Can Single Layer Centrifugation with Androcoll-Equine improve freezability in poor freezing stallions?

Despite much research to improve cryosurvival of stallion sperm, it is not always possible to obtain acceptable postthaw motility i.e. ≥ 30%, from all ejaculates. In some studies, colloidal centrifugation has been shown to improve cryosurvival and to increase the number of stallion ejaculates that can be frozen with acceptable post-thaw semen parameters. The use of Single Layer Centrifugation (SLC) and the inclusion of a prolonged equilibration step prior to freezing were tested on sperm samples from stallions known to be poor freezers. An ejaculate was collected from six “poor-freezing” stallions using an artificial vagina; Gent extender (Minitub GmbH; Tiefenbach, Germany) was added, adjusting to a concentration of 100x106 sperm/mL. The samples produced were treated as follows: control samples were either centrifuged immediately, or after 8h storage at 5°C or 15°C; in each case the sperm pellet was resuspended in EquiPlus Freeze (EF; Minitub GmbH; Tiefenbach, Germany) and frozen. For the SLC samples, the SLC was either carried out immediately after semen collection or after storage of the samples for 8h at 5°C or 15°C, followed by freezing. All sperm pellets were resuspended in EF at a concentration of 200x106 /mL for freezing in 0.5mL straws. After thawing, the percentage of sperm with progressive motility was assessed by AndroVision (Minitub GmbH; Tiefenbach, Germany) and treatment means were compared using Kruskal-Wallis and Tukey tests (P < 0.05). None of the control samples gave acceptable post-thaw motility regardless of whether they were processed immediately (mean 8.41%± 2.1) or after storage (5o C, 4.63%± 2.1; 15o C 3.87% ± 1.9). However, all SLC samples processed immediately had a post-thaw motility ≥ 30% with a significant different average of 46.02%± 5.3 in comparison with all other means. None of the samples that were stored before SLC had acceptable post thaw motility (5o C, 11.55%± 2.9; 15o C 15.16%± 1.5). In summary, for some poor freezing stallions, processing by SLC and freezing immediately results in samples with acceptable post-thaw motility