C.B.A.R. Silva1; G.A.M. Santos2; R.S. Yamatogi3; J.P. Araújo Jr.3; F.F. Souza2
The objectives to sort canine sperm are obtain offspring sex-specific for commercial purposes, and as a tool for endangered species conservation. Currently, the most feasible way to separate the sperm cells according to the sex chromosome is flow cytometry, but in the dog it is still not an applicable method. Thus, aim of this study was to evaluate the effectiveness of PercollTM density gradient and canine sperm cell viability after separation. The discontinuous gradient was composed of three PercollTM densities (90, 85, and 80%). The spermatozoa were evaluated pre- and post-centrifugation as to motility, vigor, number of spermatozoa, membrane integrity, and sperm morphology. An aliquot of each sample (19 ejaculates) was deposited on the surface of the gradient and centrifuged at 560 x g/30 minutes. Pre- and post-centrifugation aliquots were analysed by quantitative PCR using a sequence from F9-X chromosome and SRY-Y chromosome genes.
After centrifugation there was a decrease in sperm quality with reduced sperm motility (85.3 ± 8.3 versus 40.6 ± 22.6%, p < 0.001), vigor (4.5 ± 0.6 versus 2.2 ± 1.1, p < 0.001) and sperm number (133.3 ± 62.6 x 106 versus 15.3 ± 10.1 x 106 spermatozoa, p < 0.001). Nevertheless, there is no change in membrane integrity (80.8 ± 8.9 versus 80.9 ± 9.5%, p = 0.95) and percentage of morphologically normal cells (66.2 ± 12.9 versus 69.5 ± 14.5%, p = 0.19). The qPCR identified 41.2% cells X and 58.8% Y pre-centrifugation and an increase (p = 0.019) in the ratio X/Y after centrifugation (49.3% X and 50.7% Y). Density gradient centrifugation in PercollTM maintains canine spermatozoa with acceptable quality to artificial insemination, but there is no enrichment of cells containing X-chromosome to be a commercially viable method.
Financial support: FAPESP-2012/20670-8.