Single layer centrifugation improves stallion sperm motility after storage at 15ºC

Cooling of stallion semen for transport and subsequent artificial insemination has become widespread in equine reproduction. Cooled storage prolongs the lifespan of spermatozoa, however, this condition can also induce irreversible damages to structural integrity, biochemistry and biophysics of the sperm cell (Aurich C, Anim Repro Sci, 107:268-275, 2008). In addition, there is considerable variability in sperm survival of individual stallions during the process. Single layer centrifugation (SLC) is a colloid centrifugation technique designed to select spermatozoa with good motility, membrane integrity, normal morphology and intact chromatin from the rest of the ejaculate (Morrell JM & Rodriguez-Martinez H, Open Andro J, 1:1–9, 2009), improving sperm quality. The aim of this experiment was to evaluate the effect of SLC in stallion sperm motility after cooled storage at 15oC for 8 hours. During reproductive season and after two days of sexual arrest, ejaculates were collected from ten Crioulo breed stallions (n=10) with artificial vagina and an estrus mare. After collection, the ejaculate was filtered and diluted with commercial semen extender (EquiPlus®, Minitub GmbH, Tiefenbach – Germany) to 50x106 sptz/mL. Semen samples were refrigerated for 8 hours at 15oC and then submitted to two treatments: Conventional centrifugation (600G x 20’) and SLC (Androcoll Equine®, Minitub GmbH, Tiefenbach – Germany) (300G x 20’). In both protocols, the pellet was ressuspended with the same extender. Progressive motility (%) was evaluated by Computer Asssited Sperm Analyzer (AndroVision® Minitub GmbH, Tiefenbach – Germany) in three moments: 1) after collection and dilution, 2) after 8h of cooling (pre-centrifugation) and 3) after centrifugation and ressuspension (post-centrifugation). Descriptive statistic and mean comparison by Kruskal-Wallis test were performed in the program Statistix9® (p<0.05). The semen diluted had shown a 68.7±4.2 mean of percentage progressive motility. Along the cooled storage period, the sperm motility mean dropped significantly (p<0.05) to 48.7±6.2 (Conventional centrifugation) and 49.9±6.0 (SLC). The sperm percentage motility after conventional centrifugation and ressuspension (45.2±5.6) had no difference in comparison with the previous moment, while the SLC treatment resulted in 58.5±5.7. The mean of sperm motility after SLC was significantly higher (p<0.05) when compared with the moment pre-centrifugation . There was no statistical difference between the moment post-SLC when compared to the initial motility measured prior cooled storage demonstrating the efficiency of this method to improve the semen quality selecting the better sperm cells. Semen samples submitted to SLC had 29.4% higher progressive motility than the sample with normal centrifugation. It is suggested that these semen samples could be used for subsequently artificial insemination of mares or posterior methods to increase its shelf life. In the present experiment, it is demonstrated the beneficial effect in progressive motility of SLC after cooled storage at 15oC for a short period of time.