Abstract. Use of sperm separation methods such as density gradient centrifugation for selecting the best spermatozoa for animal breeding is constrained by the problem of dealing with the large volumes of ejaculate produced by the males of some species, such as boars. The purpose of this study was to compare density gradient centrifugation (DGC) with centrifugation on a single layer of colloid (SLC) for the preparation of ejaculated boar spermatozoa using Androcoll TM -P. There was no difference between the two techniques in terms of sperm motility or duration of motility after selection, and sperm motility was retained for at least 24 h longer in the centrifuged sperm preparations than in controls (uncentrifuged aliquots). Sperm motility was significantly better (P<0.001) in the centrifuged sperm preparations (means ± sd: SLC 79.6 ± 8.1% and DGC 74.2 ± 12.0%) than in the uncentrifuged controls (62.9 ± 12.7%). The mean yield of motile spermatozoa for SLC was 67.5 ± 25.6%, and for DGC it was 59.6% ± 22.3% (not significant, ns). Sperm survival was significantly increased by colloid centrifugation (control 3.1 ± 0.3 days, SLC 5.5 ± 0.79 days, DGC 5.75 ± 0.62 days; P<0.001 for uncentrifuged versus centrifuged; SLC vs. DGC, ns). Moreover, boar spermatozoa could be stored for 24 h before centrifugation without any detrimental effect on sperm motility or duration of motility. In a further experiment, larger volumes of ejaculate were processed easily on a modified SLC, indicating that this method may be practical for processing large volumes of boar ejaculates. Key words: Boar spermatozoa, Colloids, Density gradient centrifugation, Single layer centrifugation (J. Reprod. Dev. 55: [547][548][549][550][551][552] 2009) ecently, density gradient centrifugation (DGC) has been suggested as a means of selecting animal spermatozoa for artificial breeding [1][2][3]. It is one of the methods recommended by the World Health Organisation for preparing human spermatozoa for assisted reproduction [4]. DGC has been used to prepare bovine spermatozoa for in vitro fertilization [5] and to prepare stallion spermatozoa for research purposes [6,7]. However, widespread application of the DGC-technique in processing animal spermatozoa has been hampered by a lack of animal-specific colloid formulations and the difficulty of processing sufficient spermatozoa for the large insemination doses required in domestic animals. Percoll TM DGC has been used previously to prepare animal spermatozoa for IVF, but the manufacturer (Amersham Biosciences, Uppsala) withdrew the product from the market for clinical use in 1996, reportedly because the polyvinylpropylene (PVP) it contains may damage spermatozoa [8,9].Recently, colloid formulations for use with animal spermatozoa, based on silane-coated silica, have been developed at the Swedish University of Agricultural Sciences (SLU) [10]. Preliminary studies with stallion spermatozoa have shown that the new colloid formulation could be used successfully as a single colloid layer for processing stallion sper...

Single-Layer centrifugation has been used to improve the quality of sperm samples in several species. However, where stallion or boar semen is to be used for AI, larger volumes of semen have to be processed than for other species, thus limiting the effectiveness of the original technique. The objective of the present study was to scale up the SLC method for both stallion and boar semen. Stallion semen could be processed in 100 mL glass tubes without a loss of sperm quality, and similarly, boar semen could be processed in 200 mL and 500 mL tubes without losing sperm quality. The results of these preliminary studies are encouraging, and larger trials are underway to evaluate using these methods in the field.

The objective of this study was to scale-up the procedure for Single Layer Centrifugation (SLC) through AndrocollTM-P, as a preliminary step towords processing the whole ejaculate. The first experiment compared Single Layer Centrifugation using 4.5 mL and 15 mL extended ejaculate (SLC-4.5 and SLC-15, resp.), assessing sperm quality by objective motility analysis, morphology, viability, and the production of reactive oxygen species (ROS). In the second experiment, SLC-4.5 was compared to Single Layer Centrifugation with 25 mL extended ejaculate (SLC-25) using motility analysis and morphology. In both experiments, normal morphology and linear motility were significantly higher in the SLC-selected samples than in the uncentrifuged controls (P < .001), whereas total motility and membrane integrity were unchanged. Although ROS production was higher in the SLC-selected samples than in the controls (P < .01), this might have been due to the presence of antioxidants in seminal plasma in the latter. In conclusion, there was no difference in sperm quality between SLC-4.5 and SLC-15 samples, or between SLC-4.5 and SLC-25 samples, indicating that the SLC method can be scaled-up successfully.