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- Carefully remove the sperm pellets from all the tubes and combine.
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- Remove 10 µl of sperm and mix with 90 µl of the clear centrifugation buffer in a small tube. DO NOT USE A MILK EXTENDER FOR THIS STEP! Determine the concentration of this sample and multiply by 10 to get the concentration of the sperm pellet.
In this example, the concentration of the centrifuged sample is 211 million sperm/ml X 10 = 2110 million sperm per ml. If a breeding dose is 1000 million sperm then 0.479 ml is diluted in 20 ml of extender. If you want to round up, you could give the client 0.5 ml as this would only give extra sperm.
The breeding dose, 1000 X 106 sperm, is then diluted up to 20 ml with the extender. Alternatively you could also just add 20 ml to the breeding dose. This is all done at room temperature. The diluted semen is then packaged as you would a noncentrifuged dose in a storage bag, tube or syringe.
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