Centrifugation Protocol

Printable Version

Centrifuge tube size: 50 ml
Pre-warm centrifuge shields, centrifuge tubes and skim milk extender to body temperature (37° C.)

1. Pour 20 ml raw semen in each centrifuge tube.
2. Add 20 ml skim milk extender pre-warmed to 37° C (body temperature).
Total volume extended semen = 40 ml per tube
3. Replace caps, then invert tubes gently 2-3 times.
4. Place tubes in centrifuge shields, then place in centrifuge.
   CAUTION - make sure tubes within the centrifuge are balanced.
5. Set timer for 10-12 minutes.
6. Set speed at 1000 rpms or 400 g's.
Click here to for RPM calculator
7. Allow centrifuge to slow without braking.
8. Remove tubes from centrifuge.
9. Pour off supernatant from each tube to 5-10 ml. and re-suspend pellet of sperm by gently agitating.
10. Add enough extender to bring concentration of progressively motile cells to 25-50 million/ml. Use worksheet to calculate volume of extender. Divide volume by number of centrifuge tubes used.

Worksheet

Stallion

• Volume of Centrifuged Semen: ml.
  
  
• Progressive Motility: %
  
  
• Count: million/ml.
  
  

A.	Total Cells (Count x Volume) / 1000 = billion cells.
B.	Assume 70% recovery from centrifuge: (.70 x Total Cells) = billion Total Cells after centrifugation.
C.	Divide Total Cells after Centrifugation (B) x 1000 by 25-501. to get Total Extended Volume. (B x 1000) / 25-50) = ml. of semen + extender.
D.	(B) / 1 billion = Cooled breeding doses: doses.

^1.Range of concentration after final extension needs to be between 25-50 million cells/ml. You may use any number within this range. We would suggest using a higher number for stallions with lesser quality semen or stallions that have a history of not cooling well.