After semen is centrifuged, how do you calculate it for extender? I know how to calculate for cooled semen but I am confused on how to do it with the small pellet. If you can give me an example on how it is calculated, that would help.
You need to determine (a) the number of sperm that were present originally, and (b) the recovery rate.
If you had an ejaculate that was 40 ml in volume, and 80 million sperm/ml, then there were 3.2 billion sperm present in the ejaculate.
If the recovery rate was 75%, then there were 2.4 billion sperm present in the recovered portion.
If you are shipping semen, then you want the final sperm concentration to be between 25 and 50 million per ml. With non-centrifuged semen, getting it as close to 25 m/ml is best, but that is less of an issue with centrifuged semen, as you have removed (most of) the seminal plasma, so we can take it to - say - 35 million/ml.
If we had a sperm pellet that was 6 ml left behind after centrifugation, and we want a final concentration of 35 m/ml that contains 2.4 billion sperm, then we would need to extend to a final volume of (about) 68 ml, so add 62 ml of extender and you're good to go!
An alternative route is to add some extender to your sperm pellet, and then determine the concentration again (using a hemacytometer), and calculate how much more extender to add to get to your final desired concentration.
Jos, using the above figures are you talking about progressivley motile sperm? Also, if you wanted to ship 2 baggies with 1 billion per bag you would use 29 ml of the extended semen per bag? Thanks for having such an informative board.
No - we're talking total sperm. We're not interested in progressive motility until you reach the point of insemination. If you ship a 1 billion (total) sperm, and have 50% motility at the time of insemination then you have 500 million progressively motile sperm (PMS), which is what one is shooting for. It requires knowledge of what that stallions sperm survival rate is after - say - 24 hours in order to be able to send a suitable shipment, but bear in mind the 500 million PMS is the upper end number - check out the "poor semen" article for more details on required numbers.
In the above example, you had a sperm pellet of 6 ml volume containing 2.4 billion (2400 million) sperm, so the concentration was 400 million/ml (2400/6=400). If you want to send 1 billion sperm, then you would need to send 2.5 ml of raw (centrifuged) semen. You want a final concentration of 25 million/ml, so you would dilute it 16 times (400/25=16), or which 1 part is the semen and 15 parts extender. Hence, 2.5 ml of semen and 37.5 ml extender = total final volume 40 ml for a single insemination dose.
Add extender either at a 1:1 rate, or to dilute to between 25-50 million sperm/ml, as is desired or deemed suitable.
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