there are many different ways to do this test, but which one is best ? thus I'd like to know yr opinion about that. thank u
In my experience, bull spermatozoa with damaged acrosome are dead, too, so any viability assay would give youe the same info as the acrosome probes. So I woudln't bother about acrosome integrity.
You can assess the acrosome integrity using Calcium ionophore. it's indicator of fertilizability of sperm.
Hi,
You have 2 solutions :
microscopy (with a DIC analyse)...but you need a great experience in this field to analyse your sperm cells
flow cytometry : with or without calcium ionophore...You will use as probes : PI (live or dead), PNA (damage acrosome or not). There are a lot of publications talking about this assessment.
But if you whant more details, don't hesitate !
bye
Hi Eli, good to see you!
My 2 cents on the Ca-ionophore - you can induce acrosome damage with it but it isn't physiological, so it's hard to interpret. You induce a non-physiological damage the detect it - I'm not sure it's meaningful...
Hi !
yes it's true, the induction with Ca iono is not "physiological"....but it's depand of what do you want to do !! if we are talking about relationship with fertility.....the differences among ejaculates (from bulls) are not so important. So the use of stresfull conditions can increase this differences, so it's easier to establish a link with your fertility !
if you are always working in the same conditions (for all your ejaculates) you can interpret your results by making comparison amongs them...
By my experience the flow cytometry is the most precise one.
PI and PSA/PNA in fluorescence microscopy or cytofluorimeters are reliable markers for integrity of acrosome and cytoplasmic membranes.
Dear Falah;
I would suggest Naphthol Yellow S plus Aniline Blue stain for Bright Field Microscopy while for WET-fixed samples Phase Contrast or DIC microscopy would be of choice as a practical and affordable option.
EXPERIENCE compulsory.
Acrosome Reaction (AR) test using Ca ionophore surely provide better and more reliable results that would be related with fertilisability of sperm cell in question (namely the bull and rams).
The sole presence of acrosome on the sperm cell would not be sufficient enough for the cells integrity. I always recommend you to include functional tests (e.g. the AR, IVF or even thermoresistance at 23 or 37 degrees C, etc.) instead of using only classical parameters used in practice (motility, morphology, abnormal sperm, etc.)
Regards & good luck.
P.S.: Please find the following AR papers on this subject that would be of help.
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