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Centrifuging Methods

Equine-Reproduction.com Bulletin Board » General Stallion Questions » Centrifuging Methods « Previous Next »


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Carl Wood (Unregistered Guest)
Unregistered guest
Posted From: 69.21.9.118
Posted on Tuesday, November 22, 2005 - 10:37 am:   Edit Post Delete Post    Move Post (Moderator/Admin Only)

I have an older stallion whose motility is very low. We have been centrifuging his semen but feel that the method is not working well. We take the ejaculate and divide it into 4 centrifuge tubes and add as much extender as it takes to get to 40 mls in each tube. Then run for 15 minutes at 3800 rpm. We pull off all but 10 mls in the bottom of each and dispose of it. The remaining we add as much extender as we can to make an insemination dose of no more than 70mls. We find under the microscope many very motile sperm in the part we are disposing of. Is there a way to loose less of those sperm cells? Should we change what we are doing in the centrifuging process?
 

Jos
Board Administrator
Username: Jos

Post Number: 10356
Registered: 10-1999
Posted on Tuesday, November 22, 2005 - 03:31 pm:   Edit Post Delete Post    Move Post (Moderator/Admin Only)

There is an article on the site about centrifugation of semen that might help.

A couple of things spring to mind:
  • What G-force are you spinning at? 3800 rpm for most centrifuges is going to result in quite a high G-force. Obviously the actual force will depend upon the radius of the unit, but (for example) our IEC CL2 unit running at 2,600 and gives us 1,000 G! If you spin too hard you will damage the sperm membrane. Without use of a cushion, spinning at 400 G for between 6-12 minutes is the normal starting point.
  • In connection with above - how hard is the pellet you are ending up with? You don't really want a "pellet" (bad term although it's the one that's used) - more of a "sludge layer". You are better off losing sperm in the supernatant rather than spinning them too hard resulting in that membrane damage!
  • Have you tried fractionation of the ejaculate? With this technique, one captures only the first three ejaculatory spurts, which is where the ejaculated sperm are contained. The seminal plasma (which bulks out the volume, not the sperm numbers) follows in the ensuing spurts. Capturing only those first three spurts will increase the concentration and decrease the volume (discard the subsequent spurts that are captured in the lumen of the AV). This technique also reduces the potential for damage to the sperm as you're not centrifuging.
If you would like to answer the above, we can see if we can get things running a little smoother for you!
 

Carl Wood (Unregistered Guest)
Unregistered guest
Posted From: 69.21.9.81
Posted on Thursday, December 01, 2005 - 11:08 am:   Edit Post Delete Post    Move Post (Moderator/Admin Only)

I am using a Dynac centrifuge. It has four vials and it is 6 inches from center to center across the arms. I have no idea of what the g force is. This is the protocol I got from a vet at CSU. Although he did not know what type I had. The slurry in the bottom is not real tight but needs quite a bit of agitation to mix it all up again. It is not a pill but holds very tightly to the bottom of the tube. My worry was that when I look at the part that I draw off to throw away that there is quite a few very motile sperm cells in that. I can certainly slow it all down if that will help me out. Thanks Carl



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