349 IMPROVING THE FUNCTIONALITY OF STORED FLOW CYTOMETRICALLY SEX-SORTED BOAR SPERMATOZOA: SEDIMENTATION VS. CENTRIFUGATION

Abstract

Centrifugation has been the routine procedure used to concentrate the highly diluted spermatozoa obtained after sorting. However, this harmful procedure has a detrimental effect on the lifespan of the spermatozoa, particularly when a short-term storage period is required to perform the inseminations in the farms. The aim of the study was to evaluate the procedure of concentration by sedimentation of the flow cytometrically sex-sorted boar spermatozoa after a 24-h storage period, in terms of sperm functionality and recovery efficiency. Spermatozoa were sex-sorted by flow cytometry (Johnson and Welch 1999 Theriogenology 52, 1323-1342) and collected in 50-mL tubes containing 5 mL of Beltsville thawing solution (BTS) with 10% seminal plasma (SP). The collected samples were split into two aliquots. The first aliquot was concentrated by centrifugation (800g/5 min) just after sorting and stored at 17°C for 24 h at a concentration of 6 × 10⁶ sperm/mL. The second aliquot was directly stored (0.5 × 10⁶ sperm/mL) at 17°C for 24 h. In this case, after storage the supernatant was discarded and the pellet obtained by sedimentation was adjusted to 6 × 10⁶ sperm/mL. For assessment of the sperm functionality after the storage, motility and viability of the spermatozoa were analyzed using a CASA system (Sperm Class Analyzer; Microptic, Barcelona, Spain) and a EPICS XL flow cytometer (Coulter Corporation, Inc., Miami, FL, USA) after the sperm were stained with propidium iodide and SYBR-14, respectively. For assessment of the recovery efficiency, the total number of spermatozoa in each sample was counted. The results are expressed as percentages of five replicates. Statistical analysis was performed using a Dz test. Percentages of motile and viable spermatozoa just after sorting were 80% and 83.3%, respectively. After storage at 17°C for 24 h, the percentage of viable spermatozoa was significantly higher (P < 0.05) in settled spermatozoa (82.4%) compared to centrifuged spermatozoa (63.3%). Similarly, the percentage of motile spermatozoa was significantly higher (P < 0.05) in settled spermatozoa (78.3%) compared to centrifuged spermatozoa (58.3%), as was the percentage of progressive motility (63.6% vs. 35.5%, respectively; P < 0.05). Interestingly, the percentage of cell recovery was significantly higher (P < 0.05) after sedimentation (80% of the sorted population) than after centrifugation (60.3% of the sorted population). The results show that sedimentation is more beneficial than centrifugation when short-term storage of sex-sorted boar sperm is required.

This work was supported by CDTI and Fundación Seneca.