Sperm Preparation

  1. Swimup
  2. Percoll

 

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Percoll Separation Procedure

As we will be using this procedure today

  • thaw two straws of bovine semen at 35°C for 60 seconds.
  • Pool the contents of the straws in a 12 x 55 mm tube and cap it closed.

The Percol method of sperm separation is an alternative to the swim-up method and utilizes a density gradient.  That gradient is made of Percoll which is small beads (they can not be seen in the microscope) coated with a chemical that does not interact with cells.  You will now attempt to use this technique. 

Layer 2 ml of 45% (v/v) Percoll on top of the 2 ml of 90% Percoll.  You must be very careful in doing this so as not to mix the two solution in the tube.  To begin:

  • Pipette 2 ml of 90% Percoll in to a 15 ml conical tube. 

  • Next pipette 2 ml of 45% Percoll into a 12 X 55 mm plastic tube. This may already be provided to you by your instructor.

  • Suck the 45% Percoll into a Pasteur pipette and carefully layer this over the 90% Percol.  Do this while tipping the 90% Percoll tube at a 45° angle and letting the 45% Percoll slowly go down the side of the tube. 

  • Using the same technique, layer the semen thawed from the 2 straws over the 45% Percoll.

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