Step by Step Directions for the use of MaxiFreeze Step 1: Collect your stallion, filter semen and determine the concentration of the semen. Step 2: Dilute the semen 1:1 with the Solution A. Solution A should be added as soon as possible after collection. The semen and solution A should be in a 50ml centrifugation tube. Make sure to leave air in the tube, if the tube is filled too full, the sperm will be damaged. Step 3: The addition of MaxiFreeze Using the pipette provided, pass 3 - 3.5mls of Maxi Freeze through the extended semen and slowly place the MaxiFreeze on the very bottom of the 50ml tube. You will see the top layer rise. MaxiFreeze should sufficiently fill the curved section of the centrifuge tube. Example of placing MaxiFreze in the centrifuge tube. Pictures on the right show examples of this method and the pictures on the left demonstrate the traditional method without a cushion. Step 4: Centrifuge up to 1000g for 20 minutes at 22oC After Centrifugation, a sperm-rich layer just above the interface can be seen. Examples shown have the MaxiFreeze sperm pellet suspended between the two solutions in the left tube. Examples in the right tube, show conventional method with the sperm pellet packed on the bottom of the tube, compared to the floating pellet Step 5: Remove Solution above the sperm pellet Step 6: Remove MaxiFreeze by using a syringe and pipette, stab through the sperm pellet and remove solution as pictured below. Step 7: Add desired volume of freezing extender at room temperature in order to obtain 100 million spz/ml. No fastidious hematocymeter counting is necessary because you have essentially removed most of the sperm. Note: This centrifuge method can be used for both fresh, cooled semen and for freezing semen!