably greater than those dependent on the ejaculate (9 – 12%). It was also shown that the results of the flow cytometric sperm quality assessment did not vary significantly in stallions which had a period (four months) of sexual rest before providing ejaculate compared to stallions which ejaculates were collected with a regular frequency. In terms of the animals’ age there was only a weak relationship (r = 0.43; p < 0.01) with integrity of the sperm chromatin structure. The results of the flow cytometric methods for assessing the viability (SYBR® 14/PI, JC-1 and SYTO® 17/FITC-PNA/PI combination staining) were comparable (r > 0.96; p < 0.0001). The latter is the only one of these tests which provides additional information about the integrity of the acrosome of the sperms. The combination of this triple staining with the sperm chromatin structure analysis (SCSA™) also provides further important information about the sperm quality. To check the relationships between fertility of the stallions and the various sperm quality parameters obtained by flow cytometry, only seasonal pregnancy rates from fresh semen insemination were available. In terms of the viability and the acrosomal status of the sperms after thawing, these showed no correlations (p > 0.05) with the fertility of the stallions. On the other hand, a negative relationship was established between the integrity of the chromatin structure and the fertility of the stallions (r = - 0.51 / - 0.59). This study shows that the flow cytometric assessment of the quality of cryopreserved sperm from stallions represents a reliable and objective method. In addition to the routine assessment of sperm quality, it provides important additional information, for example about the intactness of the acrosome and the integrity of the sperm chromatin structure. Ziel der vorliegenden Untersuchungen war es, verschiedene durchflusszytometrische Verfahren dahingehend zu überprüfen, ob sie geeignete objektive Methoden zur Beurteilung der Qualität von kryokonserviertem Hengstsperma darstellen. Es wurde die Plasmamembranintegrität, das Mitochondrienmembranpotential, der akrosomale Status und die Integrität der Chromatinstruktur bei kryokonserviertem Hengstsperma beurteilt. Hierfür wurden verschiedene Fluoreszenzfärbungen sowohl direkt nach dem Auftauen der Samenproben als auch nach dreistündiger Inkubation bei 37 C mit SYBR® 14, JC-1 und SYTO® 17 / Propidiumjodid / FITC-PNA und eine Spermachromatinstrukturanalyse (SCSA™) durchgeführt und anschließend ausgewertet. Die durchflusszytometrisch erhobenen Spermaqualitätsparameter wiesen eine hohe Reproduzierbarkeit auf. Die Ergebnisse der unabhängig von einander durchgeführten Messungen von 3 verschiedenen Pailletten eines Ejakulats unterschieden sich nicht signifikant voneinander. Die Intra-Class-Korrelationskoeffizienten lagen zwischen 0,88 und 0,98. Daher sind die in dieser Untersuchung evaluierten durchflusszytometrischen Tests als zuverlässig zu bezeichnen Um die Zusammenhänge zwischen den herkömmlichen Untersuchungsmethoden zur Beurteilung der Spermaqualität und den in dieser Arbeit evaluierten Analyseverfahren darzustellen, wurde mit Hilfe von Bromphenolblau-Nigrosin- Ausstrichen lichtmikroskopisch die Vitalität und die Morphologie der Spermien nach dem Auftauen beurteilt. Zusätzlich wurde eine computergestützte Motilitätsanalyse durchgeführt. Zwischen der lichtmikroskopisch beurteilten Vitalität und dem durchflusszytometrisch erfassten prozentualen Anteil an vitalen Spermien bestanden mittelgradige Korrelationen (0,60 > r > 0,58; p < 0,0001). Der prozentuale Anteil an vorwärtsbeweglichen Spermien korrelierte positiv (r = 0,50; p < 0,01) mit dem prozentualen Anteil an Spermien mit hohem Mitochondrienmembranpotential. Die Beziehungen zwischen den Ergebnissen des SCSA-Tests und den prozentualen Anteilen sowohl an primären als auch an sekundären Spermienanomalien waren schwach ausgeprägt (r = 0,24; p < 0,01). Bei der flowzytometrischen Beurteilung der Vitalität, des Mitochondrienmembranpotentials und der Integrität des Akrosoms zeigte sich, dass die ejakulatbedingten Schwankungen in etwa gleich groß wie die Schwankungen zwischen den Hengsten waren. Nur bei der Beurteilung der Spermachromatinstruktur waren die individuellen Schwankungen (88 - 91 %) erheblich größer als die ejakulatbedingten (9 – 12 %). Weiter zeigte sich, dass die Ergebnisse der durchflusszytometrischen Spermaqualtitätsbeurteilung bei Hengsten, die eine mehrmonatige Deckpause vor der Ejakulatgewinnung einhielten, sich nicht signifikant von denen der Hengste, die im regelmäßigen Deckeinsatz standen, unterschieden. Bezüglich des Alters der Tiere bestand nur eine schwache Beziehung (r = 0,43; p < 0,01) zu der Integrität der Spermachromatinstruktur. Die für die Beurteilung der Vitalität der Spermien verwendeten durchflusszytometrischen Verfahren (SYBR® 14/PI, JC-1 und SYTO® 17/FITC-PNA/PI-Kombinationsfärbung) führten zu vergleichbaren Ergebnissen (r > 0,96; p < 0,0001). Die letztere lieferte als einzige dieser drei Tests zusätzliche Informationen über die Integrität des Akrosoms. Die Kombination dieser Dreifachfärbung mit der Spermachromatinstrukturanalyse (SCSA™) bringt darüber hinaus weitere wichtige Informationen über die Spermaqualität. Zur Überprüfung der Zusammenhänge zwischen der Fertilität der Hengste und den verschiedenen durchflusszytometrisch ermittelten Spermaqualitätsparametern standen in der vorliegenden Untersuchung nur die saisonalen Trächtigkeitsraten der Hengste aus der Frischsamenübertragung zur Verfügung. Hier ergaben sich hinsichtlich der Vitalität und des akrosomalen Status der Spermien nach dem Auftauen keine Zusammenhänge (p > 0,05) zu der Fertilität der Hengste. Dagegen war zwischen der Integrität der Chromatinstruktur und der Fertilität der Hengste eine negative Beziehung festzustellen. (r = - 0,51 bzw. r = - 0,59; p < 0,01). Die hier durchgeführten Untersuchungen zeigten, dass die durchflusszytometrische Beurteilung der Qualität von kryokonserviertem Hengstsperma eine zuverlässige und objektive Methode darstellt. Sie liefert in Ergänzung der herkömmlichen Spermaqualitätsbeurteilung weitere wichtige Informationen, wie z.B. über die Unversehrtheit des Akrosoms und die Integrität der Spermachromatinstruktur.
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