Some Tips and Discussion about Centrifuging Semen

WHY CENTRIFUGE SEMEN?

There are a number of reasons why one would choose to centrifuge stallion semen other than the obvious reasons such as during preparation prior to freezing.

One of the commonest of reasons is that the sperm concentration of the raw ejaculate is so low that it makes processing of a suitable insemination dose difficult for transported use. Ideally one ships an insemination dose of 1 billion progressively motile sperm (at the time of shipment) which, allowing for an arbitrary 50% die-off rate, provides the desired insemination dose of 500 million progressively motile sperm. In order to achieve a successful shipment of fertile sperm however other parameters must be considered. To achieve a minimum die-off rate sperm should be shipped at a final concentration of between 25 and 50 million per ml; it is also necessary (if using a NFDMS such as Kenney extender) to extend the raw semen at a minimum rate of one part of semen to three parts of extender (1:4 or greater may be preferable); and finally, it is desirable that the total volume of the insemination dose be no more than approximately 80 ml (and 50 ml is more desirable yet). All of these parameters may not be obtainable if the initial sperm concentration of the ejaculate is too low - typically if below 100 million sperm/ml at the time of collection.

Another common reason for centrifugation of semen is to remove seminal plasma which is harmful to sperm over an extended period of exposure. It is one of nature's little jokes that all equine seminal plasma is toxic to sperm over an extended period, however with some stallions this negative effect is far greater than others, and with those animals it becomes essential to not just dilute semen with extender, but to actually remove the majority of the seminal plasma prior to extender addition. With many so-called "poor longevity" stallions, this will result in semen that may be successfully stored or transported, whereas without centrifugation the semen must be used almost immediately.

CENTRIFUGE SIZE

While the slightly more advanced breeder wants to avoid having to purchase too much additional equipment, dual use of a centrifuge for semen and serum (for progesterone or other blood-hormone assays) may be a little tricky, although technically possible. For semen one will ideally have a centrifuge that will hold 50 ml tubes, whereas for serum one wants one that holds 15 ml tubes. How to get around this problem will depend a little on what one is planning on using the semen for - i.e. freezing it, or just super-concentrating it for cooled or immediate use. If planning on freezing, there really is no option but to get a centrifuge that accepts 50 ml tubes, as it is essential that there is a minimum of handling done in the shortest time frame, typically at lower speeds. If one is going to use the centrifuge for super-concentrating fresh semen for cooled or immediate use, one can get away with using a centrifuge that accepts a 15 ml tube. There is a possibly convenient alternative, which would be to have two different rotors for the same centrifuge, but one would have to go with a larger size centrifuge in order for it to accept the 50 ml tubes, and the larger machines do not spin as fast as the smaller, which would means that one would probably have to spin the blood for longer - not a big issue really, just a little inconvenient.

THE PROCESS

Speed and duration of centrifuging will vary a little from stallion to stallion, and depending on what one wants to do with the resulting sperm pellet.

If one is using a 15 ml centrifuge, and the sperm pellet is not to be for frozen, then two to three minutes at a mid-point setting should be sufficient to super-concentrate. There will still be some sperm left in the supernatant (the plasma portion in the upper section of the tube), but the majority of the sperm will be at, or towards the bottom, in a loose pellet. Remove the upper 10 cc of the supernatant, and the remaining 5 cc should give a sufficient yield. One should however check the supernatant, and if there are still a lot of sperm left, one may wish to increase the duration of the spin. If a 50 ml tube is being used, then one’s either going to have to experiment, or do math....

Essentially, the starting point for producing a sperm pellet would be 400 G for 6 minutes ("G-Force", "Relative Centrifugal Force" and "RCF" are all the same thing). With experimentation, one will find the optimum time and settings for a particular stallion. Some stallion's semen will tolerate being spun harder for a shorter period, some require a lower G for a longer period. I have had some that when spun at that starting point, give me a nervous breakdown when I resuspend and look at it under a microscope, as there's only 5% progressive motility!

HOW DOES THE SETTING ON THE CENTRIFUGE EQUATE TO RELATIVE CENTRIFUGAL FORCE (RCF or G-FORCE)?

To obtain an accurate determination of RCF, one must have the centrifuge rotation speed calibrated. This can be achieved with the built in or hand-held RPM tool and performing the above calculations. If there isn't a method of checking settings available, then it is possible (although not preferable) to "play it by ear". Spinning in a 50 ml centrifuge at 400 G is not really fast. It is under the halfway setting on the old IEC "Clinical" model centrifuge (the CL2 model provides a setting to give a constant RPM). To experimentally establish the correct setting, one takes an extended semen sample and spins it in a conical bottom centrifuge tube. The desired result is a soft sperm pellet. If one ends up with a pellet that is hard to resuspend, then it was centrifuged too hard, so back off on either the speed or the duration, or both. Obviously with this method, it's a little "hit and miss", but over time one gets a pretty good idea of what the averages are with one's centrifuge. There is a view of what a centrifuged sperm pellet should look like available by clicking here.

USING CENTRIFUGATION TO REMOVE GEL FRACTION

Some people have used centrifugation to remove the gel fraction of an ejaculate, if present. I have had limited success with this method, and have found it to be a tricky process, with success rates that seem to vary from stallion to stallion. If one can get the centrifugation rate set to allow a layering effect, with the sperm pellet at the bottom, and then the layer of gel, I have found that the gel layer can be removed by sucking it off with a shortened insemination pipette on a syringe. Attempting to remove it with a syringe and needle doesn't work as it is too dense for the needle. I have even tried using an IVF catheter, which has a larger bore than a needle (but smaller than an insemination pipette), but still without success. The pipette however allows the "picking up" of the gel portion - almost in the same manner as separating an egg white out from a yolk. I did find though, that some stallions had gel that would centrifuge into the sperm pellet, or others required such a hard centrifugation rate to separate it that the sperm were damaged. I had one stallion that would ejaculate about 50 ml gel free and 200 ml of gel, and with him we would not centrifuge, but simply triple or quadruple filter the semen until we had removed all the gel fraction, using milk filters - the filters from ARS for the Colorado AV were too fine and simply clogged. Fractionation of the ejaculate at the time of collection to capture only the first three ejaculatory (sperm-rich) spurts may be an easier solution with a stallion with high gel volume.

On a final, but important note: Don't use the rotor brake on the centrifuge if it has one when stopping it after spinning semen. Allow it to slow down by itself, otherwise those lil' ol' sperm bang their heads against the centrifuge tube wall, and that really upsets 'em!