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CHAPTER 5.2
A.
Fig. 5. Different cushioned centrifugation techniques: (A) conventional cushioned centrifugation of
semen extended in an opaque extender (INRA96®) using a conical 50 mL centrifugation tube
with 3.5 mL of clear MAXIFREEZE® directly underneath the sperm pellet (green arrow), and (B)
Glass nipple-bottom tube following centrifugation of semen extended in an optically clear
extender, with 30µL of clear cushion directly underneath the sperm pellet (red arrow) (Waite
et al., 2008).
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B.
Some of the downsides of centrifugation can be circumvented by the use of a cushion. This
technique, in which a protective fluid is placed at the bottom of the centrifugation tube underneath
the extended semen, was described over 25 years ago (Cochran et al., 1984), but has been refined
more recently (Ecot et al., 2005; Knop et al., 2005; Waite et al., 2008). The cushion fluid evolved from
high concentrated glucose solutions (Cochran et al., 1984), over egg yolk extenders containing
glycerol (Amann and Pickett, 1987) to iodixanol solutions (Revell et al., 1997). Various amounts of
these cushion solutions are applied, but with the introduction of commercially available cushions,
reports mention the use of 3.5 (Fig. 5A) up to 5mL of cushion fluid (Ecot et al., 2005; Knop et al., 2005;
respectively). The use of these cushions allows for longer centrifugation times and higher g-forces (20
min, 1000 × g). These so-called cushions are fluids with a higher density than the sperm cells so that
they are not compacted against the wall of the centrifuge tube (as with normal centrifugation, Fig.
6A); instead they are layered on the cushion. Since fluids are nor easily compressed, the high forces
(from the high speed centrifugation) will force the spermatozoa into the top layer of the cushion, as