view - Department of Reproduction, Obstetrics and Herd Health

CHAPTER 4.2
resulted in an improvement of sperm motility for up to 24 hours after thawing (Sharma and Agarwal,
1996).
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Different sperm selection techniques were compared such as the swim-up procedure and
the Percoll density gradient centrifugation which could process only limited volumes of semen,
whereas glass wool Sephadex filtration (GWS) and Leucosorb ® filtration (LF) allowed filtration of
larger volumes (Sieme et al., 2003). The latter two were evaluated when used prior to
cryopreservation. An increased post-thaw progressive motility (PM) for the GWS and LF treated
samples was reported, but since Percoll centrifugation could only be used to process small volumes
of 1 mL, the effect of density gradient centrifugation prior to cryopreservation was not evaluated. In
another study, glass beads column separation resulted in an increased post-thaw sperm quality
compared to unselected samples (Klinc et al., 2003). However, this technique requires specific
material not readily available.
The main drawbacks of gradient centrifugation can now be remedied since the technique
has been simplified to a single layer centrifugation (SLC) protocol using Androcoll-E with comparable
results for sperm yield and quality as for gradient centrifugation (Morrell et al., 2009a). Moreover, a
technique using Androcoll-E-Large allows processing of 15 mL of extended semen per centrifugation
tube (Morrell et al., 2009b), hence the problem of small volumes has been solved as well.
Not all stallions produce semen that can be cryopreserved with good results. In a large
French field study freezability of stallion semen was calculated by the ratio of the number of
selected ejaculates (PM>35%) over the total number of ejaculates frozen (Vidament et al., 1997).
Over a total of 427 stallions, it was found that about 50% of the stallions were non freezable or poor
freezers , about 25% were intermediate freezers and 25% were good freezers. The implementation
of Androcoll-E in the freezing protocol might be a solution to improve sperm processing of poor
freezing stallion semen.
The aim of this study was to evaluate equine sperm selection using Androcoll-E-Large in a
SLC prior to cryopreservation, by comparing this method with a cushioned centrifugation freezing
protocol. The latter has consistently yielded the best results for freezing equine semen to date,
making it an obvious point of comparison. In addition, it was evaluated whether the effect of the
treatment (Androcoll-E-Large versus cushioned centrifugation) was modified by the freezability of
the stallion as determined by number of ejaculates with post-thaw PM ≥30%.