Sperm survival following colloid centrifugation varies according to the part of the sperm-rich fraction used.

Spermatozoa present in the first 10 ml (portion 1, P1) of the sperm-rich fraction (SRF)of boar ejaculates show increased resilience to cooling and cryopreservation compared to those in the rest of the ejaculate (Saravia et al. 2008) presumably because of differences in exposure to seminal plasma. A new technique for selecting the most robust animal spermatozoa, Single Layer Centrifugation (SLC), has recently been developed at SLU (Morrell & Rodriguez-Martinez 2009). It was shown that SLCof SRFusing Androcoll-P`m followed by storage at room temperature (22°C) in extender without antibiotics resulted in sperm preparations of high motility which survive longer than unselected sperm samples (Wallgren et al. 2008). However, it is not known how SLC using P1 instead of SRFwould affect boar sperm survival. Furthermore, the technique requires scaling-up to enable larger volumes of ejaculate to be processed. Scaling up has been shown to be possible for stallion spermatozoa (Morrell et al. unpublished data). In experiment 1, P1 from 12 ejaculates (4 boars, 3 ejaculates per boar) and 12 SRFfrom the same boars (again 3 ejaculates from each boar), collected on different occasions, were extended in Beltsville Thawing Solution (BTS) without antibiotics to achieve a sperm concentration of 100x106 mL. Aliquots (1.5 ml) of these sperm samples were prepared by SLC on Androcoll-P" by centrifugation at 300xg for 20 min. The resulting sperm pellets were washed in 5 ml BTS + bSA (5 mg/ml) by centrifugation at 500xg for 10 min. before resuspending in 1.5 ml of the same extender. The sperm suspensions were stored in a styrofoam box at room temperature (22°C) for up to 6 days. Sperm motility was assessedsubjectively on a daily basis using phase contrast microscopy, after incubating the sperm suspensions at 38°C for 30 min. Differences between means were tested for statistical significance by analysis of variance, where P < 0.05 was considered significant. In the second experiment ("scale-up"), larger volumes of extended semen were used, e.g. 3 ml or 4.5 ml were pipetted on top of 4 ml Androcoll-P" and 10 ml, 12.5 ml or 15 ml extended semen were pipetted on top of 15 ml Androcoll-P Large, an optimized formulation for larger tubes. After centrifugation, the sperm pellet was resuspended in 3 ml or 10 ml BTS with bSA (1.25 mg/ml) respectively. Sperm motility was analysed by computer assisted sperm analysis (CASA) on . 5pl aliquots of sperm samples placed in a pre-warmed Makler chamber (Sefi Medical Instruments, Haifa, Israel), depth 10 pm, using a Mika Cell Motion Analyzer (MTM Medical Technologies Montreux, Switzerland) and a microscope equipped with a warm stage and phase contrast optics (20x objective, Optiphot-2, Nikon, Japan). Two hundred spermatozoa per sample were examined. In experiment 1, the mean differences in % sperm motility between control (non-SLCselected)and SLC-selected samples were as follows: time 0 h, P1 + 13.3°/n, SRF +16.6%; at 24 h, P1 +23.7%, 5RF +23.4%; at 48 h, P1 + 10%, SRF +24.6%; at 72 h, P1 +9.6°4, SRF + 39.2%; at 96 h, P1 + 9.4°4, SRF +39.2%; at 120 h, P1 + 6.7%, SRF +25.8%. The non-SLC-