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CHAPTER 4.2
For the SLC group, 15 mL of the diluted semen was gently layered on top of 15 mL Androcoll-
E-Large (patent applied for) in a 50 mL conical bottom centrifuge tube. Two tubes were prepared as
such and centrifuged (Heraeus Multifuge 3 L-R, Kendro Laboratory Products, Waltham, USA) for 20
min at 300 × g. After centrifugation, the supernatant was removed as gently as possible without
disturbing the sperm pellet and leaving as little colloid as possible. The sperm pellet was
resuspended in 5 mL of INRA96 and washed by centrifugation for 10 min at 500 × g. Following
washing, the sperm pellet was resuspended to a final sperm concentration of 100 × 10 6 sperm /mL,
in 1.5 mL of supernatant from the cushioned group (which was filtered through tandem 5 µm and
1.2 µm sterile nylon syringe filters to remove any residual spermatozoa (Rigby et al., 2001)) and
BotuCrio. Adding back the supernatant ensured that the seminal plasma content of cushioned and
SLC treated samples was comparable. The diluted semen was loaded into 0.5 mL straws and frozen
as described above.
Fig. 2. Picture of equine semen diluted in INRA96® upon Androcoll-E for single layer centrifugation
(a) before and (b) after centrifugation (300 × g for 20 min). The picture on the right clearly
shows the selected sperm pellet.
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